# Patent

## Title
Selective inhibitors of tumor-initiating cells

## Abstract

Described herein are novel malignancy associated gene signature biomarkers, and assays and methods thereof, to classify prognosis or malignant potential of a cancer and identify cancer-initiating cells. The malignancy associated gene signature biomarkers, assays and methods described herein provide, in part, new methodologies to screen for novel drugs for treating cancers and tumors, such as, for example, triple-negative breast tumors. Using the assays and methods described herein proteasome inhibitors, histone deacetylase inhibitors, and glycolysis inhibitors, were identified as being highly effective in altering gene expression signatures specifically in malignant or cancer-initiating cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. §371 National Phase Entry Application of International Application No. PCT/US2012/027474 filed Mar. 2, 2012, which designates the U.S., and which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/449,353 filed on Mar. 4, 2011, the contents of each of which are incorporated herein by reference in their entireties.


GOVERNMENT SUPPORT
This invention was made with Government support under W81XWH-09-1-0670 awarded by the Department of Defense. The Government has certain rights in the invention.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 4, 2013, is named 033393-069632 SequenceListing.txt and is 776,090 bytes in size.
FIELD OF THE INVENTION
The present invention relates to novel malignancy associated response signatures associated with tumors, such as basal-like breast tumors, and methods and assays of diagnosis, prognosis, and treatment thereof.
BACKGROUND
Despite basic and clinical research directed at understanding and controlling breast cancer, breast cancer is still a major health threat worldwide. Basal-like breast tumors (BL-BTs) or triple-negative breast cancers (TNBCs) are an especially aggressive group of tumors often found in young Afro-American and Hispanic women, with the shortest survival of all breast cancer subtypes, including luminal A, luminal B, HER2, normal-like, and claudin-low tumors. These tumors comprise  ̃15% of human breast cancers, and more than 75% of breast cancers arising in women carrying a BRCA1 mutation.
TNBCs are estrogen receptor, progesterone receptor and HER2 negative (triple-negative), and can express different combinations of CK5, CK14, CK17, CK18 and/or EGFR. These tumors retain a mixed luminal/myoepithelial (i.e., progenitor-like) phenotype, and recent studies indicate that TNBCs derive from transformed mammary epithelial progenitors. Concomitant genetic ablation of RB and TP53, or BRCA1 alone, in the progenitor cell compartment of the mouse mammary gland induces the development of basal-like malignant tumors. Notably, mutations in TP53 and/or RB genes occur in over 90% of human BL-BTs.
BL-BTs have distinct morphological and cytological features. They are typically poorly differentiated lesions with high mitotic counts, central areas of necrosis, pushing borders and prominent stromal lymphocytic infiltrates. Basal-like cancer cells, which can be arranged in solid sheets or nests, are atypical and pleomorphic, and are highly enriched for cells with the CD44+/CD24low/− phenotype, which has been associated with tumor-initiating potential.
Despite response to front-line chemotherapy, tumors such as BL-BTs have a grim prognosis because of early relapse within the first five years of diagnosis. BL-BTs rapidly acquire resistance to chemotherapy and are refractory to endocrine therapy and HER-2 inhibitors. Therefore there is a need for identifying methods to diagnose and treat refractory tumors, such as basal-like breast tumors.
SUMMARY OF THE INVENTION
The invention provides novel malignancy associated response signatures and assays and methods thereof to classify and diagnose tumors, such as basal-like breast tumors and other poor prognosis tumors, using a newly identified gene expression signature identified in basal-like breast tumor cells. Moreover, the invention provides new methods to screen for novel drugs for treating cancers and tumors, such as basal-like breast tumors, using, for example, proteasome inhibitors, histone deacetylase inhibitors, glycolysis inhibitors, or a combination thereof, which were identified as being effective in altering the gene expression signature of malignant cells. The invention is based, at least in part, on findings of a genome-wide siRNA lethality screen in highly malignant progenitor-like breast epithelial cells, which recapitulate several basal-like features and give rise to tumors closely resembling human primary BL-BTs. As shown herein, we identified core survival pathways and a “malignancy associated response signature” that are selectively associated with tumor-initiating potential that are useful for prognostic and diagnostic applications and reveal novel therapeutic targets for more effective and enhanced treatment of poor prognosis tumors, such as BL-BTs.
Accordingly, in some aspects, provided herein are, malignancy associated response signatures for a cancer-initiating cell consisting essentially of an expression pattern of a set of biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151, where at least 5 of the 23 biomarkers have increased expression compared to a reference value.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a triple-negative breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a Luminal B breast cancer-initiating cell
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is an epithelial breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the expression of the at least 5 of the 23 biomarkers is increased at least 1.8-fold compared to the reference value.
Also provided herein, in some aspects, are malignancy associated response signatures for a cancer-initiating cell comprising an expression pattern of a set of at least 10 biomarkers set forth in SEQ ID NOs: 1-154, where at least 10 of the 154 markers have increased expression compared to a reference value.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a triple-negative breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a Luminal B breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is an epithelial breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the expression of the at least 5 biomarkers is increased at least 1.8-fold compared to the reference value.
In some embodiments of these aspects and all such aspects described herein, at least 2 of the set of at least 10 biomarkers set forth in SEQ ID NOs: 1-154 is selected from the group consisting of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to proteasomal gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, such that at least 3 of the 15 biomarkers have increased expression compared to a reference value.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to mitosis gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137, where at least 3 of the 14 biomarkers have increased expression compared to a reference value.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to RNA splicing gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151, where at least 3 of the 13 biomarkers have increased expression compared to a reference value.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to molecular transport gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147, where at least 3 of the 14 biomarkers have increased expression compared to a reference value.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to metabolic gene inhibition comprising an expression pattern of a set of at least 3 biomarkers of SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153, where at least 3 of the 13 biomarkers have increased expression compared to a reference value.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a triple-negative breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is a Luminal B breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the cancer-initiating cell is an epithelial breast cancer-initiating cell.
In some embodiments of these aspects and all such aspects described herein, the expression of the at least 3 biomarkers is at least 1.8-fold increased compared to the reference value.
In other aspects, provided herein are methods of classifying a cancer in a subject in need thereof, the method comprising: a) assaying expression of ten or more of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in a biological sample obtained from the subject having a cancer, and b) comparing the expression of the ten or more of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in the biological sample obtained from the subject having a cancer with a reference value, where increased expression of 1.8-fold or greater of at least ten of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least ten of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a triple-negative breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a Luminal B breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is an epithelial breast cancer.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer and if at least two of the ten or more genes having increased expression is a proteasomal malignancy associated response signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is bortezomib.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one histone deacetylase inhibitor to the subject.
In some embodiments of these methods and all such methods described herein, the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one glygolytic inhibitor to the subject. In some such embodiments, the at least one glycolysis inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer, relative survival rate, relative risk of metastasis, treatment option, or any combination thereof is determined for the subject.
In some aspects, provided herein are methods of classifying a cancer in a subject in need thereof, the method comprising: a) assaying expression of at least five biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151 in a biological sample obtained from the subject having a cancer, and b) comparing the expression of the five or more of the 23 malignancy associated response signature biomarkers in the biological sample obtained from the subject having a cancer with a reference value, where increased expression of 1.8-fold or greater of at least ten of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least ten of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a triple-negative breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a Luminal B breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is an epithelial breast cancer.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer and if at least one of the five or more genes having increased expression is a proteasomal malignancy associated response signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is bortezomib.
In some aspects, provided herein are methods of classifying a cancer in a subject in need thereof, the method comprising: a) assaying expression of at least three malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149 in a biological sample obtained from the subject having a cancer, and b) comparing the expression of the at least three malignancy associated response signature biomarkers in the biological sample obtained from the subject having a cancer with a reference value, where increased expression of 1.8-fold or greater of at least three of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least three of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a triple-negative breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is a Luminal B breast cancer.
In some embodiments of these methods and all such methods described herein, the cancer is an epithelial breast cancer.
In some embodiments of these methods and all such methods described herein, if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is bortezomib.
Also provided herein in some aspects, are assays comprising the steps of: a) measuring expression of ten or more of the 154 malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 in a biological sample obtained from a subject having cancer, and b) comparing the expression of the ten or more of the 154 malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 in the biological sample obtained from the subject having a cancer with a reference value, where increased expression of at least ten of the measured biomarkers in the biological sample obtained from the subject relative to the reference value diagnoses the patient as having a poor prognosis or malignant cancer, and absence of increased expression of at least ten of the measured biomarkers relative to the reference value diagnoses the patient as not having a poor prognosis or malignant cancer.
In some embodiments of these assays and all such assays described herein, the increased expression of the ten or more of the 154 malignancy associated response signature biomarkers in the biological sample determines a prognosis for the subject having the cancer.
In some embodiments of these assays and all such assays described herein, the prognosis comprises relative survival rate, relative risk of metastasis, treatment option, or any combination thereof.
In some embodiments of these assays and all such assays described herein, the cancer is a breast cancer.
In some embodiments of these assays and all such assays described herein, the cancer is a triple-negative breast cancer.
In some embodiments of these assays and all such assays described herein, the cancer is a Luminal B breast cancer.
In some embodiments of these assays and all such assays described herein, the cancer is an epithelial breast cancer.
In some embodiments, if the subject is diagnosed as having a poor prognosis or malignant cancer, the subject is administered at least one proteasome inhibitor.
In some embodiments of these assays and all such assays described herein, if the subject is diagnosed as having a poor prognosis or malignant cancer and if at least two of the ten or more genes having increased expression is a proteasomal malignancy associated response signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, the subject is administered at least one proteasome inhibitor.
In some embodiments of these assays and all such assays described herein, the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these assays and all such assays described herein, the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
In some embodiments of these assays and all such assays described herein, the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these assays and all such assays described herein, the at least one proteasome inhibitor is bortezomib.
In some embodiments of these assays and all such assays described herein, if the subject is diagnosed as having a poor prognosis or malignant cancer, the subject is administered at least one histone deacetylase inhibitor.
In some embodiments of these assays and all such assays described herein, the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these assays and all such assays described herein, the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
In some embodiments of these assays and all such assays described herein, if the subject is diagnosed as having a poor prognosis or malignant cancer, the subject is administered at least one glygolytic inhibitor.
In some embodiments of these assays and all such assays described herein, the at least one glycolysis inhibitor is an siRNA or antisense RNA agent.
Provided herein, in other aspects are assays comprising the steps of: (a) dividing a cell culture grown from a biopsy obtained from a subject having cancer into at least 5 separate cultures; (b) exposing each of the at least 5 separate cultures to a different inhibitory agent, wherein each of the different inhibitory agents specifically inhibits a different malignancy associated response signature biomarker set forth in SEQ ID NOs: 1-154; (c) growing each of the at least 5 separate cell cultures of step (b) for at least 12 hours; and (d) measuring viability of the cells from each of the cultures of step (c), where if the total viability of the cells in at least 60% of the cultures is decreased by at least 25%, then the biopsy obtained from the subject comprises cancer-initiating cells.
In some embodiments of these assays and all such assays described herein, the inhibitory agents are selected from siRNA agents, antisense RNA agents, or small molecules, that specifically inhibit any of the cancer gene signature biomarkers set forth in SEQ ID NOs: 1-154.
In some embodiments of these assays and all such assays described herein, the inhibitory agents specifically inhibit any of the malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the mitosis cancer gene signature biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the RNA splicing malignancy associated response signature biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the molecular transport malignancy associated response signature biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153.
Also provided herein, in some aspects, are methods for treating a cancer in a subject in need thereof comprising the step of administering to a subject having a cancer classified or diagnosed as having poor prognosis or being malignant using any of the methods or assays described herein, at least one proteasome inhibitor in a pharmaceutically acceptable carrier.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the proteasome inhibitor specifically inhibits a proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these methods and all such methods described herein, the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
In some embodiments of these methods and all such methods described herein, the proteasome inhibitor is bortezomib.
Provided herein, in some aspects, are methods for treating a cancer in a subject in need thereof comprising the step of administering to a subject having a cancer classified or diagnosed as having poor prognosis or being malignant, using any of the methods or assays described herein, at least one histone deacetylase inhibitor in a pharmaceutically acceptable carrier.
In some embodiments of these methods and all such methods described herein, the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
Provided herein, in some aspects, are method for treating a cancer in a subject in need thereof comprising the step of administering to a subject having a cancer classified or diagnosed as having poor prognosis or being malignant, using any of the methods or assays described herein, at least one metabolic inhibitor in a pharmaceutically acceptable carrier.
In some embodiments of these methods and all such methods described herein, the at least one metabolic inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these methods and all such methods described herein, the at least one metabolic inhibitor specifically inhibits a metabolic malignancy associated response signature biomarker set forth in SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153.
Also provided herein, in some aspects, are systems for obtaining data from at least one sample from a subject having a cancer, the systems comprising:
a determination module configured to receive the at least one sample from a subject having a cancer and perform an expression analysis of ten or more malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 on the at least one sample to generate an expression data output;
a storage device configured to store the expression data output from the determination module;
a comparison module configured to receive the expression data output of the sample from the subject having a cancer and perform at least one expression analysis on the expression data output to determine the presence or absence of one of the following conditions and produce a comparison data output where:

    
    
        (i) the sample from the subject having a cancer has increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154; or
        (ii) the sample from the subject having a cancer does not have increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154; and
    
    


an output or display module for displaying a content based in part on the comparison data output from the comparison module, where the content comprises a signal indicative that the sample from the subject having a cancer has increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154, or a signal indicative that the sample from the subject having a cancer does not have increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154.
In some embodiments of these systems and all such systems described herein, the content displayed on the display module further comprises a signal indicative of the subject being recommended to receive a particular treatment regimen.
In some aspects, provided herein are methods of identifying a candidate therapeutic agent against a cancer initiating cell comprising the steps of: a) exposing a BPLER cell culture to a test agent, where the BPLER cell culture comprises human breast primary epithelial cells (BPE) transformed with a defined set of genetic elements; b) measuring expression of at least ten of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in the culture, c) comparing the expression of the same at least 10 biomarkers of malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 as was measured in step (b) to an expression signature reference from a BPLER cell culture that has not been exposed to the test agent, wherein a decrease of expression of at least 5 of the at least 10 genes in the test culture compared to the expression signature reference indicates that the test agent is a candidate therapeutic agent against a cancer initiating cell.
Also provided herein are therapeutic agents identified by the methods of identifying a candidate therapeutic agent against a cancer initiating cell, where the agent is an siRNA agent, an antisense RNA agent, an antibody or antigen-binding fragment thereof, or a small molecule compound.
In other aspects, provided herein are pharmaceutical compounds comprising a therapeutic agent identified using any of the screening methods described herein and a pharmaceutically acceptable carrier.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1E demonstrate that BPLER cells are tumor-initiating epithelial progenitor cells that give rise to triple negative breast tumors. (1A) Schematic depicting the method of Ince and Weinberg10 used to generate pairs of genetically identical breast cancer cell lines at different stages of transformation from normal breast epithelial cells. Breast organoids maintained in chemically defined media (WIT and MEGM) were sequentially transformed with retroviral vectors encoding hTERT, SV40 early region and RASV12. (1B) Both BPLER and HMLER are triple negative by qRT-PCR. Luminal MCF7, mesenchymal MDA-MB-231 and HER2+ SKBR3 cells were used as controls. (1C) CellTiterGlo assay showing similar proliferation rates of BPLER, HMLER and BPE cells in WIT medium. (1D) Tumor incidence in immunocompromised mice 8 weeks after injection of the indicated numbers of BPLER, HMLER or MCF7 cells in Nu/J mice. (1E) qRT-PCR quantification of epithelial TNBC markers in BPLER and HMLER cells. MCF7 and MDA-MB-231 cells were used as controls for luminal and mesenchymal lineage, respectively. Data for each gene here and in (1B) were normalized to β-actin and then to the highest value in the cells tested. Data in (1B, 1C, 1E) represent the mean+/−SD of 3 (1B, 1E) or 6 (1C) replicates. Immunofluorescent staining showed selective co-expression of CK14 and CK18 in BPLER cells. Data in (FIGS. 1B-1E) are representative of at least three independent experiments. Unsupervised hierarchical clustering of mRNA expression of six BPLER tumors that arose in immunodeficient mice and a set of 337 human breast primary tumors (UNC337 dataset12) was performed. BPLER tumors clustered with TNBC.
FIGS. 2A-2G demonstrate identification of genes selectively required for survival of BPLER cells. (2A) Distribution of R value and BPLER Z score for all 17,378 genes in the siRNA primary screen library. R, ratio of viability of BPLER vs HMLER. BPLER Z score, a measure of the deviation of BPLER viability from the plate mean. A Z score outside of the range of −1 to +1 is significant. Genes were considered hits if R<0.75 and the BPLER Z score was <−1.5. Shading indicates the relative selectivity of BPLER vs HMLER lethality. (2B) Validation rates in the secondary siRNA screen for which at least one individual siRNA from the library pool scored positive. (2C) List of confirmed highly selective BPLER dependency genes or malignancy associated response signature genes. (2D) Single sample gene set enrichment analysis (GSEA) of the or malignancy associated response signature and highly selective hit list in 295 human breast primary cancers in the NKI dataset. A Z-score for the expression of the signature genes was calculated for each sample. The scores are shown as bean-plots to compare the distributions in the tumor subtypes (Basal, basal-like; Lum, luminal; NL, normal-like). Each bean consists of a line for each sample with the overall distribution for the subtype represented as a gray density shape and a black line indicating the median Z score. The basal-like and luminal B tumors as a group had significantly enhanced expression of TGS genes or the highly selective genes. (2E-2G) Patients were divided into two groups based on their tumor's GSEA enrichment score. The high malignancy associated response signature tumors were defined by enrichment scores with p-value <0.1, and the remaining tumors were classified as low. (2E) Kaplan-Meier survival curves for breast cancer patients in the NKI dataset, showing shorter survival of patients with higher tumor expression of all malignancy associated response signature genes (left) or the highly selective subset (right). (2F) Kaplan-Meier survival curve for luminal B breast cancer patients in the NKI dataset showing shorter survival of patients with higher tumor expression of all malignancy associated response signature genes (left) or the highly selective subset (right). (2G) Kaplan-Meier curve depicting metastasis-free survival for breast cancer patients in the NKI dataset, showing longer metastasis-free survival in patients with lower tumor expression of all malignancy associated response signature genes (left) or the highly selective subset (right).
FIG. 3 depicts a malignancy associated response signature functional interaction network. The malignancy associated response signature interaction network was constructed by incorporating physical and predicted interactions, co-localization, shared pathways and shared protein domains, compiled from a variety of genomic and proteomic sources using GeneMANIA20. Genes were shaded according to their participation in the indicated processes. The network was constructed using Cytoscape40.
FIGS. 4A-4H demonstrate that Epithelial TNBC cells are selectively sensitive to proteasome inhibitor drugs. (4A) Proteasome activity in BPLER and HMLER 6 hr after treatment with the indicated dose of bortezomib, as determined by ProteaGlo assay. At this time most cells are viable. (4B-4C) Viability of BPLER and HMLER cells, 24 hr after treatment with the indicated doses of bortezomib (4B) or doxorubicin (4C). (4D) Propidium iodide DNA staining showing accumulation of tetraploid cells (BPE and HMLER) or subdiploid cells (BPLER) 24 hours after treatment with bortezomib (12.5 nM). (4E) Immunoblot of lysates treated with bortezomib at the indicated dose for 24 hr. Each sample was assessed in duplicate independent samples. (4F) Viability of normal breast epithelial cells (BPE), ER+ (MCF7), HER2+ (BT-474), mesenchymal (MDA-MB-231, MDA-MB-436) or epithelial TNBC (4T1-E, BPLER, HCC-1143, HCC-1937) cells treated for 24 hr with bortezomib (12.5 nM) relative to vehicle control, as assessed by CellTiterGlo. (4G) Colony assay of HCC-1143 and 4T1-E cells treated with bortezomib (12.5 nM) for 18 hours and cultured for 2 weeks in drug-free medium. (4H) Tumorigenicity assay of 4T1-E cells treated with bortezomib (12.5 nM) or DMSO for 18 hours in vitro and injected orthotopically in the mammary fat-pad of Balb/c mice.
FIGS. 5A-5H demonstrate Bortezomib targets Mcl-1 dependency in TNBC through induction of Noxa. (5A) Immunoblot analysis of protein lysates from BPLER and HMLER treated with bortezomib (12.5 nM) or DMSO for 24 hours in the presence of ZVAD-fmk (20 uM). (5B) Representative DilC1(5) staining of BPLER and HMLER treated with bortezomib (12.5 nM) for 18 hours, as determined by flow cytometry. CCCP was used as positive control. The graph (right) shows median intensity value of DilC1(5) staining +/−SD from 3 independent experiments. (5C) Cell viability of BPLER cells 48 hours after transfection with siRNAs (50 nM) against the indicated genes and treated with bortezomib (12.5 nM) or DMSO for 24 hours. Data for each siRNA are normalized to 1. (5D) Baseline levels of Noxa mRNA, relative to ACTB, by qRT-PCR in normal breast epithelial cells at different stages of transformation (BPE, BPLE, BPLER (FIG. 1A)) and in breast cancer cell lines of indicated subtype. (5E) Immunoblot of protein lysates from MCF7 (luminal) and HCC-1143 or HCC-1937 (TNBC) breast cancer cells treated with bortezomib (12.5 nM) for 24 hr. (5F) Immunoblot of protein lysates from BPLER or HMLER treated with bortezomib (12.5 nM) or DMSO for 24 hours in the presence of ZVAD and immunoprecipitated with antibodies against Mcl-1 or Bcl-XL or rabbit IgG, showing similar and specific Mcl1/Noxa binding in BPLER and HMLER. (5G-5H) Cell viability of human breast cancer cell lines of different subtype 48 hours after transfection with siRNAs (50 nM) against the indicated genes or non-targeting siRNA (control). Data in (5B, 5C, 5D, 5G, 5H) represent the mean+/−SD of 3 replicates. All data are representative of at least 3 independent experiments.
FIGS. 6A-6J demonstrate proteasome inhibition suppresses TNBC growth in vivo. (6A) Proteasome activity in protein lysates from BPLER tumors 18 hours after intratumoral (i.t.), intraperithoneal (i.p) or intravenous (i.v.) treatment with bortezomib at the indicated dose, as determined by ProteasomeGlo assay. Proteasome activity in BPLER treated with bortezomib in vitro is shown as control. (6B-6E) Mean tumor volume (6B, 6D) in BPLER tumor-bearing mice after treatment with bortezomib or DMSO at the indicated dose, schedule and route of administration. Median tumor weight was measured at time of sacrifice (6C, 6E). Immunohistochemistry of BPLER tumors treated intratumorally with 0.8 mg/kg bortezomib or DMSO q3d, showed caspase-3 cleavage in residual epithelial cells in bortezomib treated tumors. (6F) Immunoblot analysis of protein lysates from BPLER tumors 18 hours after 1 intravenous dose of bortezomib at 1.6 mg/kg bortezomib or DMSO, showing efficient Noxa induction and caspase-3 and PARP1 cleavage after bortezomib treatment. (6G) Mean tumor volume in MB-468 tumor-bearing mice after intravenous treatment with 1.6 mg/kg bortezomib weekly. (6H) Schematic diagram illustrating generation of mouse TNBC primary tumor fragments for bortezomib-based therapy studies. Histological analysis of V22 mouse tumors sections showed typical features of human TNBC, including epithelial differentiation, scant stroma and CK-14 positive staining. 4T1-E tumors are shown as control. (6I, 6J) Mean V22 tumor volume (6I) after weekly intravenous infusion of bortezomib (1.6 mg/kg) or DMSO. Treatment was started 2 days after implantation of primary tumor fragments. Box-and-whisker plot (6J) shows median tumor weight at time of sacrifice. The top and the bottom of each box represent the 75th and 25th percentile of tumor weight, respectively. Upper and lower whiskers represent maximum and minimum tumor weight, respectively. The black horizontal band in each box corresponds to median tumor weight.
FIGS. 7A-7F demonstrate HDAC inhibitors target BPLER selectively. (7A) Connectivity map (CMAP) barview of changes in expression of malignancy associated response signature genes following in vitro treatment of cancer cell lines. Each black line represents an independent experiment that measured gene expression changes following treatment of an individual cancer cell line with the indicated drugs. Shaded areas are significantly enriched or depleted in malignancy associated response signature gene expression after drug exposure (p-values in Table 5). (7B) Viability of BPLER and HMLER cells treated with Trichostatin A, Vorinostat, Loperamide, Triamterene, Resveratrol or 4-5-dianilinophthalimide for 24 hr at the indicated dose. Data represent the mean+/−SD of 6 replicates. All data are representative of at least three independent experiments. (7C, 7D) mRNA expression, analyzed by qRT-PCR relative to ACTB, of malignancy associated response signature genes, predicted to be negatively regulated by TSA, in BPLER (7C) and HMLER (7D) cells 24 hr after treatment with 1 μM TSA. (7E-7F) Percentage of apoptotic cells (7E) and immunoblot analysis of PARP1 cleavage (7F) from BPLER 24 hours after treatment with DMSO, bortezomib (12.5 nM), vorinostat (10 uM) or bortezomib/vorinostat combination.
FIGS. 8A-8C demonstrate BPLER express epithelial breast tumor-initiating cell markers, retain an epithelial phenotype and form tumors that resemble TNBC in vivo (8A, 8B) Most BPLER and HMLER cells are CD44+/CD24low (8A) and ESA+ (8B) as assessed by flow cytometry. Luminal MCF7 and mesenchymal MDA-MB-231 were used as controls. (8C) Vimentin mRNA, assessed by qRT-PCR relative to β-actin, is poorly expressed, but clearly present, in BPLER compared to MDA-MB-231 and HMLER cells. MCF7 cells have no detectable vimentin mRNA. Data represent the mean+/−SD of 3 replicates. Data are representative of at least three independent experiments. Unsupervised hierarchical clustering of mRNA expression profiles of 6 BPLER tumors grown in immunodeficient mice, 40 human breast primary tumors (classified as either basal-like or non-basal-like), and 7 normal breast tissues (non-cancer, Richardson dataset13) was performed. BPLER tumors cluster with basal-like tumors, confirming data obtained with another tumor data set. Representative immunohistochemical staining of a BPLER tumor was performed. Intense staining indicated positivity for the indicated antigen.
FIGS. 9A-9G depict siRNA screen optimization. (9A) Reverse-transfection efficiency of BPLER and HMLER cells in WIT medium in 384-well standard tissue culture plates was assessed by flow cytometry using 12 commercially available liposomal formulations complexed with Cy3-labeled siRNAs. Four transfection reagents were able to introduce Cy3-labeled siRNAs into both cell lines with  ̃90% efficiency. (9B) Only Dharmafect #1 (Dharmacon) and Lipofectamine 2000 (Invitrogen) had acceptable toxicity (<25% decrease in cell viability) when delivering control non-targeting siRNAs and induced cytotoxicity after transfection with a cytotoxic siRNA targeting PLK1. Titration of Dharmafect #1 reduced toxicity in both cell lines to <15% without loss of transfection efficiency (data not shown). Thus Dharmafect #1 was chosen for the screen and used in all subsequent experiments. (9C) Using Dharmafect #1, 13 pools of siRNAs from the Dharmacon library were transfected in both cell lines to identify additional positive controls besides PLK1. Transfection of PLK1 and KIF11 siRNA pools similarly reduced viability, measured by CellTiterGlo assay, of both BPLER and HMLER after 72 hr, whereas the other siRNAs had little effect. (9D) Optimal time for assessing cell viability was evaluated by assessing viability at indicated times after transfection. 72 and 96 hr gave similar results, preserving viability of cells treated with non-targeting siRNAs and inducing maximal cytotoxicity with PLK1 and KIF11 siRNAs. (9E) Durability of gene knockdown in BPLER/HMLER cells, transfected with 3 different non-cytotoxic siRNAs and assessed by qRT-PCR analysis of mRNA expression, showed potent silencing in both cell lines after 48 and 72 hr, which was reduced after 96 hr. Based on data in (9D, 9E), 72 hr was chosen as the optimal time to perform the viability assay for the screen. Values in (9A-9E) represent the mean+/−SD of three replicates. (9F) To determine an optimal cell density for the screen, a CellTiterGlo viability assay of BPLER (upper) and HMLER (lower) cells plated for 24 hr at the indicated cell numbers in 384-well plates. The CellTiterGlo signal was linear in the range from 0 to 3000 cells/well. Cells were plated at 1000 cells/well for all screening experiments (primary and secondary screens). All data in (9A-9F) are representative of at least three independent experiments. To assess reproducibility of the high throughput screening conditions, a prescreen was performed by transfecting cells with a custom-made siRNA library containing non-targeting siRNAs (192 wells/plate) or si-PLK1 (192 wells/plate). The Z′ factor was calculated44. For both cell lines, Z′>0.7 in each of 6 microplates transfected in two separate experiments. An additional pre-screen was performed using a 500-siRNA library (Mitchison library), which returned 4 significant positive hits (data not shown). No edge effect was detected in any of these experiments. (9G) Ranked BPLER Z score values for all 17,378 genes represented in the siRNA primary screen library. A Z score with |Z|>1 is significant. We required hits to score with Z≦−1.5.
FIG. 10 demonstrates that enrichment of malignancy associated response signature genes in primary breast tumors predicts shorter metastasis-free survival. Kaplan-Meier curves depicting metastasis-free survival for breast cancer patients in the EMC286 dataset15-16, showing shorter metastasis-free survival in patients with higher tumor expression of all malignancy associated response signature genes (left) or the highly selective subset (right). Patients from the EMC286 dataset were divided into two groups based on their tumor's GSEA enrichment score. High TGS tumors were defined by enrichment scores with p-value <0.1, and the remaining tumors were classified as low.
FIGS. 11A-11C demonstrate BPLER cells are selectively sensitive to proteasome inhibitor drugs. (11A) Immunoblot analysis of protein lysates from BPE, HMLER and BPLER 24 hours after treatment with bortezomib (12.5 nM) or DMSO showing similar induction of proteasome-regulated genes. (11B) Immunoblot of lysates from BPE cells treated with bortezomib (12.5 nM) for 24 hr. BPLER were used as control. (11C) Flow cytometry analysis of Annexin V/Propidium Iodide (PI) staining of BPE, BPLER or HMLER treated with bortezomib (12.5 nM) and/or zVAD-fmk (20 μM) for 24 hr. The percentage of double-positive cells is indicated.
FIGS. 12A-12C demonstrate bortezomib induces Noxa protein expression in TNBC cells. (12A, 12B) Noxa mRNA (12A) and protein (12B) levels assessed by qRT-PCR and immunoblot, respectively, in BPLER and HMLER treated with bortezomib (12.5 nM) for 24 hr. Values in (12A) represent mean+/−SD of 3 replicates. (12C) Noxa protein levels in human breast cancer cell lines of different subtype after treatment with bortezomib (12.5 nM) or DMSO for 24 hours. All data are representative of at least three independent experiments.
FIG. 13 demonstrates bortezomib suppresses TNBC growth in vivo. Mean tumor volume in MB-468 tumor-bearing mice upon intratumoral infusion of bortezomib (0.8 mg/kg) or DMSO q3d.
FIG. 14 depicts additional candidate drugs targeting BPLER. Relative viability, measured by CellTiterGlo, of BPLER and HMLER cells treated for 24 hr with Deferoxamine, Nabumetone, L-cavananine and Hesperetin. Viability is relative to cells grown in the absence of drug. These drugs were not active. Data represent the mean and standard deviation of six replicates. All data are representative of at least three independent experiments.



DETAILED DESCRIPTION
Provided herein are novel malignancy associated gene signature biomarkers, and assays and methods thereof, to classify prognosis or malignant potential of a cancer and identify cancer-initiating cells. Moreover, the gene signature biomarkers, assays and methods described herein provide, in part, new methodologies to screen for novel drugs for treating cancers and tumors, such as, for example, triple-negative breast tumors. Using the assays and methods described herein proteasome inhibitors, histone deacetylase inhibitors, glycolysis inhibitors, or any combination thereof, were identified as being highly effective in altering gene expression signatures specifically in malignant or cancer-initiating cells.
The invention is based, at least in part, on results from a genome-wide siRNA lethality screen using highly malignant progenitor-like human breast epithelial cells, which recapitulate several basal-like features and give rise to tumors closely resembling human primary triple-negative breast cancer cells, or TNBCs, such as basal-like breast tumors. As demonstrated herein, core survival pathways and a “malignancy associated response gene signature” selectively associated with cancer-initiating potential were identified that are useful for prognostic and diagnostic applications and reveal novel therapeutic targets for more effective and enhanced treatment of poor prognosis tumors, such as triple negative breast cancers.
Triple-negative breast cancers (TNBCs), such as basal-like breast tumors (BL-BTs), are an especially aggressive group of tumors with the shortest survival of all breast cancer subtypes. These tumors are enriched, in part, in breast tumor-initiating cells (BT-ICs), rapidly acquire resistance to chemotherapy, and are refractory to endocrine therapy and HER-2 inhibitors. No biologic therapy is currently available for these tumors.
As demonstrated herein, we discovered that a defined population of human progenitor-like breast primary epithelial cells acquire cancer initiating properties and give rise to triple-negative, basal-like breast tumors upon transformation with defined genetic elements, i.e., expression of certain specific genes. As shown herein, the induction of a cancer-initiating cell phenotype, such as that of a basal-like breast tumor initiating cell, depends on the cell of origin, because transformation of syngeneic myoepithelial-like cells with the same set of genetic elements does not induce such phenotypes.
We performed a genome-wide siRNA lethality screen to identify factors or biomarkers on which these two cell types selectively depend for survival. As demonstrated herein, these studies identified a “malignancy associated gene signature” (MARS), or “triple negative breast cancer gene signature” (TGS), comprising an expression pattern of 10-154 biomarkers for cancer initiating cells, such as triple negative breast cancer initiating cells. We further determined that most of the genes required for survival of triple negative breast cancer initiating cells are typically up regulated in poor prognosis human primary breast tumors, including basal-like tumors and some Luminal B tumors, thus providing novel diagnostic and prognostic signatures, assays and methods, and therapeutic targets, as described herein. Core cancer-initiating cell specific survival pathways and biomarkers identified herein include, but are not limited to, the proteasome-ubiquitin system, metabolic genes, histone deacetylases, RNA splicing, and the glycolytic system, the transient inhibition of which, as shown herein, impacts triple negative breast cancer initiating cells, but did not impact normal breast epithelial cells or transformed luminal, myoepithelial or mesenchymal cells. Accordingly, also provided herein are malignancy associated response gene signatures, and assays and methods thereof, comprising subsets of biomarker genes belonging to each of these core survival pathways.
It was also determined that triple negative breast cancer initiating cells are selectively and specifically sensitive to proteasome inhibitor drugs, such as bortezomib. Proteasome inhibitor drugs were demonstrated to interfere with mitosis exit and induce Noxa-dependent apoptosis. Further, the results described herein demonstrate that the selective response of cancer initiating cells to proteasome inhibition is dependent on Noxa-mediated inactivation of MCL1 (SEQ ID NO: 134), a gene belong to the malignancy associated response gene signature of 154 biomarkers. As shown herein, transformed mammary epithelial cells as well as primary human epithelial breast cancer cells selectively depend on MCL1 or MCL-1, which is antagonized by Noxa, for survival and that proteasome inhibitor target Mcl-1 dependency in these cells. Silencing of MCL1 expression in breast cancer cell lines led to similar lethality of specific breast cancers subtypes as was shown herein with bortezomib. Thus, MCL-1 was identified as a novel target for cancer initiating cells that recapitulates the effects of proteasome inhibition. Histone deacetylase inhibitors were also found to be selectively cytotoxic for triple-negative breast cancers. As proteasome and histone deacetylase inhibitors are already in the clinic, the findings described herein can be further developed for use in new indications and targeted diagnosis of and therapeutic treatments for poor prognosis cancers, such as triple negative breast cancers, in clinical settings.
Malignancy Associated Response Signatures and Assays and Methods Thereof
Provided herein are malignancy associated response signature for identification of cancer initiating cells and malignancy potential and cancer prognosis. These malignancy associated response signatures provide novel and unique combinations of biomarkers specifically required for the survival of cancer-initiating cells, such as triple-negative breast cancer initiating cells. The signatures also provide novel assays and methods for classifying cancers and determining cancer prognosis.
As used herein, a “malignancy-associated response signature” (“MARS”) (also referred to herein as a “triple negative breast cancer gene signature” (TGS)) refers to a functional module of increased gene expression associated with cancer-initiating potential comprising at least two or more of the biomarkers provided in Table 3 (SEQ ID NOs: 1-154), termed herein as “malignancy associated response signature genes” or “malignancy associated response biomarkers.” As described herein, a cancer or tumor, such as a breast cancer, can be classified as MARS (+) or MARS(−), or classified as comprising cancer initiating cells, having poor prognosis or as being malignant, by assessing the expression levels of at least two or more, preferably at least 10, of the 154 malignancy associated response signature genes or biomarkers provided herein in Table 3 (SEQ ID NOs: 1-154) in any combination, or in combination with any other prognostic or diagnostic biomarker known to one of skill in the art. Preferably, expression levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, or more, or all of the 154 malignancy associated response signature genes described herein are assessed. In some embodiments of all the aspects described herein, 5-20, 10-15, 10-20, 10-25 malignancy associated signature genes are used to analyze the unknown cancer cell for its prognosis. If at least 60% of the signature genes are expressed more than a control non-poor prognosis cell, i.e., the expression is increased, the tumor or cancer from which the cell or sample originated is considered to have poor prognosis. It is also well within the ability of one skilled in the art, using the teachings provided herein, to identify additional genes having prognostic or predictive value for use with signatures and assays and methods thereof described herein.
It is envisioned that any of the genes listed in Table 3 (SEQ ID NOs: 1-154, SEQ ID NOs: 181-184) can be used in the assays and methods described herein, either alone or in combination with any other malignancy associated response signature biomarker or other prognostic marker. Although information concerning the expression of as few as one biomarker is expected to provide useful information, confidence in the accuracy of the classification of a cancer or tumor as MARS(+) or MARS(−) increases when more biomarkers are included. Preferably at least 10 or more of the 154 TGS biomarkers listed in Table 3 (SEQ ID NOs: 1-154, SEQ ID NOs: 181-184) are used in prognostic and diagnostic analyses of a cancer sample as described herein. Cancers can be analyzed with respect to the expression of specific groups or subsets, of MARS biomarkers, including from SEQ ID NO: 1 to SEQ ID NO: 154 of the genes listed in Table 3, in any combination.
For example, in some aspects and embodiments, a malignancy associated response signature can comprise only or consist essentially of genes or biomarkers involved in a specific pathway, such as the exemplary survival pathways depicted in FIG. 3, e.g., the proteasomal pathway. In some aspects and embodiments, a malignancy associated response signature can comprise or consist essentially of the 23 biomarkers of Table 2A (SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151) described herein as having high specificity or high selectivity for cancer-initiating potential or cells. It is well within the ability of one of skill in the art to select specific combinations of MARS genes for analysis from among the genes listed in Table 3 (SEQ ID NOs: 1-154). In the interest of brevity, every possible combination of genes suitable for use in the methods described herein are not expressly listed. Nevertheless, it should be understood that every such combination is contemplated and is within the scope of the methods described herein. It is specifically envisioned that any combination of MARS biomarkers found to be differentially expressed in poor prognosis cancers, for example, triple negative breast cancer samples, are particularly useful for analysis and in the methods described herein.
Accordingly, in some aspects provided herein are malignancy associated response signatures for a cancer initiating cell, such as a breast cancer initiating cell, comprising or consisting essentially of an expression pattern or expression pattern analysis of a set of at least ten biomarkers set forth in SEQ ID NOs: 1-154, where at least 10 of the 154 biomarkers have increased expression or are increased compared to a reference value from a reference tissue or sample. In those embodiments of the aspects described herein, where a malignancy associated response signature is said to consist essentially of a particular set of biomarkers, one of skill in the art will understand that additional control genes, control well, replicates, or the like biomarkers can be added to the signature for the purposes described herein.
In some embodiments of these aspects, at least two of the set of at least ten biomarkers set forth in SEQ ID NOs: 1-154 is selected from the group consisting of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
In some embodiments of these aspects and all such aspects described herein, the expression of the at least ten biomarker is increased at least 1.8 fold compared to the reference value.
The 23 biomarkers of Table 2A (SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151) were determined to have high specificity or high selectivity for cancer-initiating potential or malignant potential of cells, as described herein. Accordingly, in some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell comprising or consisting essentially of an expression pattern of a set of at least five biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151, where at least five of the 23 biomarkers have increased expression compared to a reference value. The number of biomarkers selected for expression analysis can be at least 4, at least 5, at least 6, at least 7, at least 8, least 9, at least 10, at least 11, at least 12, at least 13, least 14, at least 15, at least 16, at least 17, at least 18, least 19, at least 20, at least 21, at least 22, or include all 23 biomarkers, in different embodiments.
In some embodiments of these aspects and all such aspects described herein, the expression of the at least five of the 23 biomarkers is increased at least 1.8 fold compared to the reference value.
The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. “Expression products” include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene, and such expression can be detected using methods known to one of skill in the art. Accordingly, an “expression pattern,” as used herein, refers to a specific, defined subset of genes or biomarkers of a given malignancy associated response signature being expressed or not expressed by a cell or cellular sample.
The terms “increased,” “increase” or “enhance” in connection with expression of the malignancy associated response signature markers are all used herein to generally mean an increase by a statically significant amount. For the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference value or level, or at least about a 1.5-fold, at least about a 1.6-fold, at least about a 1.7-fold, at least about a 1.8-fold, at least about a 1.9-fold, at least about a 2-fold, at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, at least about a 10-fold increase, any increase between 2-fold and 10-fold, at least about a 25-fold increase, or greater as compared to a reference level. In some embodiments, an increase is at least about 1.8-fold increase over a reference value.
As is understood by one of ordinary skill in the art, the degree to which increased expression can be detected is dependent on the methodology used for measuring expression of a signature biomarker. Microarray platforms, for example, which can comprise many thousands of probes corresponding to a particular biomarker, can detect even subtle changes in gene expression, such that a 1.5-fold or greater change in expression is deemed significant, as described herein. PCR based platforms, including real-time quantitative analyses, are also highly sensitive to such small changes in gene expression. Other methods of detecting or measuring changes in gene expression, such as, for example, Northern blot analyses are not as sensitive, such that small increases are generally not thought to be reliable or valid. Similarly, protein based assays used to detect changes in expression of proteins corresponding to the malignancy associated response signatures have varying sensitivities. Thus, the degree to which increased expression is considered significant can vary depending on the nature of the techniques used to measure expression, and one of ordinary skill in the art will understand this and choose a suitable platform and statistical analysis when detecting expression of the signature biomarkers, using the methods and assays described herein.
Similarly, the terms “decrease,” “reduced,” “reduction,” or “decrease” in connection with expression of the malignancy associated response signature markers are all used herein generally to refer to a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level or non-detectable level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
The results described herein revealed that the 154 genes found in the siRNA lethality screen to be selectively required for survival of cells with malignant potential or cancer initiating cells, fall into a specific groups of pathways, such as those shown in FIG. 3. Thus, in some aspects and embodiments, a malignancy associated response signature can comprise or consist essentially of specific subsets of the 154 biomarkers all related to a specific cellular function or pathway. Examples of such pathways or functions include, but are not limited to, proteasome degradation, gene expression, DNA binding, DNA repair, RNA splicing, apoptosis, cell adhesion, cell signaling, molecular transport, inflammation, mitosis, G1/S transition, metabolism, and the like. By determining expression patterns of genes belonging to particular pathways, targeted therapies can be selected, as described elsewhere herein. For example, in some aspects and embodiments described herein, malignancy associated response signature biomarkers for expression analysis include subunits of the ubiquitin-proteasome complex listed in Table 3 including, but not limited to, PSMA1 (SEQ ID NO: 2), PSMA2 (SEQ ID NO: 33), PSMA3 (SEQ ID NO: 7), PSMB4 (SEQ ID NO: 1), PSMC1 (SEQ ID NO: 38), PSMC3 (SEQ ID NO: 4), PSMD2 (SEQ ID NO: 15), PSMD7 (SEQ ID NO: 34), PSMD14 (SEQ ID NO: 16), UBL5 (SEQ ID NO: 29), NEDD1 (SEQ ID NO: 149), NEDD8 (SEQ ID NO: 76), ANAPC2 (SEQ ID NO: 68), and ANAPC4 (SEQ ID NO: 101).
Accordingly, in some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to proteasomal gene inhibition comprising or consisting essentially of an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, where at least 3 of the 15 biomarkers have increased expression compared to a reference value. The number of biomarkers selected for expression analysis can be at least 4, at least 5, at least 6, at least 7, at least 8, least 9, at least 10, at least 11, at least 12, at least 13, least 14, or include all 15.
In some aspects, provided herein are malignancy associated response signature for a cancer-initiating cell susceptible to mitosis gene inhibition comprising or consisting essentially of an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137, where at least 3 of the 14 biomarkers have increased expression compared to a reference value. The number of biomarkers selected for expression analysis can be at least 4, at least 5, at least 6, at least 7, at least 8, least 9, at least 10, at least 11, at least 12, at least 13, or include all 14.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to RNA splicing gene inhibition comprising or consisting essentially of an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151, where at least 3 of the 13 biomarkers have increased expression compared to a reference value. The number of biomarkers selected for expression analysis can be at least 4, at least 5, at least 6, at least 7, at least 8, least 9, at least 10, at least 11, at least 12, or include all 13.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to molecular transport gene inhibition comprising or consisting essentially of an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147, where at least 3 of the 14 biomarkers have increased expression compared to a reference value. The number of biomarkers selected for expression analysis can be at least 4, at least 5, at least 6, at least 7, at least 8, least 9, at least 10, at least 11, at least 12, at least 13, or include all 14.
In some aspects, provided herein are malignancy associated response signatures for a cancer-initiating cell susceptible to metabolic gene inhibition comprising or consisting essentially of an expression pattern of a set of at least 3 biomarkers of SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153, where at least 3 of the 13 biomarkers have increased expression compared to a reference value. The number of biomarkers selected for expression analysis can be at least 4, at least 5, at least 6, at least 7, at least 8, least 9, at least 10, at least 11, at least 12, or include all 13.
Expression levels of a malignancy associated response signature biomarker can be determined by comparison to a suitable reference value, such that each of the expression levels of the at least two or more, e.g., 5 or more, 10 or more, malignancy associated response signature biomarker genes measured in a sample, such as a cellular sample, are compared to a specific reference level that acts a standard of comparison. The reference level is obtained or measured in a reference biological sample, such as a reference sample obtained from an age-matched normal control (e.g., an age-matched subject not having a breast cancer, or having a non-malignant or high prognosis breast cancer), or a reference sample from the same subject not from the tumor or cancer site, e.g., healthy breast tissue cells. A “reference value” is thus typically a predetermined reference level, such as an average or median of expression levels of each of the, for example, at least 2 of the 154 malignancy associated response signature associated biomarkers (SEQ ID NOs: 1-154) obtained from, for example, biological samples from a population of healthy subjects that are in the chronological age group matched with the chronological age of the tested subject. In some embodiments, the reference biological samples can also be gender matched. For example, as explained herein, malignancy associated response signature expression levels in a sample can be assessed relative to normal breast tissue from the same subject or from a sample from another subject or from a repository of normal subject samples. If the expression level of a malignancy associated response signature biomarker is greater or less than that of the reference or the average expression level of, for example, non-basal-like breast tumors of a particular type, the malignancy associated response signature biomarker expression is said to be “increased” or “decreased,” respectively, as those terms are defined herein. Exemplary analytical methods for classifying expression of a malignancy associated response signature biomarker, determining a malignancy associated response signature status, and scoring of a sample for expression of a malignancy associated response signature biomarker are explained in detail herein.
Accordingly, in some embodiments of the malignancy associated response signatures described herein, an increased expression over a reference value is at least about a 1.5-fold increase, at least about a 1.6-fold increase, at least about a 1.7-fold increase, at least about a 1.8-fold increase, at least about a 1.9-fold increase, at least about a 2-fold increase, at least about a 3-fold increase, at least about a 4-fold increase, at least about a 5-fold increase, at least about a 10-fold increase, any increase between 1.5-fold and 10-fold, at least about a 25-fold increase, or greater as compared to a reference level. In some embodiments, an increase is at least about 1.8-fold increase over a reference value.
The malignancy associated response signatures and assays and methods thereof described herein are useful for identifying cancer-initiating cells and classifying cancers as having poor prognosis, high malignant potential, or comprising cancer-initiating cells, for example. “Cancer-initiating cells,” which also include cancer stem cells, refer to cells that are neoplastic and can undergo self-renewal as well as abnormal proliferation and differentiation. Functional features of cancer-initiating cells are that they are tumorigenic, i.e., cause cancers or tumors. Such cancer-initiating cells can give rise to additional such neoplastic cells by self-renewal, and they can give rise to non-tumorigenic neoplastic cells. Without being bound to any particular theory, cancer initiating cells contribute to the development of metastatic cancer and relapses. The unique malignancy associated response signature biomarkers, described herein, permit specific therapies targeted at cancer-initiating cells.
Accordingly, in some embodiments of the malignancy associated response signatures, the cancer-initiating cell is a breast cancer-initiating cell. In some embodiments of the malignancy associated response signatures, the cancer-initiating cell is a triple-negative breast cancer-initiating cell. In some embodiments of the malignancy associated response signatures, the cancer-initiating cell is a Luminal B breast cancer-initiating cell. In some embodiments of the malignancy associated response signatures, the cancer-initiating cell is an epithelial breast cancer-initiating cell.
A “cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastatses. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
By “metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.
Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans, in addition to the monitoring of specific symptoms.
Examples of cancer for use in the methods of diagnosis, prognosis, and treatment described herein, include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, breast cancer (including, e.g., triple negative breast cancers, basal-like breast cancers, Luminal B breast cancers); basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
Also provided herein are assays and methods for classifying cancers in a subject based on expression analyses of various combinations of the biomarkers of the malignancy associated response signatures described herein. By classifying a cancer as having poor prognosis or malignant potential, and identifying pathways that are associated with poor prognosis or malignant potential, specific genes and biomarkers, such as those described herein, can be targeted, and specific therapies can be administered to a subject. A cancer or tumor, such as a breast cancer, can be classified as highly malignant or having poor prognosis by assessing and determining increased expression levels of at least two or more, preferably at least 10, of the 154 malignancy associated response signature genes or biomarkers listed in Table 3 (SEQ ID NOs: 1-154) in a biological sample in any combination, or in combination with any other prognostic biomarker known to one of skill in the art. Preferably, expression levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 125, or more of the 154 malignancy associated response signatures genes described herein are assessed. It is well within the ability of one skilled in the art, using the teachings provided herein, to identify additional genes having prognostic or predictive value for use with the assays and methods described herein.
Accordingly, in some aspects, provided herein are methods of classifying a cancer in a subject in need thereof, such methods comprising:

    
    
        a. assaying expression of ten or more of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in a biological sample obtained from the subject having a cancer, and
        b. comparing the expression of the ten or more of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in the biological sample obtained from the subject having a cancer with a reference value, such that increased expression of 1.8-fold or greater of at least ten of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least ten of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
    
    


In some aspects, provided herein are methods of classifying a cancer in a subject in need thereof, such methods comprising:

    
    
        a. assaying expression of at least five biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151 in a biological sample obtained from the subject having a cancer, and
        b. comparing the expression of the five or more of the 23 malignancy associated response signature biomarkers in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of 1.8-fold or greater of at least ten of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least ten of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
    
    


In some aspects, provided herein are methods of classifying a cancer in a subject in need thereof, the method comprising:

    
    
        a. assaying expression of at least three malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149 in a biological sample obtained from the subject having a cancer, and
        b. comparing the expression of the at least three malignancy associated response signature biomarkers in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of 1.8-fold or greater of at least three of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least three of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
    
    


Also provided herein, in some aspects, are assays for determining cancer prognosis in a subject. Such assays comprise the steps of:

    
    
        a. measuring expression of ten or more of the 154 malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 in a biological sample obtained from a subject having cancer, and
        b. comparing the expression of the ten or more of the 154 malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of at least ten of the measured biomarkers in the biological sample obtained from the subject relative to the reference value diagnoses the patient as having a poor prognosis or malignant cancer, and absence of increased expression of at least ten of the measured biomarkers relative to the reference value diagnoses the patient as not having a poor prognosis or malignant cancer.
    
    


In some embodiments of these methods and assays, the cancer is a breast cancer. In some embodiments of these methods and assays, the cancer is a triple-negative breast cancer. In some embodiments of these methods and assays, the cancer is a Luminal B breast cancer. In some embodiments of these methods and assays, the cancer is an epithelial breast cancer.
In some embodiments of these assays and methods, increased expression of the two or more of the 154 malignancy associated response signature biomarkers, or a subset thereof, in the biological sample classifies the cancer or determines a prognosis for the subject having the cancer. In some such embodiments, the prognosis comprises relative survival rate, relative risk of metastasis, treatment option, or any combination thereof.
As demonstrated herein, the described malignancy associated response signature biomarker genes have prognostic value. The term “prognosis,” as used herein, refers to prediction of a cancer attributable progression. Prognosis includes, but is not limited to the survival rate and risk of recurrence, metastatic spread, treatment resistance, and local-regional failure. In the examples described herein, increased expression and enrichment of malignancy associated response signature biomarkers in luminal B subtype breast tumors was found to correlate with decreased survival. In breast cancer patients, enriched expression of malignancy associated response signature genes was found to correlate with increased risk and onset of metastasis, and decreased metastasis-free survival. Accordingly, expression of malignancy associated response signature biomarkers in a tumor sample can be used clinically as both a predictive and prognostic marker to provide information concerning appropriate treatment modalities and likely treatment outcome. The expression of malignancy associated response signature biomarkers can be used in conjunction with other prognostic factors, in some embodiments, to allow a clinician to predict the outcome of a cancer treatment. The stratification of patients based on enriched expression of malignancy associated response signature biomarkers can be used to determine a treatment protocol for the patients. For example, patients can be divided into those who have a low risk for metastasis and can be spared specific therapies, those with high risk for metastasis who are likely to benefit from standard treatments, and those with a high risk for metastasis and who are resistant to standard treatments. Such stratification permits tailoring of patient treatment protocols to the individual patient.
The terms “subject,” “patient,” and “individual” are used interchangeably herein, and refer to an animal, for example a human. For treatment of those disease states which are specific for a specific animal such as a human subject, the term “subject” refers to that specific animal. The terms ‘non-human animals’ and ‘non-human mammals’ are used interchangeably herein, and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. In some embodiments of the aspects described herein, a subject refers to a human subject having a breast cancer or at increased risk for a breast cancer. A subject that has a breast cancer or a tumor is a subject having objectively measurable breast cancer cells present in the subject's body.
As used herein, the term “biological sample” or “subject sample” or “sample” refers to a cell or population of cells or a quantity of tissue or fluid obtained from a subject. Most often, the sample has been removed from a subject, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e. without removal from the subject. A biological sample or tissue sample includes, but is not limited to, blood, plasma, serum, lymph fluid, bone marrow, tumor biopsy, urine, stool, sputum, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, lung tissue, adipose tissue, connective tissue, sub-epithelial tissue, epithelial tissue, liver tissue, kidney tissue, uterine tissue, respiratory tissues, breast tissue, gastrointestinal tissue, and genitourinary tract tissue, tears, saliva, milk, cells (including, but not limited to, blood cells), biopsies, scrapes (e.g., buccal scrapes), tumors, organs, and also samples of an in vitro cell culture constituent. Often, a “biological sample” will contain cells from the subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine. In some embodiments, the sample is from a resection, or core needle biopsy of a primary or metastatic tumor, such as a breast tumor, or a cell block from pleural fluid. In some embodiments, fine needle aspirate samples are used. Samples can be paraffin-embedded or frozen tissue.
In another aspect, provided herein are methods of determining the type or prognosis of a tumor from a patient, the methods comprising detecting expression of ten or more of the 154 triple negative breast cancer gene signature genes of Table 3 (SEQ ID NOs: 1-154) in a biological sample from a subject affected with or having a tumor, wherein increased expression of the genes relative to a reference sample indicates that the tumor is a poor prognosis tumor, such as a triple-negative tumor or basal-like breast tumor, and absence of increased expression of the genes relative to the reference indicates that the tumor is not a poor prognosis tumor.
In other aspects, provided herein are methods of determining the type of a breast tumor from a patient comprising detecting expression of ten or more of the 154 triple negative breast cancer gene signature genes of Table 3 (SEQ ID NOs: 1-154) in a biological sample from a subject affected with or having a triple-negative breast cancer, wherein increased expression of the genes relative to a reference sample indicates that the triple-negative breast cancer is a basal-like breast tumor or poor prognosis luminal B breast tumor, and absence of increased expression of the genes relative to the reference indicates that the tumor is not a basal-like breast tumor or a poor prognosis luminal B breast tumor.
In some embodiments of these aspects and all such aspects described herein, the methods and assays further comprise the step of administering at least one proteasome inhibitor to the subject having been determined to carry a poor prognosis tumor, a triple-negative tumor, a basal-like breast tumor, or a poor prognosis luminal B breast tumor. In some embodiments, the proteasome inhibitor is bortezomib. In some embodiments, the proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these aspects and all such aspects described herein, the method further comprises the step of administering at least one histone deactylase inhibitor to the subject having been determined to carry a poor prognosis tumor, a triple-negative breast cancer, a basal-like breast tumor, or a poor prognosis luminal B breast tumor. In some such embodiments, the histone deactylase inhibitor is Trichostatin A. In some such embodiments, the histone deactylase inhibitor is Vorinostat. In some embodiments, the histone deactylase inhibitor is an siRNA or antisense RNA agent.
In some embodiments of these aspects and all such aspects described herein, the method further comprises the step of administering at least one glycolysis inhibitor to the subject having been determined to carry a poor prognosis tumor, a triple-negative breast cancer, a basal-like breast tumor, or a poor prognosis luminal B breast tumor. In some such embodiments, the glycolysis inhibitor is 3-bromo-pyruvic acid (BRPA). In some embodiments, the glycolysis inhibitor is an siRNA or antisense RNA agent.
In various embodiments of the aspects described herein, expression levels of the TGS biomarkers in a biological sample can be evaluated by any suitable means known to one of skill in the art. For example, in some embodiments, expression can be evaluated using DNA microarrays. Alternatively, in other embodiments, RNA transcripts can be measured using real time PCR, or protein products can be detected using suitable antibodies. Methods of determining expression levels of genes by these and other methods are known in the art.
In some embodiments of these aspects and all such aspects described herein, the expression levels of the at least two or more, e.g., 10 or more, malignancy associated response signature biomarker genes measured in a biological sample are compared to a reference level, or a reference biological sample, such as biological sample obtained from an age-matched normal control (e.g., an age-matched subject not having a breast cancer, or having a non-malignant or high prognosis breast cancer), or a biological sample from the same subject not from the tumor or cancer site, e.g., healthy breast tissue cells. For diagnosing or determining a prognosis of a subject having a breast cancer in a subject using the methods and systems as disclosed herein, a “reference value” is typically a predetermined reference level, such as an average or median of expression levels of the at least 2 of the 154 malignancy associated response signature biomarkers (SEQ ID NOs: 1-154) obtained from biological samples from a population of healthy subjects that are in the chronological age group matched with the chronological age of the tested subject. As indicated earlier, in some situations, the reference biological samples can also be gender matched.
Also provided herein are assays for determining or identifying whether a sample from a subject, such as a biopsy sample, comprises cancer initiating cells. Such assays are based, in part, on the high-throughput siRNA lethality screen described herein, where different cancer types were assessed for differential susceptibility to gene inhibition using a library of different siRNA sequences targeted to different genes.
Such assays comprise the steps of: (a) dividing a cell culture grown from a biopsy obtained from a subject having cancer into at least 5 separate cultures; (b) exposing each of the at least 5 separate cultures to a different inhibitory agent, such that each of the different inhibitory agent specifically inhibits a different malignancy associated response signature biomarker set forth in SEQ ID NOs: 1-154; (c) growing each of the at least 5 separate cell cultures of step (b) for at least 12 hours; and (d) measuring viability of the cells from each of the cultures of step (c), such that if the total viability of the cells in at least 60% of the cultures is decreased by at least 25%, then the biopsy obtained from the subject comprises cancer-initiating cells.
Viability, as used herein refers, to the number of living cells present in a culture at a given timepoint. Viability can be measured using any of the assays set forth herein, such as those described in the Examples section. Exemplary viability assays include Trypan Blue staining, flow cytometric analyses of annexin V, propidium iodide staining and the like.
Cells can be cultured according to standard methods known to one of skill in the art, and the duration of culture can vary. Preferably a culture for identifying whether a sample comprises cancer-initiating cells is grown for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, or more. As understood by one of ordinary skill in the art, culture itself can cause loss in viability of cells, due, for example, to overcrowding in a dish or well, and thus the cultures should be grown for long enough to identify and measure any effect(s) on the cells from the inhibitory agent to which the cells have been exposed, but not so long as to mask any such effects due, for example, to overcrowding.
In some embodiments of these assays and all such assays described herein, the inhibitory agents are selected from siRNA agents, antisense RNA agents, or small molecules, that specifically inhibit any of the cancer gene signature biomarkers set forth in SEQ ID NOs: 1-154.
In some embodiments of these assays and all such assays described herein, the inhibitory agents specifically inhibit any of the malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the mitosis cancer gene signature biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the RNA splicing malignancy associated response signature biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the molecular transport malignancy associated response signature biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147.
In some embodiments of these assays and all such assays described herein, at least one inhibitory agent specifically inhibits any of the metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153.
In some embodiments of the aspects described herein, the assays and methods for classifying a cancer, determining whether a sample comprises cancer-initiating cells, determining or diagnosing the type of a cancer or tumor from a patient, providing a prognosis to a cancer patient, or treating a cancer patient, further comprise the step of selecting or identifying the subject having the cancer. In such embodiments, a subject is identified as having cancer by objective determination of the presence of cancer cells or a tumor in the subject's body by one of skill in the art. Such objective determinations can be performed through the sole or combined use of manual examination, tissue biopsies, blood and platelet cell counts, mammograms, ultrasound, urine analyses, magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms associated with the cancer.
Expression levels of a malignancy associated response signature biomarker can be determined by comparison to a suitable reference. For example, as explained herein, malignancy associated response signature biomarker expression levels in a sample can be assessed relative to normal breast tissue from the same subject or from a sample from another subject or from a repository of normal subject samples. Alternatively, the relative expression level of a malignancy associated response signature gene in a particular tumor can be determined with reference to the expression levels of the same gene in for example, non-basal-like breast tumors, e.g., HER2, luminal, or claudin-low breast cancers. If the expression level of a malignancy associated response signature gene is greater or less than that of the reference or the average expression level of non-basal-like breast tumors of a particular type, the malignancy associated response signature gene expression is said to be “increased” or “decreased,” respectively, as those terms are defined herein.
In other aspects, provided herein are methods for diagnostic or prognostic analysis of a cancer, such as a breast cancer, in a subject comprising the step of measuring expression of at least 10 of the 154 genes of a malignancy associated response signature (SEQ ID NOs: 1-154) for a cancer-initiating cell in a biological sample, wherein said triple negative breast cancer gene signature comprises an expression pattern of a set of 10-154 biomarker genes set forth in Table 3 (SEQ ID NOs: 1-154), and wherein an increase of expression of at least 10 of the biomarker genes is indicative of poor prognosis of the cancer in the subject.
In some embodiments of this aspect, the expression of the at least 10 of the 154 biomarkers of the malignancy associated response signature in the biological sample determines a prognosis for the subject having the cancer. In some such embodiments, the prognosis comprises relative survival rate, relative risk of metastasis, treatment option, or any combination thereof.
In further embodiments of the prognostic methods described herein, the relative survival rate of a patient can be predicted by determining the expression of at least one, but preferably at least 10, malignancy associated response signature biomarkers in a tumor sample derived from a patient. Survival in cancer patients is often quantified as a “relative cancer survival rate.” The relative cancer survival rate is the percentage of patients who survive a certain type of cancer for a specified amount of time. Cancer survival rates are often given in 5, 10 or 20 year survival rates. As seen in the examples described herein, enriched expression of malignancy associated response signature biomarkers can predict differential survival rates of patients with subgroups of Luminal B cancer, and metastasis-free survival rates in primary breast cancer samples.
Pathway Inhibitors & Therapeutic Methods
The various combinations of malignancy associated response signature biomarkers identified in the screens described herein provide novel therapeutic targets and pathways, such as the pathways highlighted in FIG. 3, e.g., ubiquitin-proteosome pathway, histone deactylase pathway, metabolic pathway, for treatment of subjects having cancers classified as malignant or poor prognosis, or having cancer-initiating cells, such as, for example, triple negative breast cancers.
Accordingly, in some embodiments, if a cancer is classified as having a poor prognosis or being a malignant cancer, or comprising cancer-initiating cells using, for example, the assays and methods for classifying cancers provided herein, at least one proteasome inhibitor is administered to the subject.
For example, in some embodiments, if the cancer is classified as having a poor prognosis or being a malignant cancer, or comprising cancer-initiating cells using, for example, the assays and methods for classifying cancers provided herein, such that at least 10 of the 154 genes of a malignancy associated response signature (SEQ ID NOs: 1-154) for a cancer-initiating cell in a biological sample have increased expression over a reference value, then at least one proteasome inhibitor is administered to the subject. In some such embodiments, if, in addition to the requirement that at least 10 of the 154 genes of a malignancy associated response signature (SEQ ID NOs: 1-154) are increased relative to reference value, at least two of the ten or more genes having increased expression are required to be proteasomal malignancy associated response signature biomarkers of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, then at least one proteasome inhibitor is administered to the subject.
Similarly, in some embodiments, if the cancer is classified as having a poor prognosis or being a malignant cancer, or comprising cancer-initiating cells using, for example, the assays and methods for classifying cancers provided herein, such that at least five of the biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151 have increased expression relative to a reference value, then at least one proteasome inhibitor is administered to the subject. In some such embodiments, if, in addition to the requirement that at least five of the 23 genes of the malignancy associated response signature are increased relative to reference value, at least one of the five or more genes having increased expression are required to be proteasomal malignancy associated response signature biomarkers of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38, then at least one proteasome inhibitor is administered to the subject.
The term “proteosome inhibitor,” as used herein, relates to a compound or an agent which targets, decreases or inhibits one or more components of the proteosome or proteosomal pathway. The proteasome is an intracellular structure which is a multicatalytic proteinase which is a highly conserved. Proteasomes are responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. Thus, the proteosome is a regulatory element in cell growth and differentiation. Several steps are involved in protein degradation via the proteasome or “ubiquitin-proteasome” pathway. Initially, a protein is marked for destruction with a chain of small polypeptides known as ubiquitin. Ubiquitinylation guides the protein into the proteosome's enclosed proteolytic chamber. Three enzymatic activities, E1, E2, and E3, are required for ubiquitinylation. The ATP-dependent E1 enzyme activates ubiquitin and links it to the ubiquitin-conjugating enzyme, E2. The E3 enzyme, an ubiquitin ligase, then links the ubiquitin molecule to the protein. This process is repeated until the designated polypeptide trails a long chain of ubiquitin moieties and the proteasome finally degrades the protein into small fragments. The ubiquitin-proteasome pathway is responsible for the degradation of 90% of all abnormal, misfolded proteins and all of the short-lived, regulatory proteins in the cell.
Examples of targets of a proteosome inhibitor include, but are not limited to, O(2)(−)-generating NADPH oxidase, NF-κB farnesyltransferase, and geranylgeranyltransferase I.
A variety of inhibitors of the proteasome complex have been reported, e.g., Dick, et al., Biochem. 30: 2725 (1991); Goldberg, et al., Nature 357: 375 (1992); Goldberg, Eur. J. Biochem. 203: 9 (1992); Orlowski, Biochem. 29: 10289 (1989); Rivett, et al., Archs. Biochem. Biophys. 218: 1 (1989); Rivett, et al., J. Biol. Chem. 264: 12, 215 (1989); Tanaka, et al., New Biol. 4: 1 (1992). Proteasome inhibitors are also discussed in U.S. Pat. No. 5,693,617, the disclosure of which is incorporated herein by reference. In addition to antibiotic inhibitors originally isolated from actinomycetes, a variety of peptide aldehydes have been synthesized, such as the inhibitors of chymotrypsin-like proteases described by Siman et al. (WO91/13904).
Specific proteasome inhibitors fall into five classes distinguished by the pharmacophore that interacts with the active site threonine in the proteasome: peptide aldehydes such as CEP1612 and MG132, peptide boronates such as bortezomib, peptide vinyl sulfones, peptide epoxyketones and β-lactone inhibitors such as lactocystin. In some embodiments of the aspects and embodiments provided herein, a proteasome inhibitor is bortezomib.
Examples of proteosome inhibitors or analogues thereof contemplated for use in the methods described herein include, but are not limited to, aclacinomycin A; MG-132; gliotoxin; PS-341; MLN 341; peptide aldehydes, e.g., N-acetyl-leucinyl-leucynil-norleucynal, N-acetyl-leucinyl leucynil-methional, carbobenzoxyl-leucinyl-leucynil-norvalinal, carbobenzoxyl-leucinyl-leucynil-leucynal, lactacystine, b-lactone; boronic acid peptides; ubiquitin ligase inhibitors; PS-519 (1R-[1S,4R,5S]]-1-(1-hydroxy-2-methylpropyl)-4-propyl-6-oxa-2-azabicyclo[-3.2.1.]heptane-3,7-dione); clasto-lactacystin beta-lactone; lactacystin; epoxomicin; CVT634 (-5-methoxy-1-indanone-3-acetyl-leucyl-D-leucyl-1-indanylamide); TMC96 ((3-methylbutanoyl-L-threonine N-(1-(2-(hydroxymethyl)-oxiran-2-ylcarbonyl)-3-methylbut-3enyl)amide); MG-115; CEP161; cyclosporin A; deoxyspergualin; bortezomib and/or compounds having structure similar to that of bortezomib; and Velcade. Proteasome inhibitors having structure similar to bortezomib include those compounds disclosed in U.S. Pat. Nos. 7,119,080; 6,747,150; 6,617,317; 6,548,668; 6,465,433; 6,297,217; 6,083,903; 5,780,454; 7,422,830; 7,109,323; 6,958,319; 6,713,446; and 6,699,835, the contents of each of which are herein incorporated by reference in their entireties.
In addition to the proteasome protease inhibitors, the ubiquitin-proteasome pathway can be blocked by inhibitors of the facilitating enzymes Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligases (E3 enzymes). E1 inhibitors have been identified such as himeic acid A (Tsukamoto, et al. 2005, Bioorgan Med Chem Lett 15(1):191-194. Other methods known in the art, such as RNA mediated silencing or inhibition, can also be used to reduce or eliminate the activities of specific ubiquitinylation-related enzymes.
MCL1 (SEQ ID NO: 134) is a myeloid cell leukemia protein of the Bcl-2 family of proteins. Two alternatively spliced transcripts encoding distinct isoforms have been identified. The longer gene product (isoform 1) enhances cell survival by inhibiting apoptosis while the alternatively spliced shorter gene product (isoform 2) promotes apoptosis and is death-inducing. The results described herein identify MCL-1 as a malignancy associated response signature biomarker, and demonstrate that inhibition of MCL-1 has the same lethal effects as proteasome inhibition in poor prognosis cancer cells, such as triple-negative breast cancer cells. Accordingly, in some embodiments, a proteasome inhibitor is chosen that specifically inhibits MCL-1 of SEQ ID NO: 134 for use with the assays and methods described herein.
In some embodiments, the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149. In some embodiments, the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NO: 134 (MCL-1).
In some embodiments, the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
In some embodiments, the at least one proteasome inhibitor is bortezomib.
In some embodiments of the methods and assays described herein, if a cancer is classified as having a poor prognosis or being a malignant cancer, at least one histone deacetylase inhibitor is administered to the subject having cancer. In some such embodiments, the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent. In some such embodiments, the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
The terms “histone deacetylase inhibitor” or “inhibitor of histone deacetylase,” as used herein, relate to a compound or an agent which targets, decreases, or inhibits the activity or expression of one or more histone deacetylases or components of the histone deacetylase pathway. Inhibiting histone deacetylase enzymatic activity means reducing the ability of a histone deacetylase to remove an acetyl group from a histone or another protein substrate. Preferably, such inhibition is specific, i.e., the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a histone or another protein substrate at a concentration that is lower than the concentration of the inhibitor that is required to produce some other, unrelated biological effect. The terms “histone deacetylase” and “HDAC” are intended to refer to any one of a family of enzymes that remove acetyl groups from the E-amino groups of lysine residues at the N-terminus of a histone. Unless otherwise indicated by context, the term “histone” is meant to refer to any histone protein, including H1, H2A, H2B, H3, H4, and H5, from any species. Human HDAC proteins or gene products, include, but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10 and HDAC-11. The histone deacetylase can also be derived from a protozoal or fungal source.
Any histone deacetylase inhibitor can be used in the context of the present invention. Histone deacetylase inhibitors from various chemical classes have been described, with four most important classes, namely (i) hydroxamic acid analogs, (ii) benzamide analogs, (iii) cyclic peptides (generally tetrapeptides)/peptolides and (iv) fatty acid analogs. Histone deacetylase inhibitors differ in their specificities towards the various histone deacetylases. These enzymes are divided into four main classes according to their sequence homology and expression patterns. Histone deacetylase inhibitors differ in their specificities towards the four main classes of histone deacetylases. These enzymes are divided into four classes according to their sequence homology and expression patterns. Generally hydroxamic acid analogs are effective on classes I, II, IV enzymes, benzamide analogs on class I and some also on classes II, III and/or IV, cyclic peptides/peptolides on class I and fatty acid analogs on classes I and IL Brief overviews on histone deacetylase inhibitors have recently been given by Smith and Workman (International Journal of Biochemistry & Cell Biology (2008) doi:10.1016/j.biocel.2008.09.008) and, in a broader context, by Szyf (Annu. Rev. Pharmacol. Toxicol. (2009) 49, 243-263).
Suitable examples of a histone deacetylase inhibitor for use in the methods described herein include, but are not limited to, trichostatin A (TSA), N′-hydroxy-N-phenyl-octanediamide (suberoylanilide hydroxamic acid, SAHA), pyroxamide, M-carboxycinnamic acid bishydroxamide (CBHA), trichostatin C, salicylihydroxamic acid (SBHA), azelaic bishydroxamic acid (ABHA), azelaic-1-hydroxamate-9-anilide (AAHA), 6-(3-chlorophenylureido) carpoic hydroxamic acid (3C1-UCHA), oxamflatin, A-161906, scriptaid, PXD-101, LAQ-824, cyclic hydroxamic acid-containing peptide (CHAP), ITF-2357, MW2796, MW2996, trapoxin A, FR901228 (FK 228 or Depsipeptide), FR225497, apicidin, CHAP, HC-toxin, WF27082, chlamydocin, sodium butyrate, isovalerate, valerate, 4-phenylbutyrate (4-PBA), 4-phenylbutyrate sodium (PBS), arginine butyrate, propionate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, tributyrin, valproic acid, valproate, CI-994, MS-27-275 (MS-275 or SNDX-275), 3′-amino derivative of MS-27-275, MGCD0103 or Depudecin, and SNDX-275. A number of histone deacetylase inhibitors are currently being clinically tested and SAHA (VORINOSTATTM) has recently been approved by the FDA for treatment of cutaneous T cell lymphoma.
In some embodiments of the methods and assays described herein, if a cancer is classified as having a poor prognosis or being a malignant cancer, at least one glygolytic or metabolic inhibitor is administered to the subject. In some such embodiments, the at least one glycolysis inhibitor is an siRNA or antisense RNA agent.
The terms “metabolic inhibitor,” “glycolysis inhibitor” or “glycolytic inhibitor,” as used herein, relates to a compound or an agent which targets, decreases, stops, or inhibits one or more components of the glycolytic or glycolysis pathway. Glycolysis refers to the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO—+H+. The free energy released in this process is used to form the high-energy compounds ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). Glycolysis involves a sequence of ten reactions involving ten intermediate compounds (one of the steps involves two intermediates). The intermediates provide entry points to glycolysis. For example, most monosaccharides, such as fructose, glucose, and galactose, can be converted to one of these intermediates. The intermediates can also be directly useful. For example, the intermediate dihydroxyacetone phosphate (DHAP) is a source of the glycerol that combines with fatty acids to form fat.
The most common type of glycolysis is the Embden-Meyerhof-Parnas pathway (EMP pathway). Glycolysis also refers to other pathways, such as the Entner-Doudoroff pathway and various heterofermentative and homofermentative pathways. Suitable examples of a glycolysis inhibitor for use in the methods described herein include, but are not limited to, 3-bromo-pyruvic acid (bromoethylpyrruvate), Hypoglycin A, 2-deoxy-D-glucose, dichloro-acetate, oxamate and other pyruvate analogs, staurosporine, oligomycin, 6-dichloro-1,6-dideoxy-2-deoxyglucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), 2-fluor-2-deoxy-D-glucose (2FG), 2-deoxy-D-galactose, 3H-2-deoxyglucose or analogs thereof. In addition, examples of 2-deoxyglucose compounds useful in the methods described herein include, but are not limited to, 2-deoxy-D-glucose, 2-deoxy-L-glucose; 2-bromo-D-glucose, 2-fluoro-D-glucose, 2-iodo-D-glucose, 6-fluoro-D-glucose, 6-thio-D-glucose, 7-glucosyl fluoride, 3-fluoro-D-glucose, 4-fluoro-D-glucose, 1-O-propyl ester of 2-deoxy-D-glucose, 1-O-tridecyl ester of 2-deoxy-D-glucose, 1-O-pentadecyl ester of 2-deoxy-D-glucose, 3-O-propyl ester of 2-deoxy-D-glucose, 3-O-tridecyl ester of 2-deoxy-D-glucose, 3-O-pentadecyl ester of 2-deoxy-D-glucose, 4-O-propyl ester of 2-deoxy-D-glucose, 4-O-tridecyl ester of 2-deoxy-D-glucose, 4-O-pentadecyl ester of 2-deoxy-D-glucose, 6-O-propyl ester of 2-deoxy-D-glucose, 6-O-tridecyl ester of 2-deoxy-D-glucose, 6-O-pentadecyl ester of 2-deoxy-D-glucose, and 5-thio-D-glucose, and mixtures thereof.
In some aspects, provided herein are methods for treating cancer comprising the step of administering to a subject diagnosed as having the tumor, using, for example, the diagnostic and prognostic methods and systems described herein, at least one proteosome inhibitor in a pharmaceutically acceptable carrier. In other aspects, provided herein are methods for treating a tumor comprising the step of administering to a subject diagnosed as having the tumor at least one histone deactylase inhibitor in a pharmaceutically acceptable carrier. In another aspect, provided herein is a method for treating a tumor comprising the step of administering to a subject diagnosed as having the tumor, using, for example, the diagnostic and prognostic methods and systems described herein, at least one glycolytic inhibitor in a pharmaceutically acceptable carrier. In some embodiments of these aspects and all such aspects described herein, the diagnosis of the tumor in the subject was performed according to determination of the TGS signature using the methods, systems, and kits described herein.
In other aspects, provided herein are methods for treating a basal-like breast tumor comprising the step of administering to a subject diagnosed as having the basal-like breast tumor, using, for example, the diagnostic and prognostic methods and systems described herein, at least one proteosome inhibitor in a pharmaceutically acceptable carrier. In some aspects, provided herein are methods for treating a basal-like breast tumor comprising the step of administering to a subject diagnosed as having the basal-like breast tumor, using, for example, the diagnostic and prognostic methods and systems described herein, at least one histone deactylase inhibitor in a pharmaceutically acceptable carrier. In other aspects, provided herein are method for treating a basal-like breast tumor comprising the step of administering to a subject diagnosed as having the basal-like breast tumor, using, for example, the diagnostic and prognostic methods and systems described herein, at least one glycolytic inhibitor in a pharmaceutically acceptable carrier.
The “agents” and “inhibitors” used in the therapeutic methods described herein can be selected from a group of a chemical, small molecule, chemical entity, nucleic acid sequences, nucleic acid analogues or protein or polypeptide or analogue of fragment thereof specific for one or more a malignancy associated response signature biomarkers or genes encoding a malignancy associated response signature biomarker. Nucleic acid sequences include, for example, but not limited to, nucleic acid sequence encoding proteins that act as repressors in the various target pathways described herein, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc., specific for or directed against one or more genes encoding a malignancy associated response signature biomarker, such as for example, one or more of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93; one or more of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38; or SEQ ID NO: 134, for example. One of skill in the art, knowing the sequence a desired malignancy associated response signature biomarker to target, can design suitable inhibitory nucleic acid sequences using techniques known in the art or obtain ones from a commercial vendor, as described herein.
A protein and/or peptide agent or fragment thereof, can be any protein of interest, for example, but not limited to; mutated proteins; therapeutic proteins; truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins of interest can be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. A therapeutic agent also includes any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments, the agent is a small molecule having a chemical moiety. Such chemical moieties can include, for example, unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups, including macrolides, leptomycins and related natural products or analogues thereof. Therapeutic agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
As used herein, the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
The term “inhibitor” refers to an agent that inhibits expression of a polypeptide or polynucleotide, or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of the polypeptide or the polynucleotide. Inhibitors are agents that, e.g., inhibit expression, e.g., translation, post-translational processing, stability, degradation, or nuclear or cytoplasmic localization of a polypeptide, or transcription, post transcriptional processing, stability or degradation of a polynucleotide or bind to, partially or totally block stimulation, DNA binding, transcription factor activity or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide. An inhibitor can act directly or indirectly. Inhibition is achieved when the activity value of a polypeptide or polynucleotide is about at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or absent or undetectable in comparison to a reference or control level in the absence of the inhibitor.
Accordingly, in some embodiments, an inhibitor for use in the therapeutic methods described herein is an siRNA agent, an antisense RNA agent, a small molecule agent, an antibody or antigen-binding fragment thereof specific for or directed against one or more malignancy associated response signature biomarkers of Table 3 (SEQ ID NOs: 1-154). In some such embodiments, the malignancy associated response signature biomarker is a proteasomal pathway biomarker, a histone descetylase pathway biomarker, a glycolysis pathway biomarker, or any combination thereof. In some embodiments, an inhibitor for use in the therapeutic methods described herein is an siRNA agent, an antisense RNA agent, a small molecule agent, an antibody or antigen-binding fragment thereof specific for or directed against one or more of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93. In some embodiments, an inhibitor for use in the therapeutic methods described herein is an siRNA agent, an antisense RNA agent, a small molecule agent, an antibody or antigen-binding fragment thereof specific for or directed against one or more of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38. In some embodiments, an inhibitor for use in the therapeutic methods described herein is an siRNA agent, an antisense RNA agent, a small molecule agent, an antibody or antigen-binding fragment thereof specific for or directed against SEQ ID NO: 134,
Therapeutic compositions comprising inhibitors, such as small molecules, drugs, siRNA or antisense RNA agents, of malignancy associated response signature biomarkers useful for practicing the therapeutic methods described herein comprise a physiologically tolerable carrier together with an active compound, such as a proteosome inhibitor, histone deacetylase inhibitor, or glycolytic inhibitor, as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired. As used herein, the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable or unacceptable physiological effects such as toxicity, nausea, dizziness, gastric upset, immune reaction and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Saline-based carriers are most useful for the administration of cells or cell preparations. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
Dosage and administration of the therapeutic compositions comprising, for example, inhibitor agents that specifically inhibit one or more of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, described herein vary with the subject to be treated and the therapeutic approach taken in a given instance. The success of a treatment can be evaluated by the ordinarily skilled clinician by monitoring one or more symptoms or markers of the breast cancer being treated. Effective treatment includes any statistically significant improvement in one or more indicia of the disease. Where appropriate, a clinically accepted grade or scaling system for the given disease or disorder can be applied, with an improvement in the scale or grade being indicative of effective treatment. Depending upon the therapeutic composition, various subject parameters, and the mode of delivery, effective dosages can include, for example, 1 ng/kg of body weight up to a gram or more per kg of body weight and any amount in between. Preferred amounts can be, for example, in the range of 5 μg/kg body weight to 500 mg/kg of body weight or any amount in between. Dosages in such ranges can be administered once, twice, three times, four times or more per day, or every two days, every three days, every four days, once a week, twice a month, once a month or less frequently over a duration of days, weeks or months, depending on the breast cancer being treated, and as determined by a clinician. Sustained release formulations of the therapeutic compositions targeting one or more TGS biomarkers are specifically contemplated herein. Continuous, relatively low doses are also contemplated after an initial higher therapeutic dose.
The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Exemplary modes of administration of the therapeutic compositions described herein, include, but are not limited to, injection, infusion, inhalation (e.g., intranasal or intratracheal), ingestion, rectal, and topical (including buccal and sublingual) administration. The phrases “parenteral administration” and “administered parenterally” as used herein, refer to modes of administration other than enteral and topical administration, usually by injection. As used herein, “injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. The phrases “systemic administration,” “administered systemically”, “peripheral administration” and “administered peripherally” as used herein refer to administration of a therapeutic composition other than directly into a target site, tissue, or organ, such as the lung, such that it enters the subject's circulatory system and, thus, is subject to metabolism and other like processes.
Systems
Also provided herein, in other aspects and embodiments are systems (and computer readable media for causing computer systems) to perform methods for classifying, determining or diagnosing the type of a cancer or tumor, such as a breast tumor from a patient, or providing a prognosis to a cancer patient.
Accordingly, in some aspects, provided herein are systems for classifying, determining or diagnosing the type of a cancer or tumor, such as a breast tumor from a patient, or providing a prognosis to a tumor patient. Such systems comprise: (a) a determination module configured to receive a biological sample, measure expression levels in the biological sample of at least 2 of the 154 biomarkers of the triple negative breast cancer gene signature biomarker genes provided in Table 3 (SEQ ID NOs: 1-154), and to output information of the expression levels of the at least 2 of the 154 biomarkers of the triple negative breast cancer gene signature biomarker genes in the biological sample; (b) a storage device configured to store the output information of the expression levels of the at least 2 of the 154 biomarkers of the triple negative breast cancer gene signature biomarker genes from the determination module; (c) a comparison module adapted to receive input from the storage device and compare the data stored on the storage device with reference expression level data of each of the at least 2 of the 154 biomarkers of the triple negative breast cancer gene signature biomarker genes, wherein if the expression level data of the at least 2 of the 154 biomarkers of the triple negative breast cancer gene signature biomarker genes in the biological sample is increased relative to the reference expression level data, the comparison module provides information to an output module that the biological sample is associated with a subject that has an epithelial progenitor cell tumor, a poor prognosis tumor or a highly malignant tumor, or a subject having a poor prognosis of breast cancer; and (d) an output module for displaying the information to the user.
Embodiments of the systems provided herein can be described through functional modules, which are defined by computer executable instructions recorded on computer readable media and which cause a computer to perform method steps when executed. The modules described herein are segregated by function for the sake of clarity. However, it should be understood that the modules/systems need not correspond to discreet blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times. Furthermore, it should be appreciated that the modules can perform other functions, thus the modules are not limited to having any particular functions or set of functions.
The computer readable storage media can be any available tangible media that can be accessed by a computer. Computer readable storage media includes volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer readable storage media includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (erasable programmable read only memory), EEPROM (electrically erasable programmable read only memory), USB memory, flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, cloud server memory systems, other types of volatile and non-volatile memory, and any other tangible medium which can be used to store the desired information and which can accessed by a computer including and any suitable combination of the foregoing.
Computer-readable data embodied on one or more computer-readable storage media can define instructions, for example, as part of one or more programs, that as a result of being executed by a computer, instruct the computer to perform one or more of the functions described herein, and/or various embodiments, variations and combinations thereof. Such instructions can be written in any of a plurality of programming languages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof. The computer-readable storage media on which such instructions are embodied can reside on one or more of the components of either of a system, or a computer readable storage medium described herein, or can be distributed across one or more of such components.
The computer-readable storage media can be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein. In addition, it should be appreciated that the instructions stored on the computer-readable medium, described above, are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions can be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a computer to implement aspects of the present invention. The computer executable instructions can be written in a suitable computer language or combination of several languages. Basic computational biology methods are known to those of ordinary skill in the art and are described in, for example, Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001).
The functional modules of certain embodiments of the systems described herein include, at minimum, a determination module or device, a storage module or device, a comparison module or device, and an output module or device or display module or device. The functional modules can be executed on one, or multiple, computers, or by using one, or multiple, computer networks. The determination system has computer executable instructions to provide e.g., expression information in computer readable form.
The determination system can comprise any system for determining or assaying expression levels of two or more genes of SEQ ID NOs.: 1-154. Such systems can include, but are not limited to, PCR or quantitative PCR machines or devices, microarray devices or systems, Northern blot analysis systems, ELISA etc., as known to one of ordinary skill in the art.
The information determined in the determination system can be read by the storage device. As used herein the “storage device” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of an electronic apparatus suitable for use with the present invention include a stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, local and remote servers, and local and distributed computer processing systems. Storage devices also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, remote or local servers, magnetic tape, optical storage media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, general hard disks and hybrids of these categories such as magnetic/optical storage media. The storage device is adapted or configured for having recorded thereon nucleic acid sequence information. Such information can be provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication.
As used herein, “stored” refers to a process for encoding information on the storage device. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising information relating to immune stimulating microbes.
In some embodiments of the aspects and embodiments described herein, the reference data stored in the storage device to be read by the comparison module is e.g., expression data of two or more genes of Table 3 (SEQ ID NOs: 1-154) obtained from a patient sample, such as a patient having a triple-negative breast cancer.
The “comparison module” can use a variety of available software programs and formats for the comparison operative to compare expression information data of the two or more TGS genes or biomarkers of Table 3(SEQ ID NOs: 1-154) determined in the determination system to one or more reference samples and/or stored reference data. In some embodiments of the systems described herein, the comparison module is configured to use pattern recognition techniques to compare information from one or more entries to one or more reference data patterns. The comparison module can be configured using existing commercially-available or freely-available software for comparing patterns, and can be optimized for particular data comparisons that are conducted. The comparison module provides computer readable information related to the expression levels of two or more of the TGS genes of Table 3 (SEQ ID NOs: 1-154).
The comparison module, or any other module of the invention, can include an operating system (e.g., UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server. World Wide Web application includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) statements). Generally, the executables will include embedded SQL statements. In addition, the World Wide Web application can include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests. The Configuration file also directs requests for server resources to the appropriate hardware—as may be necessary should the server be distributed over two or more separate computers. In one embodiment, the World Wide Web server supports a TCP/IP protocol. Local networks such as this are sometimes referred to as “Intranets.” An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in some embodiments, users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers.
The comparison module provides a computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide a content based in part on the comparison result that may be stored and output as requested by a user using a display module or output device.
The content based on the comparison result, can be, for example, the type of tumor, or the prognosis of the tumor. Alternatively, or additionally, the content based on the comparison result can be a further treatment step indicated for the patient, e.g., administration of a proteasome inhibitor or histone deacetylase inhibitor.
In some embodiments of the systems described herein, the content based on the comparison result is displayed on a computer monitor. In some embodiments of the systems described herein, the content based on the comparison result is displayed through printable media. The display module can be any suitable device configured to receive from a computer and display computer readable information to a user. Non-limiting examples include, for example, general-purpose computers such as those based on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, Calif., tablet or mobile phone devices, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types.
In some embodiments of the systems described herein, a World Wide Web browser is used for providing a user interface for display of the content based on the comparison result. It should be understood that other modules of the invention can be adapted to have a web browser interface. Through the Web browser, a user may construct requests for retrieving data from the comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces.
The modules of the machine, or those used in the computer readable medium, can assume numerous configurations. For example, function may be provided on a single machine or distributed over multiple machines.
Accordingly, the methods described herein therefore provide for systems (and computer readable media for causing computer systems) to perform methods for determining or diagnosing the type of a cancer or tumor in a patient, such as a breast tumor from a patient, or providing a prognosis to a tumor patient.
Systems and computer readable media described herein are merely illustrative embodiments of the invention for performing methods of diagnosis in an individual, and are not intended to limit the scope of the invention. Variations of the systems and computer readable media described herein are possible and are intended to fall within the scope of the invention.
Kits for Detection of Malignancy Associated Response Signature Biomarkers
Conveniently, in some aspects, expression of malignancy associated response signature biomarkers in the methods described herein can be evaluated using a kit comprising at least one malignancy associated response signature biomarker probe suitable for detecting one or more malignancy associated response signature biomarkers. As used herein, a “malignancy associated response signature probe” can include any molecule capable of detecting a malignancy associated response signature biomarker including, but not limited to, monoclonal and polyclonal antibodies and fragments thereof, and oligonucleotides. For example, the kit can comprise an antibody specific for an epitope of a malignancy associated response signature biomarker protein encoded by an malignancy associated response signature biomarker gene, an oligonucleotide probe complementary to at least a portion of a malignancy associated response signature biomarker gene or to at least a portion an RNA (e.g., mRNA) encoded by a malignancy associated response signature biomarker gene, or primer pairs suitable for evaluating gene expression of a malignancy associated response signature biomarker by a polymerase chain reaction (PCR)-based method, such as real time PCR or reverse transcription PCR. Other methodologies for measuring expression of a malignancy associated response signature biomarker include ribonuclease protection assays, 51 nuclease assays, and Northern blot analyses. Optionally, the kits include instructions for detecting malignancy associated response signature biomarker detection or for performing the methods or diagnosis and prognosis described herein
In some embodiments, the kit can comprise a microarray that can be used to determine expression of at least one malignancy associated response signature biomarker in a tumor sample and instructions for analyzing the information for use in the methods described herein. The microarray includes at least one oligonucleotide comprising a sequence of at least one of the malignancy associated response signature biomarker from the group of markers listed in Table 3 (SEQ ID NOs: 1-154). More preferably, the microarray includes oligonucleotides comprising a sequence of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 150, or all 154 of the 154 malignancy associated response signature biomarkers described in Table 3 (SEQ ID NOs: 1-154). The term “microarray,” as used herein, refers to an ordered arrangement of hybridizable array elements, e.g. oligonucleotide probes, on a substrate, e.g. glass slide or silica. Suitably, the microarray comprises control probes to allow for detection of expression levels that can be used to determine enrichment of malignancy associated response signature biomarker genes relative to a control or reference sample.
Screening Methods
The malignancy associated response signature biomarkers described herein are also useful in screening and identifying novel therapeutic agents against malignant and poor prognosis breast cancers, such as basal-like breast tumors.
Accordingly, in one aspect, provided herein is a method of identifying a candidate therapeutic agent against a tumor, such as a basal-like breast tumor, the method comprising the steps of (a) exposing a BPLER cell culture to a test agent, wherein said BPLER cell culture comprises human breast primary epithelial cells (BPE) transformed with a defined set of genetic elements (BPLER cells); (b) measuring the expression of at least 10 of the 154 of the genes of a malignancy associated response signature in the culture, wherein the malignancy associated response signature comprises an expression pattern of a set of 10-154 biomarkers set forth in Table 3 (SEQ ID NOs: 1-154); (c) comparing the expression of the same at least 10 genes of malignancy associated response signature as was measured in step (b) to an expression signature reference from a BPLER cell culture that has not been exposed to the test agent, wherein a decrease in expression of at least 5 of the at least 10 genes in the test culture compared to the expression signature of the reference culture indicates that the test agent is a candidate therapeutic agent against the tumor, e.g., basal-like breast tumor.
The term “screening” as used herein refers to the use of cells and tissues in the laboratory to identify agents with a specific function, e.g., inhibiting activity against a malignancy associated response signature biomarker. “High-throughput screening technologies” refer to platforms and assays used to rapidly test thousands of test compounds. For example, reporter systems used in cell lines can be used to assess whether compounds activate or inhibit particular signaling pathways of interest, such as the ubiquitin-proteasome pathway.
The “candidate agent” used in the screening methods described herein can be selected from a group of a chemical, small molecule, chemical entity, nucleic acid sequences, an action; nucleic acid analogues or protein or polypeptide or analogue of fragment thereof. Nucleic acid sequences include, for example, but not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide agent or fragment thereof, can be any protein of interest, for example, but not limited to; mutated proteins; therapeutic proteins; truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins of interest can be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. A candidate agent also includes any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments, the candidate agent is a small molecule having a chemical moiety. Such chemical moieties can include, for example, unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups, including macrolides, leptomycins and related natural products or analogues thereof. Candidate agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
As used herein, the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
The term “inhibitor” refers to an agent that inhibits expression of a polypeptide or polynucleotide, or binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity of the polypeptide or the polynucleotide. Inhibitors are agents that, e.g., inhibit expression, e.g., translation, post-translational processing, stability, degradation, or nuclear or cytoplasmic localization of a polypeptide, or transcription, post transcriptional processing, stability or degradation of a polynucleotide or bind to, partially or totally block stimulation, DNA binding, transcription factor activity or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide. An inhibitor can act directly or indirectly. Inhibition is achieved when the activity value of a polypeptide or polynucleotide is about at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or absent or undetectable in comparison to a reference or control level in the absence of the inhibitor.
Also included as candidate agents are pharmacologically active drugs, genetically active molecules, etc. Such candidate agents of interest include, for example, chemotherapeutic agents, hormones or hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof. Exemplary of pharmaceutical agents suitable for use with the screening methods described herein are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all of which are incorporated herein by reference in their entireties. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992), the contents of which is herein incorporated in its entirety by reference.
Candidate agents, such as chemical compounds, can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the candidate compounds for use in the screening methods described herein are known in the art and include, for example, those such as described in R. Larock (1989) Comprehensive Organic Transformations, VCH Publishers; T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof, the contents of each of which are herein incorporated in their entireties by reference.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233, the contents of each of which are herein incorporated in their entireties by reference.
Libraries of candidate agents can be presented in solution (e.g., Houghten (1992), Biotechniques 13:412-421), or on beads (Lam (1991), Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.), the contents of each of which are herein incorporated in their entireties by reference.
High through-put screening is a process in which libraries of compounds are tested for a given activity. High through-put screening seeks to screen large numbers of compounds rapidly and in parallel. For example, using microtiter plates, robotic equipment, and automated assay equipment, a pharmaceutical company may perform as many as 100,000 assays per day in parallel.
The compound screening assays described herein can involve more than one measurement of an observable reporter function, expression or activity of a malignancy associated response biomarker gene. Multiple measurements allow for following the biological activity over incubation time with the test compound. In one embodiment, the expression or activity of a malignancy associated response biomarker gene is measured at a plurality of times to allow monitoring of the effects of the test compound at different incubation times.
Accordingly, in some embodiments of the screening aspects, provided herein is a high throughput automated screening system for identifying a candidate therapeutic agent against a tumor, such as a basal-like breast tumor, the system comprising:
a) a high throughput candidate agent screening culture, connected to
b) a computer processor and a computer-readable physical medium with software instructions encoded thereupon for a process, executable by said processor, said instructions comprising:
(i) instructions for receiving data regarding the measurement of the expression of at least 10 of the 154 of the genes of a malignancy associated response in a high-throughput screening culture, wherein the malignancy associated response comprises an expression pattern of a set of 10-154 biomarkers set forth in Table 3 (SEQ ID NOs: 1-154); (ii) instructions for comparing the expression data received in (i) with reference signature expression data of the same at least 10 genes of malignancy associated response from a cell culture that has not been exposed to the test agents, wherein said comparing identifies whether there is a decrease in expression of at least 5 of the at least 10 genes in the test culture compared to the expression signature of the reference culture and thus indicates that the test agent is a candidate therapeutic agent against the tumor, such as basal-like breast tumor; and (iii) outputting the result of said comparison to a user interface.
The high-throughput screening and other system methods described herein be implemented using any device capable of implementing the methods. Examples of devices that can be used include but are not limited to electronic computational devices, including computers of all types. When the methods described herein are implemented in a computer, the computer program that can be used to configure the computer to carry out the steps of the methods can be contained in any computer readable medium capable of containing the computer program. Examples of computer readable medium that can be used include but are not limited to diskettes, CD-ROMs, DVDs, ROM, RAM, USB devices, and other memory and computer storage devices. The computer program that can be used to configure the computer to carry out the steps of the methods can also be provided over an electronic network, for example, over the internet, world-wide web, an intranet, or other network.
In one such example, the methods described herein can be implemented in a system comprising a processor and a computer readable medium that includes program code means for causing the system to carry out the steps of the methods described in the present invention. The processor can be any processor capable of carrying out the operations needed for implementation of the methods. The program code means can be any code that when implemented in the system can cause the system to carry out the steps of the methods described in the present invention. Examples of program code means include but are not limited to instructions to carry out the methods described in this patent written in a high level computer language such as C++, Java, or Fortran; instructions to carry out the methods described herein written in a low level computer language such as assembly language; or instructions to carry out the methods described herein in a computer executable form such as compiled and linked machine language.
Some aspects and embodiments disclosed herein can be illustrated by, for example any of the following numbered paragraphs:

    1. A malignancy associated response signature for a cancer-initiating cell consisting essentially of an expression pattern of a set of biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151, wherein at least 5 of the 23 biomarkers have increased expression compared to a reference value.
    2. The malignancy associated response signature of paragraph 1, wherein the cancer-initiating cell is a breast cancer-initiating cell.
    3. The malignancy associated response signature of paragraph 1, wherein the cancer-initiating cell is a triple-negative breast cancer-initiating cell.
    4. The malignancy associated response signature of paragraph 1, wherein the cancer-initiating cell is a Luminal B breast cancer-initiating cell
    5. The malignancy associated response signature of paragraph 1, wherein the cancer-initiating cell is an epithelial breast cancer-initiating cell.
    6. The malignancy associated response signature of any one of paragraphs 1-5, wherein the expression of the at least 5 of the 23 biomarkers is increased at least 1.8-fold compared to the reference value.
    7. A malignancy associated response signature for a cancer-initiating cell comprising an expression pattern of a set of at least 10 biomarkers set forth in SEQ ID NOs: 1-154, wherein at least 10 of the 154 markers have increased expression compared to a reference value.
    8. The malignancy associated response signature of paragraph 7, wherein the cancer-initiating cell is a breast cancer-initiating cell.
    9. The malignancy associated response signature of paragraph 7, wherein the cancer-initiating cell is a triple-negative breast cancer-initiating cell.
    10. The malignancy associated response signature of paragraph 7, wherein the cancer-initiating cell is a Luminal B breast cancer-initiating cell.
    11. The malignancy associated response signature of paragraph 7, wherein the cancer-initiating cell is an epithelial breast cancer-initiating cell.
    12. The malignancy associated response signature of any one of paragraphs 7-11, wherein the expression of the at least 5 biomarkers is increased at least 1.8-fold compared to the reference value.
    13. The malignancy associated response signature of any one of paragraphs 7-12, wherein at least 2 of the set of at least 10 biomarkers set forth in SEQ ID NOs: 1-154 is selected from the group consisting of SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
    14. A malignancy associated response signature for a cancer-initiating cell susceptible to proteasomal gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, wherein at least 3 of the 15 biomarkers have increased expression compared to a reference value.
    15. A malignancy associated response signature for a cancer-initiating cell susceptible to mitosis gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137, wherein at least 3 of the 14 biomarkers have increased expression compared to a reference value.
    16. A malignancy associated response signature for a cancer-initiating cell susceptible to RNA splicing gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151, wherein at least 3 of the 13 biomarkers have increased expression compared to a reference value.
    17. A malignancy associated response signature for a cancer-initiating cell susceptible to molecular transport gene inhibition comprising an expression pattern of a set of at least 3 biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147, wherein at least 3 of the 14 biomarkers have increased expression compared to a reference value.
    18. A malignancy associated response signature for a cancer-initiating cell susceptible to metabolic gene inhibition comprising an expression pattern of a set of at least 3 biomarkers of SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153, wherein at least 3 of the 13 biomarkers have increased expression compared to a reference value.
    19. The malignancy associated response signature of any one of paragraphs 14-18, wherein the cancer-initiating cell is a breast cancer-initiating cell.
    20. The malignancy associated response signature of any one of paragraphs 14-18, wherein the cancer-initiating cell is a triple-negative breast cancer-initiating cell.
    21. The malignancy associated response signature of any one of paragraphs 14-18, wherein the cancer-initiating cell is a Luminal B breast cancer-initiating cell.
    22. The malignancy associated response signature of any one of paragraphs 14-18, wherein the cancer-initiating cell is an epithelial breast cancer-initiating cell.
    23. The malignancy associated response signature of any one of paragraphs 14-22, wherein the expression of the at least 3 biomarkers is at least 1.8-fold increased compared to the reference value.
    24. A method of classifying a cancer in a subject in need thereof, the method comprising:
    
        a. assaying expression of ten or more of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in a biological sample obtained from the subject having a cancer, and
        b. comparing the expression of the ten or more of the 154 malignancy associated response signature biomarkers of SEQ ID NOs: 1-154 in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of 1.8-fold or greater of at least ten of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least ten of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
    
    
    25. The method of paragraph 24, wherein the cancer is a breast cancer.
    26. The method of paragraph 24, wherein the cancer is a triple-negative breast cancer.
    27. The method of paragraph 24, wherein the cancer is a Luminal B breast cancer.
    28. The method of paragraph 24, wherein the cancer is an epithelial breast cancer.
    29. The method of any one of paragraphs 27-31, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
    30. The method of any one of paragraphs 24-29, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer and wherein if at least two of the ten or more genes having increased expression is a proteasomal malignancy associated response signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
    31. The method of any one of paragraphs 29 or 30, wherein the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
    32. The method of any one of paragraphs 29-31, wherein the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
    33. The method of any one of paragraphs 29-32, wherein the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
    34. The method of any one of paragraphs 29-30, wherein the at least one proteasome inhibitor is bortezomib.
    35. The method of any one of paragraphs 24-34, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one histone deacetylase inhibitor to the subject.
    36. The method of paragraph 35, wherein the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent.
    37. The method of paragraph 35, wherein the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
    38. The method of any one of paragraphs 24-37, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one glygolytic inhibitor to the subject.
    39. The method of paragraph 38, wherein the at least one glycolysis inhibitor is an siRNA or antisense RNA agent.
    40. The method of any one of paragraphs 24-39, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer, relative survival rate, relative risk of metastasis, treatment option, or any combination thereof is determined for the subject.
    41. A method of classifying a cancer in a subject in need thereof, the method comprising:
    
        a. assaying expression of at least five biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151 in a biological sample obtained from the subject having a cancer, and
        b. comparing the expression of the five or more of the 23 malignancy associated response signature biomarkers in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of 1.8-fold or greater of at least ten of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least ten of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
    
    
    42. The method of paragraph 41, wherein the cancer is a breast cancer.
    43. The method of paragraph 41, wherein the cancer is a triple-negative breast cancer.
    44. The method of paragraph 41, wherein the cancer is a Luminal B breast cancer.
    45. The method of paragraph 41, wherein the cancer is an epithelial breast cancer.
    46. The method of any one of paragraphs 41-45, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
    47. The method of any one of paragraphs 41-45, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer and wherein at least one of the five or more genes having increased expression is a proteasomal malignancy associated response signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 29, 33, 34, and 38, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
    48. The method of any one of paragraphs 46-47, wherein the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
    49. The method of any one of paragraphs 46-47, wherein the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
    50. The method of any one of paragraphs 46-49, wherein the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
    51. The method of any one of paragraphs 46-47, wherein the at least one proteasome inhibitor is bortezomib.
    52. A method of classifying a cancer in a subject in need thereof, the method comprising:
    
        a. assaying expression of at least three malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149 in a biological sample obtained from the subject having a cancer, and
        b. comparing the expression of the at least three malignancy associated response signature biomarkers in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of 1.8-fold or greater of at least three of the biomarkers in the biological sample obtained from the subject relative to the reference value indicates that the cancer is classified as having a poor prognosis or being a malignant cancer, and absence of increased expression of 1.8-fold or greater of at least three of the biomarkers relative to the reference value indicates that the cancer does not have poor prognosis or is not a malignant cancer.
    
    
    53. The method of paragraph 52, wherein the cancer is a breast cancer.
    54. The method of paragraph 52, wherein the cancer is a triple-negative breast cancer.
    55. The method of paragraph 52, wherein the cancer is a Luminal B breast cancer.
    56. The method of paragraph 52, wherein the cancer is an epithelial breast cancer.
    57. The method of any one of paragraphs 52-56, wherein if the cancer is classified as having a poor prognosis or being a malignant cancer, the method further comprises the step of administering at least one proteasome inhibitor to the subject.
    58. The method of paragraph 57, wherein the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
    59. The method of paragraph 57, wherein the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
    60. The method of any one of paragraphs 57-59, wherein the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
    61. The method of paragraph 57, wherein the at least one proteasome inhibitor is bortezomib.
    62. An assay comprising the steps of:
    
        a. measuring expression of ten or more of the 154 malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 in a biological sample obtained from a subject having cancer, and
        b. comparing the expression of the ten or more of the 154 malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 in the biological sample obtained from the subject having a cancer with a reference value, wherein increased expression of at least ten of the measured biomarkers in the biological sample obtained from the subject relative to the reference value diagnoses the patient as having a poor prognosis or malignant cancer, and absence of increased expression of at least ten of the measured biomarkers relative to the reference value diagnoses the patient as not having a poor prognosis or malignant cancer.
    
    
    63. The assay of paragraph 62, wherein the increased expression of the ten or more of the 154 malignancy associated response signature biomarkers in the biological sample determines a prognosis for the subject having the cancer.
    64. The assay of paragraph 63, wherein the prognosis comprises relative survival rate, relative risk of metastasis, treatment option, or any combination thereof.
    65. The assay of any one of paragraphs 62-64, wherein the cancer is a breast cancer.
    66. The assay of any one of paragraphs 62-64, wherein the cancer is a triple-negative breast cancer.
    67. The assay of any one of paragraphs 62-64, wherein the cancer is a Luminal B breast cancer.
    68. The assay of any one of paragraphs 62-64, wherein the cancer is an epithelial breast cancer.
    69. The assay of any one of paragraphs 62-68, wherein if the subject is diagnosed as having a poor prognosis or malignant cancer, the subject is administered at least one proteasome inhibitor.
    70. The assay of any one of paragraphs 62-68, wherein if the subject is diagnosed as having a poor prognosis or malignant cancer and wherein at least two of the ten or more genes having increased expression is a proteasomal malignancy associated response signature biomarker of SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149, the subject is administered at least one proteasome inhibitor.
    71. The assay of any one of paragraphs 69-70, wherein the at least one proteasome inhibitor specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
    72. The assay of any one of paragraphs 69-71, wherein the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
    73. The assay of any one of paragraphs 69-72, wherein the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
    74. The assay of any one of paragraphs 69-70, wherein the at least one proteasome inhibitor is bortezomib.
    75. The assay of any one of paragraphs 69-74, wherein if the subject is diagnosed as having a poor prognosis or malignant cancer, the subject is administered at least one histone deacetylase inhibitor.
    76. The assay of paragraph 75, wherein the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent.
    77. The assay of paragraph 75, wherein the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
    78. The assay of any one of paragraphs 69-77, wherein if the subject is diagnosed as having a poor prognosis or malignant cancer, the subject is administered at least one glygolytic inhibitor.
    79. The assay of paragraph 78, wherein the at least one glycolysis inhibitor is an siRNA or antisense RNA agent.
    80. An assay comprising the steps of:
    
        a. dividing a cell culture grown from a biopsy obtained from a subject having cancer into at least 5 separate cultures;
        b. exposing each of the at least 5 separate cultures to a different inhibitory agent, wherein each said different inhibitory agent specifically inhibits a different malignancy associated response signature biomarker set forth in SEQ ID NOs: 1-154
        c. growing each of the at least 5 separate cell cultures of step (b) for at least 12 hours;
        d. measuring viability of the cells from each of the cultures of step (c), wherein if the total viability of the cells in at least 60% of the cultures is decreased by at least 25%, then the biopsy obtained from the subject comprises cancer-initiating cells.
    
    
    81. The assay of paragraph 80, wherein the inhibitory agents are selected from siRNA agents, antisense RNA agents, or small molecules, that specifically inhibit any of the cancer gene signature biomarkers set forth in SEQ ID NOs: 1-154.
    82. The assay of any one of paragraphs 80-81, wherein the inhibitory agents specifically inhibit any of the malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
    83. The assay of any one of paragraphs 80-81, wherein at least one inhibitory agent specifically inhibits any of the malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72, 77, 78, 84, 91, 93, and 151.
    84. The assay of any one of paragraphs 80-83, wherein at least one inhibitory agent specifically inhibits any of the proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
    85. The assay of any one of paragraphs 80-84, wherein at least one inhibitory agent specifically inhibits any of the mitosis cancer gene signature biomarkers set forth in SEQ ID NOs: 9, 17, 22, 23, 31, 37, 35, 52, 68, 80, 101, 124, 130, and 137.
    86. The assay of any one of paragraphs 80-85, wherein at least one inhibitory agent specifically inhibits any of the RNA splicing malignancy associated response signature biomarkers set forth in SEQ ID NOs: 5, 10, 11, 18, 20, 26, 36, 41, 57, 61, 123, 127, and 151.
    87. The assay of any one of paragraphs 80-86, wherein at least one inhibitory agent specifically inhibits any of the molecular transport malignancy associated response signature biomarkers set forth in SEQ ID NOs: 12, 13, 32, 48, 51, 56, 64, 91, 96, 98, 108, 121, and 147.
    88. The assay of any one of paragraphs 80-87, wherein at least one inhibitory agent specifically inhibits any of the metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153.
    89. A method for treating a cancer in a subject in need thereof comprising the step of administering to a subject having a cancer classified or diagnosed as having poor prognosis or being malignant, using the method of any one of paragraphs 24-61, or the assay of any one of paragraphs 62-79, at least one proteasome inhibitor in a pharmaceutically acceptable carrier.
    90. The method of paragraph 89, wherein the at least one proteasome inhibitor is an siRNA or antisense RNA agent.
    91. The method of any one of paragraphs 89-90, wherein the proteasome inhibitor specifically inhibits a proteasomal malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1, 2, 4, 7, 15, 16, 21, 29, 33, 34, 38, 60, 74, 76, and 149.
    92. The assay of any one of paragraphs 89-90, wherein the at least one proteasome inhibitor specifically inhibits the malignancy associated response signature biomarker set forth in SEQ ID NOs: 134 (MCL-1).
    93. The method of paragraph 89, wherein the proteasome inhibitor is bortezomib.
    94. A method for treating a cancer in a subject in need thereof comprising the step of administering to a subject having a cancer classified or diagnosed as having poor prognosis or being malignant, using the method of any one of paragraphs 24-61, or the assay of any one of paragraphs 62-79, at least one histone deacetylase inhibitor in a pharmaceutically acceptable carrier.
    95. The method of paragraph 92, wherein the at least one histone deacetylase inhibitor is an siRNA or antisense RNA agent.
    96. The method of paragraph 93, wherein the at least one histone deacetylase inhibitor is trichostatin A (TSA) or Vorinostat.
    97. A method for treating a cancer in a subject in need thereof comprising the step of administering to a subject having a cancer classified or diagnosed as having poor prognosis or being malignant, using the method of any one of paragraphs 24-61, or the assay of any one of paragraphs 62-79, at least one metabolic inhibitor in a pharmaceutically acceptable carrier.
    98. The method of paragraph 97, wherein the at least one metabolic inhibitor is an siRNA or antisense RNA agent.
    99. The method of any one of paragraphs 97-98, wherein the at least one metabolic inhibitor specifically inhibits a metabolic malignancy associated response signature biomarker set forth in SEQ ID NOs: 46, 49, 65, 69, 72, 78, 104, 107, 112, 126, 138, 141, and 153.
    100. A system for obtaining data from at least one sample from a subject having a cancer, the system comprising:
    
        a. a determination module configured to receive said at least one sample from a subject having a cancer and perform an expression analysis of ten or more malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154 on said at least one sample to generate an expression data output;
        b. a storage device configured to store said expression data output from said determination module;
        c. a comparison module configured to receive said expression data output of the sample from the subject having a cancer and perform at least one expression analysis on said expression data output to determine the presence or absence of one of the following conditions and produce a comparison data output:
        
            i. the sample from the subject having a cancer has increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154; or
            ii. the sample from the subject having a cancer does not have increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154; and
        
        
        d. an output or display module for displaying a content based in part on the comparison data output from said comparison module, wherein the content comprises a signal indicative that the sample from the subject having a cancer has increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154, or a signal indicative that the sample from the subject having a cancer does not have increased expression of ten or more metabolic malignancy associated response signature biomarkers set forth in SEQ ID NOs: 1-154.
    
    
    101. The system of paragraph 100, wherein the content displayed on said display module further comprises a signal indicative of the subject being recommended to receive a particular treatment regimen.
    102. A method of identifying a candidate therapeutic agent against a cancer initiating cell comprising the step of
    
        a. exposing a BPLER cell culture to a test agent, wherein said BPLER cell culture comprises human breast primary epithelial cells (BPE) transformed with a defined set of genetic elements;
        b. measuring expression of at least ten of the 154 malignancy associated response signature biomarkers of paragraph 1 in the culture,
        c. comparing the expression of the same at least 10 biomarkers of malignancy associated response signature biomarkers of paragraph 1 as was measured in step (b) to an expression signature reference from a BPLER cell culture that has not been exposed to the test agent, wherein a decrease of expression of at least 5 of the at least 10 genes in the test culture compared to the expression signature reference indicates that the test agent is a candidate therapeutic agent against a cancer initiating cell.
    
    
    103. A therapeutic agent identified by the method of paragraph 102.
    104. The therapeutic agent of paragraph 103, wherein said agent is an siRNA agent, an antisense RNA agent, an antibody or antigen-binding fragment thereof, or a small molecule compound.
    105. A pharmaceutical compound comprising the therapeutic agent of paragraph 103 and a pharmaceutically acceptable carrier.


Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology are found in Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.), which are all incorporated by reference herein in their entireties.
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that could be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
This invention is further illustrated by the following examples which should not be construed as limiting.
EXAMPLES
Example 1
BPLER and HMLER Breast Cancer Cells Retain Progenitor-Like and Myoepithelial-Like Features, Respectively
It has been shown that different populations of human breast primary epithelial cells (BPE and HME), selected in chemically-defined media (e.g., WIT-T and MEGM, respectively), give rise to distinct tumor phenotypes upon transformation with the same set of genetic elements (e.g., hTERT, SV40 early region and h-RASV12), indicating that key properties of a tumor can be predetermined by its cell of origin. BPE transformed derivatives (BPLER) are highly malignant, whereas HME transformed derivatives (HMLER) are poorly tumorigenic compared to BPLER cells, although the two cell lines proliferate at a similar rate in vitro. As described herein, we have determined that such properties can be associated with a different state of differentiation.
To exclude any effects related to cell culture medium or substrate, all experiments described herein were performed by propagating BPLER and HMLER cells in the same medium (WIT-T) in standard tissue culture plates, which did not alter the relative rate of proliferation. Under these conditions, BPLER cells expressed a wide array of luminal and myoepithelial cytokeratins, including CK5, CK14, CK17, CK8, and CK18, consistent with an epithelial progenitor-like phenotype, whereas HMLER cells expressed lower levels of most cytokeratins except CK5. Accordingly, BPLER cells retained an epithelial morphology and co-expressed CK14 and CK18 proteins at the single-cell level, a property of mammary epithelial progenitors and terminal duct lobular unit (TDLU) cells, from which breast cancer is believed, without wishing to be bound or limited by theory, to originate. BPLER cells also expressed E-cadherin and Vimentin transcripts, whereas HMLER cells expressed higher levels of vimentin but lower levels of E-cadherin transcripts. Both cell lines were triple-negative (ER−, PR−, HER−) and expressed EGFR mRNA.
Together, these data demonstrate that BPLER cells retain a progenitor-like phenotype, expressing both luminal and myoepithelial markers, whereas HMLER cells retain a more committed myoepithelial phenotype. These findings confirm at a protein level previous microarray-based studies, which showed a mixed luminal/myoepithelial and myoepithelial-like transcriptional profile associated with BPLER and HMLER cells, respectively.
BPLER Cells have Properties of Basal-Like Breast Tumor-Initiating Cells.
The phenotypic features of BPLER cells are similar to poor prognosis human basal-like breast tumors (BL-BTs), which are enriched in breast tumor-initiating cells (BT-ICs). We found that BPLER cells retained high tumor-initiating potential, demonstrated by their ability to form tumors in mice with as few as 50 cells. In contrast, we found that HMLER cells did not initiate tumors in mice with 5×104 or fewer cells, indicating that most HMLER cells had shed their tumor-initiating potential.
The bulk of BPLER cells displayed a CD44+/CD24low/−/ESA+ antigenic configuration, which was previously associated with human BT-ICs. However, we show that these markers are not sufficient to specify the tumor-initiating state, because poorly malignant HMLER cells also displayed a similar phenotype.
To define clinical parameters, histological features of tumors originated from BPLER cells were first analyzed. BPLER xenografts stained positive for CK5 and CK14 and were negative for ER, were highly proliferative (as determined by Ki-67 staining), had a high mitotic index (as determined by phospho-Histone H3 staining), displayed a clear inflammatory infiltrate, a discernible tumor stroma, pushing borders, and had alternated areas of glandular focal differentiation with poorly differentiated areas, all of which are characteristic properties of human basal-like tumors. Further, only BPLER cells that established direct contact with the stroma retained vimentin expression, which was undetectable in the remaining cells, i.e., BPLER cells not having direct contact with the stroma. Because vimentin is typically repressed in differentiated epithelial cells, this indicates that the tumor stroma plays a role in maintaining the relatively uncommitted state of BPLER cells in vivo. Accordingly, BPLER cells give rise to tumors closely resembling human basal-like tumors at the histological level.
To further corroborate these findings, we compared the global transcriptional profile of six different BPLER tumor explants, derived from either Nude or NOD/SCID mice, with a set of 337 human primary tumors of known subtype (UNC337 dataset). To reduce background signal derived from human stroma, as opposed to BPLER mouse stroma, we adopted a principal component (PC) approach. All BPLER tumor explants clustered with human primary triple-negative breast tumors, and not with luminal, normal, or HER-2 subtypes. We further validated these findings using an independent set of 47 human primary breast tumors (Richardson-06 dataset), in which BPLER tumor explants also clustered with triple-negative tumors.
Accordingly, as demonstrated herein, BPLER cells not only have properties of BT-ICs, but also initiate tumors with global features of human primary basal-like breast tumors, thus representing a model of basal-like BT-ICs.
A Genome-Wide siRNA Screen Identifies Factors Required for Survival of Basal-Like BT-ICs
The data described herein so far demonstrate that BPLER and HMLER cells retain high and low tumor-initiating potential, respectively. Moreover, BPLER cells display several features of human basal-like breast cancer, including malignancy, cytokeratin expression, hormone receptor status and CD44/CD24 antigenic configuration, and form tumors closely resembling human basal-like breast cancer.
To identify survival factors associated with the tumor-initiating state, we performed a genome-wide siRNA lethality screen using BPLER and HMLER cell lines, which were derived from the same patient, harbored the same set of genetic elements, and proliferated at a similar rate, but were differentially malignant. By silencing one gene at a time, a comprehensive overview of the key mechanisms supporting the growth of highly malignant basal-like breast cancer cells was obtained, and revealed novel diagnostic biomarkers and therapeutic targets for more effective targeted diagnosis of and therapies against basal-like breast tumors.
The screen was organized into separate modules, each of which was developed independently. The assay was optimized to identify candidate genes required for survival of either BPLER or HMLER cells in an unbiased way on a genome-wide scale. We used siRNA libraries including 17,378 siRNA pools, each containing 4 distinct siRNAs targeting the same gene, which were screened in triplicate for each cell line.
A total of 1025 siRNA pools decreased BPLER viability, of which 780 also decreased HMLER viability. These siRNAs include hits not associated with malignancy but more generally affecting proliferation or survival, the global silencing of which has a greater likelihood of being cytotoxic to all cell types. For example, the top 20 hits in this category included PLK1, KIF11, RRM2, BCL-XL and POL2RA, all of which are well-known regulators of basic cellular functions (Table 1).
The remaining 245 pools were highly, moderately, and modestly selective siRNAs preferentially inhibiting BPLER cells as opposed to HMLER cells, and were therefore associated with tumor-initiating potential (Tables 2A-2C).
To further increase confidence in these hits, a secondary screen was performed (termed herein as a “cherry-pick screen”) transfecting the 4 siRNAs comprising each pool individually. Hits were confirmed if at least one out of four siRNAs scored positive in the secondary screen, since several siRNA pools used in the primary screen are predicted to contain only one functional siRNA. A total of 154 hits were confirmed in the cherry-pick screen, where validation rates were 88%, 75% and 52% for highly, moderately, and poorly selective hits, respectively (Table 3). These 154 high-confidence hits represent genes on which BPLER cells selectively depend for survival, and therefore comprise a functional module associated with tumor-initiating potential, referred to herein as a “Triple Negative Breast Cancer Gene Signature” (TGS) or a “Malignancy-Associated Response Signature” (MARS), as termed herein.
Poor Prognosis Human Breast Primary Tumors are Enriched for Genes Required for BPLER Survival
Having identified genes selectively required for survival of BPLER cells, we next determined that such genes were enriched in malignant human primary breast tumors. A single sample GSEA approach was used to project the TGS (MARS) gene set across a set of 47 human breast primary tumors and normal breast tissues (DFHCC3 dataset). A significant positive correlation was observed between the expression of TGS (MARS) genes and the primary tumor expression profiles (p<1.2e-05), indicating that most genes were enriched in malignant breast lesions compared to normal breast tissue.
Next, we assessed whether TGS (MARS) expression was specifically associated with poor prognosis breast tumors, which are enriched in BT-ICs, in an independent set of 295 human breast primary tumors with annotated clinical and molecular subtype data (NKI dataset). TGS (MARS) expression positively correlated with shorter survival (p<0.003), demonstrating that highly malignant cancer cells within primary tumors retain the expression of genes required for survival of BPLER cells.
To define whether basal-like primary tumors, in particular, were enriched in TGS (MARS) genes, we assessed their expression in the different subtypes of breast cancer within the NKI dataset. TGS (MARS) genes were retained in highly malignant basal-like breast cancers, as compared to HER-2, luminal A, and “normal” breast cancers (p<2.61×10−5). Notably, TGS (MARS) genes were also enriched in Luminal B tumors (p<3.79×10−6). These tumors comprise  ̃15% of human breast tumors and have variable prognosis. Luminal B tumors are ER positive, much like good prognosis luminal A tumors, but are also moderately differentiated, resembling poor prognosis basal-like tumors. We next determined whether TGS (MARS) enrichment separates tumors with different prognosis within the luminal B category. Impressively, two luminal B patient subgroups with markedly different survival were identified based on TGS (MARS) expression. No significant differences in survival were observed within the other subtypes using the same approach. These data further demonstrate the association between TGS (MARS) and malignancy, which appears, without wishing to be bound or limited by theory, to be independent from the tumor subtype.
Metastasis is the leading cause of death in breast cancer patients. We next determined whether metastatic tumors were also enriched in TGS (MARS) genes. We found that within the NKI dataset, patients with higher TGS (MARS) expression developed metastasis significantly earlier than patients with TGS expression (p<0.01). To further confirm these results, we performed an analysis of TGS (MARS) expression in 560 primary breast tumors collated from three independent datasets (EMC286, MSK82, EMC192), for which metastatic relapse data were available. In this setting, patients with higher TGS (MARS) expression also had a significant shorter metastasis-free survival (p<0.002).
In conclusion, the expression of TGS (MARS) genes was retained in a variety of malignant disease conditions, confirming the clinical relevance of the genes identified in our screens described herein as novel diagnostic and prognostic biomarkers and therapeutic targets.
BPLER Cells are Selectively Sensitive to Proteasome and Glycolysis Inhibitor Drugs
To determine the biological functions of the TGS genes, their enrichment in canonical pathways was assessed. The most significantly enriched pathways included Proteasome Degradation, Glycolysis, TNFα/NFκB signaling, IL-7 signaling, mRNA processing, Transcription Initiation, ID signaling, and GPCR Class A Rhodopsin-like. An approach based, in part, on the Pathway Expansion Analysis (PEXA) of Tu et al. was used to establish how these pathways are related to the TGS genes and identify the underlying mechanisms that drive their observed effects. This analysis leveraged pathway and interaction curation to identify a high-confidence core interaction network. The network from these analyses included a large number of TGS genes that interacted directly at the protein level, or were functionally related, supporting a coherent biological relationship within TGS genes.
To establish which of these pathways correlated with the basal-like phenotype in breast cancer patients, and select biological pathways for therapeutic intervention, we analyzed the expression of TGS-associated pathways in human primary breast tumors. Indeed, Proteasome, Proteasome degradation, Glycolysis, mRNA Processing, and TNF-alpha pathways were selectively enriched in poor prognosis basal-like tumors compared to good prognosis luminal A tumors, but not IL-7 signaling, ID signaling, GPCR Class A Rhodopsin-like and Transcription Initiation. Notably, pathways enriched in basal-like primary tumors were also enriched in Luminal B tumors.
Indeed, the integration of genome-wide functional and expression data allows us to identify and select biological pathways the inhibition of which can be therapeutically effective in the context of basal-like breast tumors, because they are required for survival of highly malignant basal-like breast cancer cells, and are also enriched in human primary BL-BTs.
To further support these results, we further validated some of these pathways functionally. Proteasome Degradation and Glycolysis pathways were particularly attractive because chemical inhibitors are available. Moreover, Proteasome Degradation was the pathway most highly represented in the TGS genes, and also the most connected, as the hits were all subunits of the 20S or 26S proteasome complexes. These included PSMA1, PSMA2, PSMA3, PSMB4, PSMC1, PSMC3, PSMD2, PSMD7, and PSMD14. Moreover, other TGS genes, such as UBL5, NEDD1, NEDD8, ANAPC2, and ANAPC4, were also involved in the ubiquitin-proteasome system. On the other hand, only two hits were associated with Glycolysis, PFKL and GAPDH pathways but their enrichment in the TGS was highly significant. HK1, which initiates glucose catabolism through the same pathway, also scored positive in the primary screen, but could not be validated in the secondary screen. Most of the screening hits in these pathways were classified as highly or intermediate BPLER selective inhibitors, and could each be validated with 2-4 individual siRNAs in the secondary screen.
We used chemical inhibitor drugs to further confirm the selective response of BPLER and HMLER cells to inhibition of proteasomal activity or glycolysis in an RNAi-independent manner. Indeed, BPLER cells were more sensitive to increasing doses of bortezomib (a proteasome inhibitor drug) and 3-bromo-pyruvic acid (BRPA, a chemical inhibitor of glycolysis) relative to HMLER cells. Treatment with bortezomib decreased proteasomal activity similarly in both cell lines, demonstrating that BPLER cells were intrinsically more sensitive to transient proteasomal inhibition. Exposure to BRPA for 4 hours, when most cells were viable, decreased ATP levels in BPLER and HMLER cells by 90% and 60% respectfully, indicating, without wishing to be bound or limited by theory, a higher dependence of BPLER cells on glycolysis for ATP generation. However, this might also indicate, without wishing to be bound or limited by theory, a differential ATP consumption rate between the two cell lines. Notably, the exquisite sensitivity of BPLER cells to bortezomib and BRPA was drug-specific, because BPLER and HMLER cells responded similarly to treatment with the anthracycline doxorubicin.
To further validate the correlation between tumor-initiating potential and response to either proteasome or glycolysis inhibition, we assessed the dependency of untransformed BPE cells on these pathways. Although BPE were as proliferative as HMLER and BPLER cells, they were resistant to both proteasome and glycolysis inhibitors, further confirming the specificity of these drugs for highly malignant breast cancer cells.
While glycolysis inhibitors are in pre-clinical development, proteasome inhibitors are already in the clinic for the treatment of multiple myeloma and mantle cell lymphoma, and can be rapidly investigated for breast cancer therapy in a clinical setting. Nonetheless, some phase I/II clinical studies have shown poor response of unselected breast cancer patients to proteasome inhibitors. Accordingly, we furthered explored and determined the molecular basis of BPLER dependence on the proteasome degradation pathway for survival.
Proteasome Inhibition Selectively Promotes Noxa-Dependent Apoptosis in Basal-Like Breast Cancer Cells
The data described herein show that highly malignant BPLER cells selectively depend on proteasomal activity for survival compared to poorly malignant HMLER cells and untransformed breast primary epithelial BPE cells. To further define factors underlying such properties of BPLER cells, we first determined the molecular mechanism by which proteasome inhibition affects the growth of these cells. Treatment with clinically relevant doses of bortezomib for 24 hours induced marked cleavage of caspase 3 and PARP in BPLER cells, but only partially in HMLER cells and not in BPE cells, indicating selective activation of apoptosis in BPLER cells. Likewise, upon treatment with low-dose bortezomib, 34%, 17% and 2% of BPLER, HMLER and BPE cells stained positive for Annexin V/PI, whereas co-treatment with the pan-caspase inhibitor ZVAD dramatically reduced the number of double-positive cells, further demonstrating the selective induction of caspase-dependent cell death in BPLER cells upon proteasome inhibition.
Other basal-like breast cancer cell lines, such as HCC-1143 and HCC-1937, were also exquisitely sensitive to proteasome inhibition compared to mesenchymal MDA-MB-231 and MDA-MB-436 or luminal MCF7 and BT-474 breast cancer cell lines. In fact, treatment with bortezomib decreased cell viability by 50-65% in basal-like cells, but only by 20-25% in mesenchymal cells and 5% in luminal cells. Moreover, bortezomib induced PARP cleavage in both HCC1143 and HCC1937 cells, but not in MCF7 or MDA-MB-436 cells. Therefore, as shown herein, the selective response to proteasome inhibitors is an intrinsic feature of human basal-like breast cancer cells, and not limited to BPLER cells.
BH3-only proteins have been shown to mediate bortezomib-induced apoptosis in multiple myeloma cells, which are also exquisitely sensitive to inhibition of the proteasome. We assessed the levels of these proteins in BPLER cells upon treatment with bortezomib. Notably, the BH3-only protein Noxa was highly increased in treated cells, whereas other BH3-only proteins were only modestly increased (e.g., Bik, Bim), unchanged (e.g., Bid, Bad), or decreased (e.g., Puma).
Indeed, we determined that Noxa was necessary to mediate apoptosis in BPLER cells, because its silencing by RNAi partially rescued the lethal effect of proteasome inhibition in BPLER cells, similar to the pan-caspase inhibitor ZVAD. On the other hand, silencing Bim or Bik did not rescue BPLER cells from cell death, whereas silencing both Bik and Noxa simultaneously did not further increase cell viability upon proteasome inhibition. Finally, Noxa was also induced in the bortezomib-responsive cell lines HCC1143 and HCC1937 cells, and not in bortezomib-resistant luminal MCF7 cells, further confirming the central role of Noxa in mediating bortezomib-induced cell death. However, Noxa did not appear sufficient to activate apoptosis, because it was similarly induced in HMLER cells and MDA-MB-231 cells upon treatment with bortezomib. Although Noxa was barely detectable at the protein level in all untreated cells, it was actively transcribed in triple-negative breast cancer cells compared to luminal breast cancer cells and untransformed breast epithelial cells, indicating, without wishing to be bound or limited by theory, that triple-negative cells have high ability to induce Noxa because of high baseline mRNA levels. Noxa mRNA was only modestly increased in BPLER and HMLER cells upon proteasome inhibition, despite a strong induction of Noxa protein in both cell lines. Together, these findings demonstrate that basal-like breast cancer cells undergo Noxa-dependent apoptosis upon proteasome inhibition.
Proteasome Inhibitors Interfere with Mitosis in Highly Malignant Basal-Like Breast Cancer Cells.
The proteasome is a key regulator of cell cycle. We next determined whether the selective response of BPLER cells to proteasome inhibitors is linked to an abnormal regulation of cell cycle dynamics. To define the physiological response of normal breast epithelial cells to proteasome inhibition, we analyzed the cell cycle distribution of BPE cells treated with bortezomib for 24 hours, which showed a marked accumulation of cells in G2/M phase. A similar response was observed in HMLER cells, and BPLER cells treated with suboptimal doses of bortezomib. Accordingly, proteasome inhibition induced a marked increase of p21 and p27 proteins, two main cell cycle inhibitors, in all the three cell lines.
To exclude secondary effects of apoptosis, and track progression through different cell cycle phases, we analyzed cell cycle distribution upon proteasome inhibition in BPE, HMLER and BPLER cells in the presence of the pan-caspase inhibitor ZVAD, which inhibits apoptosis induced by proteasome inhibition, and nocodazole, which arrests cells as they reach mitosis.
All the three cell lines underwent mitotic arrest when treated with nocodazole for 24 hours. Addition of bortezomib delayed progression to G2/M phase in BPE cells and, to a lesser extent, HMLER cells, but not in BPLER cells. Notably, BPLER cells failed to arrest in G1 or G2 phases and entered mitosis, upon proteasome inhibition, whereas BPE cells and, in part, HMLER cells remained in interphase, as determined by morphology, Histone H3 phosphorylation and DNA staining. Likewise, HCC1143 basal-like breast cancer cells entered mitosis upon proteasome inhibition.
Basal-like breast cancer cells are known to be highly sensitive to antimitotic agents. Because proteasomal activity drives exit from mitosis, we next determined whether proteasome inhibition interfered with mitosis in BPLER cells. When apoptosis was blocked by ZVAD, proteasome inhibition delayed the exit from mitosis of BPLER cells that were first synchronized with nocodazole. Notably, a mitotic arrest achieved by nocodazole treatment for 24 hours was sufficient to activate apoptosis in BPLER cells, although not as much as proteasome inhibition, indicating that disruption of normal mitosis contributed to bortezomib-induced apoptosis. Indeed, bortezomib appeared to act as an antimitotic agent, but was more potent compared to nocodazole, presumably because of concomitant induction of proapoptotic factors such as Noxa. Importantly, BPLER cells became resistant to bortezomib treatment once they reached confluence, further supporting a higher sensitivity of dividing cells to proteasome inhibitors.
Accordingly, these data show basic mechanisms of action of bortezomib in basal-like breast cancer cells, which involve, in part, inactivation of the G2/M checkpoint and disruption of normal mitosis.
Proteasome Inhibition Inhibits the Outgrowth of Basal-Like Breast Tumors
Although breast cancer patients have previously been shown to respond poorly to proteasome inhibitors, we next determined whether bortezomib exerts an antitumoral effect on basal-like breast cancer cells in vivo. For all the experiments described herein, bortezomib was given every three days (q3d), reflecting clinical protocols used in human patients, at doses of 0.5 mg/kg or 1.0 mg/kg intraperitoneally (i.p.), which were previously reported to be well-tolerated in mice. We did not observe any significant weight loss after 10-16 days of therapy, at which time points mice were sacrificed.
We first assessed the effect of bortezomib on pre-established subcutaneous BPLER xenografts in nude mice. The growth of BPLER tumors was influenced by host factors, and tumors developed between 2 and 5 weeks, even when high number of cells (5×105) cell were injected. To reduce experimental variability, we selected mice bearing tumors of homogenous size ( ̃0.5 mm in diameter after 3 weeks from injection), which were randomized into two groups. Each group received 6 doses of either vehicle (DMSO) or bortezomib at 0.5 mg/kg i.p. q3d over a 16-day period, respectively. At the end of the treatment period, mice were sacrificed and tumor weight was assessed. Treatment with bortezomib significantly reduced median tumor weight by 54% after 16 days (p=0.001). Histological examination of residual tumors revealed extensive cleaved caspase-3 positive apoptotic/necrotic areas in both treated and untreated tumors, which is a typical feature of human triple-negative breast tumors. We could not quantify with precision the relative effect of the treatment on apoptosis by immunohistochemistry alone. Although the histological picture was compatible with the induction of cell death, without wishing to be bound or limited by a theory, other mechanisms of tumor growth inhibition can also play a role.
To minimize host-related factors in inducing response to bortezomib, we decided to further validate these data in a syngeneic mouse model of basal-like breast cancer, where the influence of the immune system or other host factors is diminished.
In the course of our studies described herein, we identified a subpopulation of basal-like breast cancer cells within the 4T1 cell line, which derives from a spontaneous BALB/c mouse breast tumor. These cells, termed 4T1-E, display a CD44med/CDH1low/ESAhigh epithelial phenotype, whereas the remaining cells, termed 4T1-M, display a CD44high/CDH1−/ESAlow mesenchymal phenotype. The two populations are stable, have a similar proliferation rate, and do not appear hierarchically linked, because they did not convert into each other after extensive culture in vitro.
Indeed, 4T1-E cells expressed higher levels of epithelial markers, including CDH1, ZO-1, CK-14 and EGFR, and lower levels of myoepithelial/mesenchymal markers, including Vimentin, MMP3 and Snail, compared to both 4T1-M cells as well as 67NR cells, which originate from an established mesenchymal clone previously isolated from the same tumor. Moreover, 4T1-E over-express the murine stem-like markers CD49f, CD24 and CD29 compared to 67NR cells. Finally, 4T1-E cells are more tumorigenic than 4T1-M cells and 67NR cells when transplanted in the mammary fat-pad of syngeneic mice, can form tumors with as few as 500 cells in syngeneic BALB/c mice after 3 weeks with complete penetrance, and give rise to highly malignant CK-14 positive epithelial breast tumors. Indeed, 4T1-E had a stem/progenitor-like phenotype, and recapitulated several features of BPLER cells and human basal-like breast tumors. 4T1-E also responded to bortezomib in vitro. Therefore, we assessed the therapeutic effect of bortezomib on the outgrowth of 4T1-E tumors in syngeneic BALB/c mice. Because all tumors grew very rapidly in this model, treatments were started 2 days after tumor cell inoculation. Similarly to BPLER xenografts, treatment with bortezomib (4 doses at 1.0 mg/kg i.p. q3d) reduced the median tumor weight by 56% after 10 days of treatment (p<0.01).
In conclusion, bortezomib exerts a significant therapeutic effect on basal-like tumors in vivo, although it does not induce complete remission of the tumors. Thus, in some embodiments of the methods described herein, a patient determined to have increased or enriched expression of at least one, or more preferably, at least 10 TGS biomarker genes in a tumor sample using the methods described herein, or a patient diagnosed having a basal-like breast tumor, can be further administered a proteosome inhibitor. In some such embodiments, the proteosome inhibitor is bortezomib.
Recent studies on animal models have indicated that basal-like breast tumors derive from transformed epithelial progenitors expressing both luminal and myoepithelial markers. Herein we demonstrated that human primary epithelial progenitor-like cells (BPE) give rise to tumors closely resembling human BL-BTs upon transformation with canonical oncogenes. Our data described herein indicate that epithelial progenitors retain the potential to initiate such tumors upon transformation. This is not an artifact of in vitro transformation, because introduction of the same genetic elements into myoepithelial-like cells (HME) did not induce the development of basal-like tumors. Moreover, the oncogenes used to transform BPE cells recapitulate common genetic alterations occurring in human BL-BTs, including loss of function of p53 and Rb (which are inactivated by SV40) and activation of RAS signaling. As shown herein, transformation of human primary cells into basal-like tumor-initiating cells appears dependent on the cell of origin. However, it is possible that other combinations of oncogenes can also induce basal-like phenotypes from different cells of origin.
BPLER cells represent a novel and the first genetically-defined model of a tumor-initiating cell with a basal-like phenotype. According to the cancer stem cell (CSC) hypothesis, only a minor subpopulation of cancer cells within a tumor retain tumor-initiating potential, whereas the bulk of tumor cells undergoes differentiation, thus shedding malignant properties. The CSC model can apply to certain low-grade tumors (including, e.g., luminal A breast tumors), in which cancer cells are capable of differentiation, however, human basal-like breast tumors are typically poorly differentiated lesions with a diffuse progenitor-like phenotype. In other tumors, where differentiation is impaired by genetic alterations, such as loss of p53, such as, for example, human basal-like breast tumors, the majority of cancer cells retain malignant potential. Our data demonstrate that human progenitor-like epithelial cells expressing BT-IC markers give rise to basal-like epithelial tumors, showing that at least certain stem-like features, such as self-renewal, can be acquired by epithelial progenitors as a result of transformation.
Previous studies have shown that passage through an epithelial-mesenchymal transition (EMT) generates mesenchymal cells with cancer stem-like properties. The pure mesenchymal phenotype does not appear to be a corollary of breast cancer “stemness,” because BPLER cells retain both epithelial and mesenchymal features (i.e., a metastable phenotype) and are highly malignant, similar to 4T1-E mouse cells. Mesenchymal breast cancer cells have been shown to initiate tumors resembling claudin-low tumors or other rare mesenchymal breast cancer variants, but not basal-like epithelial tumors.
It should be noted that, since most of the established EMT markers are either luminal or myoepithelial transcripts, the metastable (mixed) phenotype is indicative of a progenitor-like state of differentiation. Therefore, metastable cells can represent early epithelial progenitors.
Unbiased forward genetic screens have been powerful tools with which to identify molecules involved in fundamental pathways in model organisms, but have been difficult to do in mammalian cells. The discovery of RNAi, coupled with the sequencing of the genome, has provided the opportunity to identify candidate genes required for a biological process in an unbiased way on a genome-wide scale by silencing one gene at a time. As described herein, we performed a genome-wide siRNA lethality screen to define functional vulnerabilities associated with the tumor-initiating state in the BPLER/HMLER system. We identified 154 therapeutic targets that can be harnessed for diagnosis, prognosis, and treatment of cancers, such as basal-like breast tumors. These targets are clinically relevant because they are enriched in human primary basal-like breast tumors and their expression correlates with poor prognosis and metastatic relapse. The prognostic value of these biomarker genes does not depend on their direct association with basal-like tumors, because they also discriminate between good and poor prognosis patients within the luminal B category.
In contrast to other breast cancer signatures, we have identified genes and pathways that are not only predictive of tumor subtype, prognosis, and risk of metastasis, but are also required for survival of basal-like BT-ICs in vitro. Importantly, the malignancy associated response signature genes described herein can be used to identify breast cancer patients who are at higher risk of death or metastasis, and who are more likely to respond to biological therapies targeting MARS-related genes or networks of genes.
Amongst the biological pathways selectively inhibiting BPLER cells, we focused, in part, on the proteasome-ubiquitin system, not just because it was one of the most significantly enriched for pathways, but also because proteasome inhibitor drugs are already in the clinic for multiple myeloma and relapsed mantle cell lymphoma. Although bortezomib is the only proteasome inhibitor approved for clinical use, 5 second-generation proteasome inhibitors are currently in phase I trials. Moreover, 10 drugs targeting specific E1-activating enzymes and E3-ubiquitin ligases are also in clinical development. It is believed that proteasome/ubiquitination inhibitors represent a novel class of drugs, whose number of potential targets may exceed protein kinases. Of note, BPLER cells also selectively responded to glycolysis inhibitors, which are currently in preclinical development. Accordingly, in some embodiments of the methods described herein, a subject having increased or enriched expression of at least one or more, preferably, at least 10, MARS (TGS) biomarker genes of SEQ ID NOs: 1-154 can be treated using any proteosome inhibitor, such as bortezomib and second-generation proteasome inhibitors in development, as well as drugs targeting specific E1-activating enzymes, E3-ubiquitin ligases, and inhibitors of glycolysis.
Treatment with bortezomib induced apoptosis in basal-like breast cancer cells, but not in luminal or mesenchymal breast cancer cells or in normal breast epithelial cells. Based on our data described herein, at least two mechanisms underlie such selectivity. First, basal-like cells are poised to undergo bortezomib-induced apoptosis, compared to luminal cells, because of active transcription of the proapoptotic factor Noxa mRNA, whose protein levels are kept in check by the proteasome. Second, consistent with their malignant nature, basal-like breast cancer cells are unable to activate cell cycle checkpoints upon proteasome inhibition, which represents the physiological response of normal breast epithelial cells. Indeed, proteasome inhibition interferes with mitosis exit, which represents a pro-apoptotic signal per se, because BPLER cells also responded to antimitotic agents.
Alteration of the G2/M checkpoint is a hallmark of human BL-BTs, for which treatment with antimitotic agents represents the standard of care. Bortezomib acted as an antimitotic agent by interfering with mitosis exit, but also induced the expression of potent pro-apoptotic molecules like Noxa. Significantly, proteasome inhibition was more effective than induction of mitotic arrest alone in eliminating basal-like breast cancer cells.
Little is known about the mechanisms linking mitotic arrest to apoptosis. It has been proposed that prolonged mitotic stall induces inactivating phosphorylation of antiapoptotic BCL2-family proteins, including BCL2, BCL-XL and MCL1. Interestingly, we observed phosphorylation of MCL1 (a main antagonist of Noxa) in mitotic BPLER cells treated with bortezomib, which can contribute to induction of apoptosis upon proteasome inhibition.
Recent studies have shown that targeting mitosis exit by inhibiting CDC20, which links proteasomal activity with mitosis progression, leads to tumor regression in vivo. Our data demonstrate that proteasome-based therapies are effective against basal-like tumors in vivo as well. These date are in agreement with eight phase I/II studies assessing chemotherapy combination regimens with bortezomib, which showed partial response rates of 10-20% in unselected breast cancer patients.
Provided herein are novel insights into mechanisms of resistance in human cancer cells. We showed herein that non-dividing BPLER cells became resistant to bortezomib. However, six out of eight clinical studies assessing the effect of bortezomib on breast tumors were based on combination regimens with other drugs, such as doxorubicin, that are known to induce G1 arrest. Interestingly, administration of bortezomib in combination with docetaxel, which induces mitotic arrest, in one study compared favorably with patients treated with docetaxel alone.
On the other hand, our data indicate that basal-like tumors, but not luminal tumors, are more likely to respond to proteasome inhibitors. Because luminal tumors comprise over 70% of human breast cancers, a targeted approach aimed at identifying and selecting patients, based on expression of at least one, but more preferably at least 10 MARS biomarker genes, with basal-like tumors can increase response rates to proteasome inhibitors in the clinic. In conclusion, we identified core survival pathways on which basal-like BT-ICs depend for survival, and identified novel diagnostic, prognostic, and therapeutic biomarker targets with documented clinical relevance. Proteasome inhibition provides one therapeutic strategy to selectively tackle basal-like breast tumors. Other therapeutic approaches, such as glycolysis inhibition, can also be effective against poor prognosis breast tumors.
Example 2
Triple negative breast cancers (TNBC) are poor prognosis cancers, frequently associated with TP53 mutation and active RAS signaling. As described herein, we show that human progenitor-like primary mammary epithelial cells (BPE) selectively acquire TNBC-initiating properties after transformation with defined genetic elements. A genome-wide siRNA screen identified 154 genes upon which BPE transformed derivatives (BPLER) selectively depend for survival, compared to genetically identical myoepithelial-like cells from the same donor (HMLER). BPLER selective dependencies comprise a malignancy associated response signature (MARS) or TNBC gene signature (TGS) whose expression correlates with poor prognosis and metastasis in breast cancer patients. The MARS is enriched in proteasome-ubiquitin system, metabolism, RNA splicing, mitosis and molecular transport related genes. BPLER and human epithelial TNBC cell lines are poised to accumulation of the Mcl-1 antagonist Noxa and selectively depend on Mcl-1 for survival. In fact, proteasome inhibitor drugs target Mcl-1 dependency in these cells. Using “chemogenetics” to compare the MARS with gene expression signatures induced by >1,300 chemicals, we also identified histone deacetylase inhibitor drugs as selectively cytotoxic for BPLER. Thus, as described herein an unbiased siRNA screen identified novel classes of candidate drugs to treat TNBC.
Triple-negative breast cancers (TNBC), defined by their lack of estrogen (ER) and progesterone (PR) receptor and Her2 expression, are an especially aggressive group of tumors often found in young Afro-American and Hispanic women, with the shortest survival of breast cancer subtypes. TNBC, which comprise  ̃15% of human breast tumors, are associated with BRCA1 mutations and/or Tp53 and Rb loss of function in over 90% of cases1. Compared to ER+ luminal tumors, TNBCs are poorly differentiated and most cells have features of epithelial progenitor cells.2,3 Targeted genetic ablation of Brca1, Tp53 and/or Rb in mammary epithelial progenitor cells in mice leads to TNBC-like malignancies, suggesting that TNBC arise from transformed epithelial progenitor cells4-6. TNBC generally express markers associated with breast tumor-initiating cells7-9, CD44+/CD24low/−/ESA+, EGFR and varied combinations of cytokeratins CK5, CK14, CK17, CK8, CK182. These tumors rapidly become resistant to chemotherapy and are refractory to hormonal therapy and HER-2 inhibitors1.
As described herein, we performed an unbiased genome-wide siRNA lethality screen to identify factors on which epithelial TNBC-initiating cells selectively depend for survival. For the screen we took advantage of a method to generate pairs of essentially genetically identical breast cancer cell lines, BPLER and HMLER, by transforming normal breast primary epithelial cells with defined genetic elements10 (FIG. 1A). We found that BPLER cells, which derive from progenitor-like epithelial cells, have the phenotype of TNBC and retain TNBC-initiating potential, uniformly generating tumors that resemble primary TNBCs after injection of just 50 cells in immunodeficient mice, while HMLER cells are myoepithelial-like and do not form tumors after injection of 5×104 cells. Thus BPLER cells are a good model for studying malignant traits of TNBC. Knockdown of 154 genes selectively reduced the survival of BPLER cells compared to genetically related HMLER cells prepared from the same donor. These hits were highly enriched for components of the proteosome and for genes that participate in the ubiquitin-proteosome system (UPS) as well as for genes important in RNA splicing, glycolysis, DNA replication and mitosis. The 154 hits constitute a malignancy associated response signature (MARS) or TNBC gene signature (TGS) of genes whose expression is associated with reduced survival and early metastasis in human primary breast tumors. The MARS predicted that TNBC would be selectively vulnerable to proteosome and histone deacetylation inhibition. Indeed BPLER and TNBC cell lines were highly susceptible to these classes of drugs, providing novel approaches to treat this deadly disease that is largely refractory to current therapeutic regimens.
Results
BPLER Cells are Tumor-Initiating Epithelial Cells that Give Rise to TNBC
At least two types of breast epithelial cells, termed BPEC and HMEC cells, can be reproducibly derived from normal human breast organoids expanded in vitro using different chemically-defined media, WIT and MEGM, respectively10 (FIG. 1A). BPEC have a progenitor-like phenotype, whereas HMEC have a myoepithelial-like phenotype. After transformation with hTERT, SV40 early region and H-RASV12 in these respective media, they give rise to BPLER and HMLER cancer cell lines. Because these cells are essentially genetically identical but differentially tumorigenic, they represent an attractive model to study epigenetic changes associated with tumor initiation. To exclude continuing contributions of the differences in the growth media to their tumor properties, we propagated both cell lines in WIT medium for two weeks before analyzing their phenotype, proliferation and tumor-forming capacity. (The phenotypic properties of the HMLER cell line once established did not change upon culture in WIT medium; therefore all subsequent experiments compared BPLER and HMLER cells both grown in WIT medium.) We first set out to determine whether either of these cell lines, which are both triple negative and EGFR+ (FIG. 1B), might be a good model for TNBC. Both BPLER and HMLER also stain with markers CD44+CD24low/−ESA+ used to define breast tumor-initiating cells (BT-IC) (FIGS. 8A-8C). Although BPLER and HMLER cells proliferated at a similar rate in vitro (FIG. 1C), BPLER cells uniformly formed tumors in mice with as few as 50 cells, whereas 5×104 or fewer HMLER cells did not (FIG. 1D). Thus only BPLER are highly enriched for breast tumor-initiating cells (BT-IC). When more cells are injected, HMLER form squamous tumors that do not resemble any of the common breast cancer subtypes10.
BPLER expressed a wide array of luminal and myoepithelial cytokeratin mRNAs, as is typical of TNBCs and untransformed bipotent epithelial progenitor cells, and intermediate levels of both E-cadherin and vimentin mRNAs, whereas HMLER cells had reduced expression of luminal cytokeratin and E-cadherin mRNA and expressed higher levels of vimentin mRNA (FIG. 1E, FIG. 8C). When examined by immunofluorescence microscopy, BPLER, but not HMLER, cells co-expressed CK14 and CK18 proteins, a property of mammary luminal epithelial progenitors2. Therefore, although both BPLER and HMLER cells were triple-negative and expressed cell surface markers that have been used to identify BT-IC, only BPLER cells had tumor-initiating potential and expressed markers of TNBCs and epithelial progenitors.
Next we asked whether tumors formed in vivo by subcutaneous injection of BPLER cells in immunodeficient mice resemble human primary TNBCs. BPLER tumor explants stained positive for CK5 and CK14 and negative for ER, had a high mitotic index and displayed a clear inflammatory infiltrate, reactive tumor stroma and pushing borders, all of which are typical of human TNBC1,11. Moreover, BPLER tumors were poorly differentiated epithelial lesions, with focal areas of glandular differentiation, consistent with their epithelial phenotype10. To examine more closely whether BPLER tumors resemble TNBCs, we compared by unsupervised hierarchical clustering the global transcriptional profile of six BPLER tumor explants that grew in either nude or NOD/SCID mice with mRNA expression profiles of 337 human primary breast tumors of known subtype (UNC337 dataset12). The BPLER tumor explants clustered with human primary TNBC and not with luminal A/B, normal-like or HER2+ subtypes. These findings were verified by analyzing an independent set of 7 normal breast samples and 40 human primary breast tumors (Richardson-06 dataset13), classified as basal-like or non-basal-like. BPLER again clustered with basal-like tumors, the most common gene expression subtype of TNBC. Thus, BPLER initiate tumors closely resembling human TNBCs and are a good model for studying TNBC.
A Genome-Wide siRNA Screen Identifies BPLER Survival Dependency Factors
Although BPLER and HMLER are essentially genetically identical, they importantly differ in their differentiation state and ability to form tumors. To identify functional vulnerabilities associated with a TNBC-initiating phenotype, we performed a high throughput genome-wide siRNA lethality screen by transfecting BPLER and HMLER cell lines derived from the same donor in triplicate wells, each with a pool of 4 siRNAs targeting distinct sites within a single gene using the Dharmacon siGenome library of 17,378 gene targets (FIG. 2A). Three days later the mean number of surviving cells was assessed by CellTiterGlo assay and the ratio (R) of viable BPLER to viable HMLER cells was calculated. Data describing the optimization of the screen are described in FIGS. 9A-9G. The majority of the pools scored within one median absolute deviation (MAD) of the plate median. 1025 siRNA pools significantly decreased BPLER viability, of which 780 decreased HMLER viability to a similar extent (Table 1). Of the remaining 245 pools, 26 were highly selectively lethal for BPLER(R≦0.55), 76 were moderately selective (0.55<R≦0.65) and 143 were modestly selective (0.65<R≦0.75) (FIG. 2B, Tables 2A-2C). To validate the candidate hits (i.e. genes selectively required for BPLER survival), we next screened each of the hits by transfecting BPLER and HMLER cells separately with each of the four individual siRNAs comprising each pool. In the validation screen, 154 of the pools (63%) reconfirmed with at least one siRNA (Tables 2A-2C, FIG. 2B). Not surprisingly, the validation rates were higher for hits with lower R values (88% of highly selective, 75% of moderately selective and 52% of modestly selective hits were validated) (FIG. 2C).
The BPLER Dependency Genes are Highly Expressed in TNBC and their Expression is Associated with Poor Prognosis in Breast Cancer
To evaluate the clinical relevance of the 154 validated BPLER dependency genes, we examined the expression of this gene signature across breast cancer subtypes using the NKI mRNA expression profiles of 295 primary breast cancers, classified as basal-like, HER2+, luminal A, luminal B, or normal-like14. The dependency genes were significantly over-expressed in both basal-like and luminal B tumors relative to other subtypes (p-value <3×10−5 and 4×10−6, respectively, by Kolmogorov-Smirnov (KS) test). When the analysis was limited to the 23 highly selective gene hits, the same two subtypes significantly overexpressed these genes (p<3×10−6 and 4×10−5) (FIG. 2D). To determine whether overexpression of these genes might correlate with clinical prognosis, we divided the NKI tumors into two groups based on their expression of either the 154 or the subset of 23 highly selective BPLER dependency genes. In both cases, patients with high expression of the dependency genes had significantly reduced survival. The survival difference was significantly greater if the analysis were restricted to the highly selective hits (FIG. 2E). Because high expression of the 154 BPLER dependency genes is linked to TNBC and to poor prognosis, we consider the 154 genes a malignancy associated response signature (MARS) or TNBC gene signature (TGS). Luminal B tumors, which comprise  ̃15% of human breast tumors, have variable prognosis. Within this category, expression of the TGS or highly selective hits clearly separated luminal B tumor patients with poor survival (p<0.003 and p<0.01, respectively), indicating that the MARS is enriched in highly malignant breast tumors, independently of tumor subtype (FIG. 2F). Indeed, it can be used to predict prognosis in luminal B tumor patients. Within the NKI dataset, patients with higher MARS or highly selective hit expression also were diagnosed with metastasis significantly earlier than patients with low MARS expression (FIG. 2G). A highly significant association of low MARS expression with metastasis-free survival was also found by analyzing an independent dataset of 286 primary breast tumors (EMC286 dataset15,16, FIG. 10). In conclusion, BPLER dependency genes are more highly expressed in highly malignant human primary breast cancers, and might serve as therapeutic targets against TNBC.
BPLER Dependency Genes are Enriched for Proteasome Subunits and Genes Involved in Mitosis, Metabolism, DNA Transcription and RNA Biogenesis
An examination of the 23 validated highly selective BPLER dependency genes (FIG. 2C) indicated to us that BPLER cells might be selectively dependent on proteasome function since 7 of these genes are proteasome components (hypergeometric p-value 1.1×10−14) and an additional hit (UBL5) encodes for a ubiquitin-like molecule. Another group of 4 genes is involved in mRNA biogenesis and nuclear export. These included the spliceosome component PRPF8 and 2 RNA helicases (DDX19B, DHX8) and the nuclear RAS protein RAN, all involved in mRNA nuclear export. In addition the hit list contained 4 Zinc finger genes (ZNF490, ZNF574, ZNF643, FIZ1), all likely transcriptional activators, and a nuclear receptor coactivator gene SNW1 associated with activation of retinoic acid and estrogen-regulated genes. Also of note were 2 genes RACGAP1 and HAUS3 that regulate the mitotic spindle and PP2CA, the gene encoding the catalytic subunit of the tumor suppressor PP2A that modulates the action of many oncogenic protein kinases.
To begin to make sense of the larger 154 member MARS, we first looked at whether the hits could be grouped into well-defined functional categories, using a combination of the pathways and processes in the Reactome17, KEGG18 and Wikipathway19 databases. Of the 154 MARS genes, 121 genes have well described annotations that could be grouped into 13 functions with at least 3 genes assigned to each function (Table 4A). The proteasome was highly over-represented in the MARShit list with 10 proteasome subunits)(p<3×10−10 and 6 other genes whose products participate in the UPS system. Of these, 5 genes are involved in ubiquitylation, including 2 components of the anaphase promoting complex (APC; ANAPC2, ANAPC4), and one gene is involved in neddylation of the cullins (NEDD8) to promote mitosis.
We next used GeneMANIA20 to build an interaction network relating the MARS set incorporating physical and predicted interactions, co-localization, shared pathways and shared protein domains. 87 genes in the MARS set formed a single interacting network. Genes that participate in the major functional categories, but are not annotated to have direct protein interactions, were added to this network to produce a core functional-interaction module (FIG. 3). Notably, the proteasome and its associated proteins constituted a core module linking multiple TGS genes. Other broad processes that stood out were metabolism, which included genes encoding 2 glycolytic enzymes (GAPDH, PFKL), the G1/S transition and mitosis steps of the cell cycle and multiple steps in mRNA expression, since the MARS captured multiple DNA binding proteins expected to regulate transcription, several RNA polymerases and genes involved in mRNA splicing.
Highly Malignant TNBC Epithelial Cells are Selectively Addicted to Proteasome Activity
One of the goals of the screen was to point to potential therapeutic approaches for TNBC. Since the proteasome is a key module in the MARS and proteasome inhibitors are available in the clinic, we first evaluated the effect of proteasome inhibitors on BPLER and other breast cancer lines.
The proteasome inhibitor bortezomib similarly inhibited proteasome activity (FIG. 4A) and induced accumulation of p21 and p27 proteins, two established biological markers of proteasome inhibition, in BPLER and HMLER (FIG. 11A). However, as anticipated, BPLER were more sensitive to bortezomib compared to HMLER (FIG. 4B). Similar results were obtained using MG132 and second-generation proteasome inhibitor drugs. The sensitivity of BPLER cells to bortezomib was specific, since BPLER and HMLER cells responded similarly to treatment with doxorubicin (FIG. 4C).
After low-dose bortezomib treatment, BPLER did not undergo cell cycle arrest and showed clear evidence of DNA fragmentation, caspase-3 activation, and PARP1/Mcl-1 cleavage, suggesting that they died of apoptosis, whereas HMLER underwent G2/M arrest with minimal signs of apoptosis. Notably, untransformed BPE underwent G2/M arrest and were resistant to bortezomib-induced apoptosis. Thus, bortezomib was not cytotoxic at this dose (FIG. 4D-4E, and FIG. 11B). This was confirmed by flow cytometry after annexin V and propidium iodide (PI) staining. Moreover, BPLER developed an annexin V+PI+ population that mostly disappeared after treatment in the presence of the pan-caspase inhibitor zVAD-fmk, confirming the induction of caspase-dependent cell death by proteasome inhibition (FIG. 11C).
Four other epithelial TNBC cells (MB-468, HCC-1143, HCC-1937 and 4T1-E) were also relatively sensitive to bortezomib, whereas ER+(MCF7), HER2+ (BT-474) and mesenchymal (MB-231, MB-436) breast cancer cell lines were resistant, suggesting that TNBC epithelial cell lines are selectively addicted to proteasome activity (FIG. 4F). In the highly malignant 4T1-E breast cancer cell line, viable cells that persisted after 18-hour bortezomib treatment neither formed colonies in vitro after 2 weeks, nor were able to initiate tumors in vivo. Similar results were obtained in HCC-1143 in vitro (FIGS. 4G-4H).
Together, these data indicate that TNBC epithelial cells with tumor-initiating potential are exquisitely sensitive to proteasome inhibition.
Noxa Mediates Bortezomib-Induced Apoptosis in Epithelial TNBC Cell Lines.
Because the proteasome was so prominent in the MARS and bortezomib was selectively lethal to highly malignant TNBC epithelial cells, we wanted to understand better the mechanism underlying BPLER and TNBC sensitivity to proteasome inhibition. Toward this goal, we first assessed the relative protein level of key modulators of apoptosis in BPLER and HMLER after treatment with bortezomib, including regulators of mithocondrial membrane depolarization and factors acting downstream of mithocondria (FIG. 5A). Because some of these factors, such as Mcl-1, are cleaved upon caspase activation, we performed these experiments in the presence of zVAD-fmk. In both cell lines, bortezomib induced expression of Mcl-1 and the BH3-only proteins Noxa, Bik and Bim, while it inhibited post-mithocondrial factors XIAP and c-IAP1. However, bortezomib induced mithocondrial membrane depolarization in BPLER but not HMLER, indicating that pre-mithocondrial factors regulated the selective response of BPLER to proteasome inhibition (FIG. 5B). To identify factors mediating apoptosis at a functional level, we silenced the expression of 24 genes that were linked with bortezomib-induced cell death in previous studies. Of these, only Noxa silencing rescued BPLER cell death after bortezomib treatment to the same extent of the pan-caspase inhibitor zVAD-fmk (FIG. 5C). Indeed, Noxa protein is up regulated after treatment with bortezomib and mediates bortezomib-induced apoptosis in BPLER. However, Noxa was not sufficient for bortezomib-induced cell death, since HMLER overexpressed Noxa after treatment with bortezomib as well.
The accumulation of Noxa in both cells appeared to be regulated both transcriptionally and post-transcriptionally, since Noxa protein increased at a low bortezomib concentration that showed no change in mRNA (FIGS. 12A-12B). Notably, Noxa mRNA increased  ̃21-fold when BPE were transduced with SV40 early region (BPLE) and did not increase further after transduction with activated RAS (BPLER). Furthermore, TNBC lines expressed  ̃5-25-fold more baseline Noxa mRNA compared to untransformed BPE cells and poorly malignant luminal MCF7 and T47D cells, suggesting that Noxa transcriptional activation in mammary epithelial cells occurs upon transformation (FIG. 5D). Accordingly, bortezomib strongly induced Noxa protein in all the TNBC cell lines tested, but only weakly in BPE, MCF7 and T47D (FIG. 12C). Thus, TNBC cells are poised to Noxa protein accumulation upon proteasome inhibition. Of note, Noxa induction upon bortezomib treatment correlated with PARP1 cleavage in BPE, luminal MCF7 and epithelial TNBC cell lines (HCC-1143, MB-468 and HCC-1937, FIG. 5E).
Together, these data indicate that Noxa is a key mediator of bortezomib-induced cell death in TNBC epithelial cells.
Mcl-1 Dependency Underlies TNBC Sensitivity to Proteasome Inhibition.
Since differential expression of Noxa did not explain the selective sensitivity of BPLER and other epithelial TNBC cell lines to bortezomib, we sought to define the molecular mechanism underlying their selective dependence upon proteasome inhibition.
Noxa is an established, direct and specific antagonist of Mcl-1, which stood out as the only bcl-2 family member among BPLER dependency genes in our screen. Thus, we wished to determine whether BPLER selective response to proteasome inhibition depends on Noxa-mediated inactivation of Mcl-1. To test this, we first assessed Noxa/Mcl1 protein binding upon proteasome and caspase inhibition in BPLER and HMLER. In fact, Noxa bound to Mcl-1, but not Bcl-XL, in the 2 cell lines to a similar extent, indicating that Noxa was not just expressed at similar levels, but also equally capable of binding Mcl-1 (FIG. 5F).
Then, we assessed BPLER and HMLER dependence upon distinct Bcl-2 family members. As expected, Mcl-1 silencing was selectively lethal to BPLER compared to HMLER, whereas BCL-XL silencing killed both BPLER and HMLER to a similar extent and BCL-2 silencing had no effect in either cell line. Hence, BPLER were selectively and specifically dependent on Mcl-1 for survival compared to HMLER, and Mcl-1 silencing was sufficient to recapitulate the lethal effect of proteasome inhibition (FIG. 5G).
In order to validate these findings in other genetic and epigenetic contexts, we silenced Mcl-1 expression in breast cancer cell lines of different subtypes. As anticipated, Mcl-1 silencing was lethal to epithelial TNBC cell lines HCC-1937, HCC-1143 and MB-468, but not to luminal MCF7 and mesenchymal MB-231 cells. Notably, MB-468 that we demonstrated are exquisitely sensitive to bortezomib were also exquisitely sensitive to Mcl-1 silencing (FIG. 5H). Thus, other human epithelial TNBC cell lines, and not just BPLER, are selectively sensitive to Mcl-1 inhibition.
These data indicate that Mcl-1 dependency underlies the selective response of TNBC cells to proteasome inhibition. Because epithelial TNBC cells are intrinsically poised to Noxa accumulation and depend on Mcl-1 for survival, proteasome inhibition represents a viable therapeutic strategy to target Mcl-1 dependency in TNBC.
Proteasome Inhibition Suppresses TNBC Outgrowth In Vivo
Our data thus far indicate that highly malignant epithelial TNBC cells are selectively susceptible to proteasome inhibition. Bortezomib is already in the clinic for treatment of multiple myeloma and other lymphomas. To start investigating potential clinical efficacy in TNBC, BPLER tumor-bearing mice were given 1 dose (0.8 mg/kg) of bortezomib intratumorally (i.t.), intraperitoneally (i.p) or intravenously (i.v.) and proteasome activity in the tumor was assessed after 18 hours (FIG. 6A). Nearly complete proteasome inhibition was achieved via i.t. injection, whereas i.p. and i.v. routes were less effective. However, increasing the dose of bortezomib to 1.6 mg/kg allowed efficient proteasome activity inhibition via systemic administration (i.v.). Thus, we used biologically active therapeutic regimens (0.8 mg/kg i.t. and 1.6 mg/kg i.v.) for subsequent experiments. To assess therapeutic efficacy, we administered bortezomib every 3 days (0.8 mg/kg i.t.) or weekly (1.6 mg/kg i.v.) after BPLER tumors, implanted in the flank of Nu/J mice, became palpable. BPLER tumor outgrowth was assessed by tumor volume during the treatment period and by tumor weight at the time of sacrifice. Compared to control mice, tumors in bortezomib-treated mice were on average from 85% (i.t.) to 59% (i.v.) smaller (p<0.01 and p<0.002, respectively), and weighed on average from 91% (i.t) to 63%% (i.v.) less compared to control mice (p<0.001 and p<0.006, respectively, FIG. 5b-e). Moreover, bortezomib induced Noxa induction and caspase 3 cleavage in vivo, as determined by immunohistochemistry and western blot analysis (FIG. 6F). Thus, bortezomib inhibited the activity of its intended target, induced Noxa accumulation, triggered apoptosis and suppressed tumor growth in BPLER tumor xenografts.
To further validate bortezomib efficacy in vivo, we used MB-468 that had shown dramatic response to proteasome inhibition in vitro. Treatment was started once mice developed palpable tumors. Using either intratumoral or intravenous therapeutic regimens, mice treated with bortezomib for 45 days had tumors that were smaller by at least 6-fold compared to control mice (p<0.0001 in both conditions, FIG. 6G and FIGS. 11A-11C).
Since most of our work was based on cancer cell lines, we sought to assess bortezomib efficacy using primary tumors from TP53 heterozygous Balb/c mice that develop TNBC with a 40-week latency. Breast tumors were resected from tumor-bearing mice and tumor fragments implanted into the mammary fat-pad of TP53+/+ Balb/c recipient mice. After implantation, mice were randomized and treated with bortezomib weekly by i.v. infusion (FIG. 6H). Mice treated with bortezomib developed secondary tumors that were  ̃90% smaller and weighed  ̃80% less on average compared to control mice (p<2.27×10^−6 and p<0.001, respectively, FIGS. 6I-6J). Histological examination confirmed that these tumors displayed an epithelial phenotype, resembled human TNBC histologically, and stained positive for CK14, thus retaining a TNBC phenotype.
Indeed, bortezomib-based therapy significantly suppressed tumor progression in a variety of epithelial TNBC models in both immunodeficient and immunocompetent mice.
A Chemogenetics Approach Reveals BPLER Selective Sensitivity to HDAC Inhibitor Drugs.
To identify additional small molecules that might be active against BPLER, we adopted a “chemogenetic” approach to compare the TGS with the connectivity map (CMAP), a collection of gene expression profiles of human cancer lines treated with bioactive molecules22,23. The CMAP contains genome-wide mRNA expression data from over 7,000 control vs drug-treated pairs of cancer cell lines that originated from diverse tissues exposed to a subset of 1,309 compounds. We reasoned that small molecules that suppress expression of TGS dependency genes might have selective activity against BPLER. 37 compounds significantly preferentially suppressed TGS gene expression in the CMAP (Table 5). We selected 10 drugs from this list for in vitro testing, based partly on their potential for clinical use (FIG. 7A). The histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) most significantly suppressed TGS gene expression, which was shown in 182 independent control/TSA-treated cell experiments (P<0.0001). Treatment with another HDAC inhibitor (Vorinostat) also significantly suppressed expression of the TGS genes. The other drugs examined were Resveratrol, a natural product active in animal cancer models24; 4,5-dianilinophthalimide, an EGFR inhibitor; L-cavananine, an amino acid analog that competes with L-arginine; Nabumetone, a non-steroidal anti-inflammatory drug; Deferoxamine, an iron chelating agent with anti-cancer properties; Loperamide, an opioid-receptor agonist; Triamterene, a potassium-sparing diuretic; and Hesperetin, a bioflavenoid found in citrus fruits. Amongst these, 5 small molecules decreased BPLER viability at relatively low doses, of which 2 (the HDAC inhibitors TSA and Vorinostat) were selectively lethal for BPLER compared to HMLER (FIG. 7B and FIG. 14). Moreover, TSA and vorinostat had no effect on BPE viability at the indicated dose.
The CMAP data predicted that TSA would suppress expression of 52 TGS transcripts by ≧1.8-fold (Table 6). We measured the change in expression of the top 11 of these transcripts in BPLER and HMLER cells treated with TSA. Ten of these, including Mcl-1 and two proteasome subunit genes (PSMC3, PSMD7), were significantly decreased in BPLER cells treated with TSA (FIG. 7C). All but two of these also had significantly reduced expression in HMLER cells treated with TSA (FIG. 7D).
TSA synergized with bortezomib and increased apoptosis in BPLER (FIGS. 7E-7F), whereas the combination was not cytotoxic in untransformed BPE cells in vitro. Thus, HDAC inhibitor drugs selectively targeted highly malignant TNBC-initiating cells in the BPLER/HMLER system by regulating the expression of multiple genes on which such cells selectively depend for survival.
Discussion
Here we performed a genome-wide siRNA lethality screen of a genetically defined human breast cancer cell line (BPLER) to identify vulnerabilities and potential drug targets of highly malignant and poorly differentiated TNBC, the breast cancer subtype with the worst prognosis and response to current treatment. To our knowledge, this is the first RNAi-based high-throughput functional screen of a clinically relevant breast cancer cell type with a high frequency of tumor-initiating cells. Previous studies have focused on highly malignant mesenchymal breast tumor-initiating cells that give rise to mesenchymal breast tumors, a rare subtype of TNBC also known as “claudin-low”. BPLER cells represent the first genetically defined model of tumor-initiating cells that give rise to human triple-negative epithelial adenocarcinomas, the most common type of TNBC encountered in the clinic.
Recent studies suggest that TNBC arises from transformed luminal epithelial progenitor cells4-6,34,35. Our data lend support to this hypothesis, because oncogenic transformation of human primary BPEC epithelial progenitor-like cells generates cancer lines enriched for BT-IC that give rise to tumors closely resembling human TNBC. Thus, BPEC-like epithelial progenitor cells may be the cell of origin of human TNBCs. This is unlikely to be an artifact of in vitro transformation, because introduction of the same genetic elements into myoepithelial-like cells (HMEC) does not induce TNBC10. The oncogenes used to transform BPE cells recapitulate common genetic alterations in TNBC, including loss of function of Tp5336 and Rb37 (which are inactivated by SV40) and activation of RAS signaling38. Indeed, transformation of human primary cells into TNBC tumor-initiating cells appears to depend on the cell of origin. However, it is possible that other combinations of oncogenes may also induce TNBC-like phenotypes from different cells of origin.
The cancer stem cell (CSC) hypothesis postulates that only a minor subpopulation of stem-cell-like cancer cells within a tumor retain tumor-initiating potential. While the CSC model may apply to certain low-grade tumors (including luminal breast tumors), it seems unlikely to describe TNBC, which are typically poorly differentiated lesions with a diffuse progenitor-like phenotype. The entire population of BPLER cells uniformly has the phenotype attributed to breast CSCs and only 50 cells are needed to initiate tumors in immunodeficient mice (we didn't try injecting smaller numbers). For poorly differentiated tumors like TNBC that arise from early progenitor cells, most of the tumor cells are likely to be tumor-initiating cells. Tumors with an early progenitor phenotype may not rely on malignant stem cells for self-renewal, an idea that has been supported by data using other poorly differentiated tumors39. Instead, poorly differentiated transformed bipotent epithelial progenitors can acquire self-renewing properties, and the bulk of cells in some poorly differentiated tumors can be capable of tumor initiation. It should be noted that HMLER cells, which do not efficiently generate tumors, also have the phenotype attributed to BT-IC. Thus there is a real need for additional BT-IC markers.
We identified 154 genes, which we termed a malignancy associated response signature (MARS) or TGS, which when silenced are selectively lethal for BPLER, but not for a closely related cell line (HMLER) derived from the same donor using the same transforming oncogenes. HMLER is myoepithelial-like and is unable to initiate tumors when injected at low numbers10. BPLER dependency genes include Mcl-1 and are highly enriched for proteasome subunits. The proteosome inhibitor bortezomib, currently approved for treatment of multiple myeloma and relapsed mantle cell lymphoma26, causes caspase-dependent apoptosis in TNBC-initiating epithelial cells. These cells are especially sensitive to proteasome inhibition, because they selectively depend on Mcl-1 for survival and are intrinsically poised to Noxa protein accumulation, a potent inhibitor of Mcl-1. In fact, a number of human epithelial TNBC cell lines, and not just BPLER, appear to be selectively sensitive to Mcl-1 inhibition and to proteasome inhibitor drugs that induce Noxa. On the other hand, TNBC cell lines that express Noxa but do not depend on Mcl-1 for survival, such as mesenchymal MDA-MB-231, are also resistant to proteasome inhibition. Indeed, proteasome inhibitor drugs can be used to target Mcl-1 dependency for TNBC therapy. Identifying specific factors that regulate Noxa expression as well as Mcl-1 addiction, using the methods and assays described herein, can also provide novel therapeutic targets against TNBC.
Although clinical studies of bortezomib for unselected breast cancer subtypes have not been promising27-29, our study indicates that further clinical evaluation of proteasome inhibitors for selected TNBC patients, which represent  ̃15% of breast cancer patients, is worthwhile. Recent studies indicate that achieving robust proteasome silencing in solid tumors can represent a major obstacle in the clinic. However, second-generation proteasome inhibitors with better pharmacokinetic profiles or targeting specific components in the ubiquitin-proteasome system, currently under clinical development, might hold promise for TNBC therapy. Our data also indicate that combining proteasome inhibitors with Mcl-1 inhibitors, also in preclinical development, can enhance clinical responses.
In this study we also used a novel approach to identify other potential drugs that are active against TNBC. We compared the CMAP that provides data on changes in global gene expression after exposure to bioactive molecules for many cancer cell lines with the list of MARS genes to identify drugs that selectively reduce expression of BPLER dependency genes. This approach, which illustrates the potential of combining siRNA screen results with CMAP or similar chemogenetics databases, identified HDAC inhibitors as selectively toxic for TNBC-initiating cells in the BPLER/HMLER system. HDAC inhibition suppressed multiple BPLER dependency genes, including Mcl-1 and proteasome subunits. In fact, TSA and bortezomib had a synergistic effect against BPLER. It is worth noting that HDAC inhibitors have been previously shown to target multiple myeloma cells and to synergize with bortezomib. In fact, 2 clinical trials for bortezomib/HDAC combination therapy (Velcade/Zolinza) in multiple myeloma are currently active. Indeed, TNBC-initiating cells appear to share selective functional dependencies with multiple myeloma cells, and indicates the activation of a related oncogenic program in distinct disease entities.
The MARS or TGS biomarkers (or the subset of the 23 biomarkers) can also be useful for identifying other potential TNBC drugs. Because the list is of a manageable size, the pathways for which there are multiple hits or the genes whose products lie at the nodes of the protein-protein interaction network can be evaluated as drug targets for TNBC, either for conventional drug development or for gene knockdown using small RNAs or antisense oligonucleotides. For example, the MARS also comprises glycolytic enzyme genes, and glycolysis inhibiting drugs are actively under development. We found that BPLER are highly reliant on glycolysis for ATP generation and are selectively sensitive to glycolysis inhibition.
A recent chemical screen identified the potassium ionophore salinomycin as a candidate drug active against highly malignant mesenchymal HMLER cells knocked down for E-cadherin30. Three hits in our screen, SLC4A5, SCL24A3 and SLC47A1, are potassium-sodium exchange transporters. It is worth investigating whether highly malignant breast cancer cells like BPLER that more closely resemble human breast tumors are also selectively sensitive to inhibition of specific ion transport.
TNBCs are sensitive to drugs like paclitaxel that inhibit mitosis and form the basis of current care. Our screen identified many genes involved in mitosis as required for BPLER survival. We verified that Mcl1 protein also declines in BPLER cells arrested in mitosis (by nocodazole, data not shown). Hence, combining proteosome inhibition with drugs that arrest cells in mitosis can be a synergistic approach.
The hits in our screen involved in the UPS and mitosis (the proteasome and the APC) overlap with genes identified in a recent genome-wide siRNA screen of synthetic lethal interactions with KRAS32. Because both BPLER and HMLER are transformed to express constitutively activated RAS, their differential dependence on these hits indicates that the effect of RAS activation is context-dependent and can be especially relevant in BT-IC or poorly differentiated tumors that arise from multipotent progenitor cells. In fact, we previously showed that RAS plays an important role in maintaining the self-renewal properties of BT-IC33.
Expression of the 154 member MARS of BPLER dependency genes or the smaller subset of 23 highly selective hits provides a useful signature for, for example, predicting cancer prognosis, such as breast cancer prognosis, response to certain therapeutic drugs or probability of resistance or metastatic recurrence, as demonstrated herein. These results are also useful for stratifying patients for clinical studies, especially for drugs expected to impact the pathways enriched in the gene set. The prognostic value of this signature can also be used in luminal B subtype tumor prognosis, as we found by retrospective analysis that it discriminates between good and poor prognosis patients with luminal B subtype tumors.
Methods
Cell Culture.
Human BPE, BPLE, BPLER and HMLER cells were provided by R. Weinberg and T. Ince and grown in WIT medium (Stemgent). All experiments were performed with pairs of cells derived from the same patient (BPLER-2 and HMLER-2). Other human cell lines were obtained from ATCC and grown in DMEM, except for MCF7 (MEM) and HCC-1943 and HCC-1937 (RPMI), all supplemented with 10% FBS unless otherwise indicated. BPLER and HMLER cells were reverse-transfected with 50 nM siRNAs for screening and functional experiments using Dharmafect#1 (Dharmacon) or LIPOFECTAMINE 2000 (Invitrogen), respectively, in WIT medium. For drug treatment, cells were plated at 50,000-80,000 cells/well in 6-well plates or 1000 cells/well in 384-well plates in WIT and treated 24 hr later. Drug treatment of transfected cells was begun 24 hr after transfection. Cell viability was assessed by CELLTITERGLO (Promega) in 384-well plates or by Trypan-Blue staining in 6-well plates. Chemoluminescence was measured using an ENVISION (PerkinElmer) high-throughput plate reader. Cell viability after treatment with BRPA was assessed by Trypan-Blue staining.
RNA Analysis.
qRT-PCR analysis was performed as described41. Briefly, total RNA was extracted with TRIZOL (Invitrogen) and cDNA prepared from 600-900 ng total RNA using THERMOSCRIPT RT kit (Invitrogen) as per the manufacturer's instructions. 2.5 ul of diluted cDNA (1:20) was used as template for qPCR using POWER SYBR-GREEN MASTER MIX (Applied Biosystems) and BIORAD C1000 THERMAL CYCLER (Biorad). Primer sequences are available upon request. Relative CT values were normalized to □-actin and converted to a linear scale using the −ΔCT method.
Protein Analysis.
Immunoblot was performed as described41. Primary antibodies were as follows: cleaved Caspase 3 (Cell Signaling, rabbit polyclonal, 1:1000), PARP1 (Santa Cruz, rabbit polyclonal, 1:500), MCL1 (Cell Signaling, Rabbit polyclonal, 1:1000), Bim (Santa Cruz, rabbit polyclonal, 1:500), Bik (Cell Signaling, rabbit polyclonal, 1:1000), Puma (Cell Signaling, rabbit polyclonal, 1:1000), Noxa (Calbiochem, mouse monoclonal, 1:500), Bid (Santa Cruz, rabbit polyclonal, 1:500), Bad (Santa Cruz, rabbit polyclonal, 1:500). Antibodies were diluted in 5% milk in TBS-T and incubated O.N. at 4° C. Secondary mouse and rabbit HRP-conjugated antibodies were from Amersham. Protein signal was detected using the ECL Plus kit (Amersham). For flow cytometry, cells were stained as previously described33, using the following fluorescent-conjugated antibodies: CD44 (BD Biosciences), CD24 (BD Biosciences), CD326 (ESA, Biolegend), Annexin V (Invitrogen). For immunofluorescence microscopy, cells were fixed in 2% PFA for 10 min, permeabilized with 1% Triton X in PBS for 5 min, and incubated with primary antibodies in 0.5% Triton X, 1% FBS in PBS for 30 min at RT. Antibodies were: CK18 (Santa Cruz, mouse monoclonal, 1:100) and CK14 (Millipore, mouse monoclonal, 1:100). After washing the cells were stained sequentially with AlexaFluor647-conjugated secondary antibody (Invitrogen, 1:200) and DAPI (Sigma, 1:5000). For immunohistochemistry, BPLER tumor sections were prepared from paraffin-embedded tissues, deparaffinized and treated with 10% citrate buffer for 15 min. Staining was performed using a BIOGENEX IHC DAB kit (Biogenex) following the manufacturer's protocol. Primary antibodies were: CK14 (Millipore, mouse monoclonal, 1:100), CK5 (Millipore, mouse monoclonal, 1:100), HH3 (Cell Signaling, rabbit polyclonal, 1:100), ER (Abcam, mouse monoclonal, 1:100), Vimentin (Invitrogen, mouse monoclonal 1:100), Ki67 (Millipore, mouse monoclonal, 1:100).
In Vivo Experiments.
All animal procedures were performed with IACUC approval. Exponentially growing cells were trypsinized with TRYPLE EXPRESS (Invitrogen), resuspended in a 1:1 WIT-Matrigel solution at the indicated numbers, and injected subcutaneously in the flank of 4-week old female Nu/J mice (Stock #002019, Jackson Laboratories). For bortezomib treatment, beginning 21 days after BPLER cells were injected (when tumors became palpable), mice bearing tumors of comparable size were randomized into two groups and treated i.p. q3d with bortezomib 0.5 mg/kg or DMSO, respectively. Mice were sacrificed 16 days after beginning treatment and tumor weight was assessed.
RNAi Screen.
Automated screening procedures were performed at the Harvard ICCB-L Screening Facility using Human siGenome siRNA libraries (Dharmacon). Procedures were optimized and validated for high-throughput screening under ICCB-L guidance. Screening conditions were identical for BPLER and HMLER cells. In the primary screen, library plates that contained siRNAs no longer supported by transcript evidence were not screened. For each cell line, each siRNA or pool of siRNAs was transfected in triplicate wells. Each microplate included 8 negative and 8 positive internal controls, which were used to monitor experimental conditions across the screen. Only microplates with a Z′ factor >0.5 were analyzed (covering 98.7% of the siRNA library). WELLMATE rapid plate dispensers and Teflon-coated manifolds (Matrix) were used to dispense the cells. siRNAs libraries were transferred into 384-well assay plates using liquid handling robots (VELOCITY 11 BRAVO). siRNAs were reverse-transfected at a final concentration of 50 nM in 384-well white/clear microplates (Corning Cat. #3707) using Dharmafect #1 (Dharmacon) in WIT medium. Fresh medium was added after 24 hours and cell viability was assessed by CELLTITERGLO (Promega) 3 days after transfection using an ENVISION high-throughput plate reader (Perkin-Elmer).
Screening Hit Selection.
Viability scores were judged based on a combination of parameters, including relative standard deviation among replicates (RSD), median absolute deviation (MAD)-based Z score (Z score), fold-change from the plate median (FC), and BPLER/HMLER FC ratio (R)42. For each set of triplicate plates, siRNAs with RSD >0.25 were excluded. Any siRNAs causing severe cytotoxicity to HMLER(HMLER Z score ≦−3 or HMLER FC ≦0.5) were also excluded from further analysis. siRNAs were considered hits if they satisfied the following criteria: BPLER Z score ≦−1.5, BPLER FC ≦0.75, R≦0.75. Positive hits were classified into highly, moderately or modestly selective based on R values. The positive hits were subjected to a secondary screen in which cells were transfected individually with the 4 siRNAs in the positive pools. Hits were considered validated if wells treated with at least one siRNA in the pool met the same criteria used in the primary screen.
Microarray Analysis.
Human breast tumor array data was obtained from two previous studies on Agilent and Affymetrix arrays (UNC dataset12, 337 cases and Richardson dataset13, 47 cases). The gene expression profile of BPLER tumor explants was acquired using Affymetrix arrays. For cross platform comparison arrays were rank normalized using only genes common to all arrays. To account for the fact that all of the stromal genes in the BPLER xenografts are of mouse origin, a principal component analysis was used to identify the major component that separated the tumor and BPLER arrays and these genes were excluded from the subsequent analysis. The samples were co-clustered based on the remaining genes that were differentially expressed across the tumor dataset by Ward's minimum variance method according to Spearman correlation coefficients (GEO references GSE18229, GSE3744 and GSE6885).
Survival Analysis of Primary Tumor Datasets.
The expression of the TGS and highly specific gene signatures were assessed in each tumor sample in the NKI dataset14 using the single-sample GSEA enrichment score, as previously described43. Briefly, each sample was rank-normalized and an enrichment score was obtained by a weighted sum of the difference between the empirical distribution functions of the genes in the signature and that of the remaining genes. The enrichment scores were classified as low and high expression, according to the p-value associated with up-regulation of expression of TGS genes. Tumors whose score ranked with p<0.1 were assigned to the high expression group and the remaining tumors were defined as low. Survival and metastasis-free survival curves were derived using the Kaplan-Meier method.
Tables for Examples 1 and 2







TABLE 1







Genes that Decreased BPLER Viability





















BPLER/


Entrez Gene


MAD
MAD
Fold
Fold
HMLER


ID
Accession #
Gene Symbol
BPLER
HMLER
BPLER
HMLER
Ratio

















79864
NM_024806
C11orf63
−1.69
−2.37
0.709
0.63
1.12


54934
NM_017822
C12orf41
−1.53
−1.15
0.652
0.84
0.78


80209
NM_025138
C13orf23
−1.78
−1.61
0.680
0.72
0.95


221150
NM_145061
C13orf3
−1.66
−0.47
0.694
0.92
0.76


283598
NM_182560
C14orf177
−2.78
−2.57
0.401
0.48
0.83


283635
NM_173607
C14orf24
−1.66
−1.94
0.638
0.60
1.06


64207
NM_024496
C14orf4
−1.63
−0.21
0.731
0.97
0.75


348110
NM_182616
C15orf38
−2.27
−2.24
0.626
0.67
0.93


90381
NM_152259
C15orf42
−1.66
−1.47
0.666
0.78
0.85


283897
NM_175900
C16orf54
−1.80
−1.98
0.611
0.60
1.02


146850
XM_375404
C17orf38
−2.01
−1.40
0.643
0.79
0.82


79018
NM_024052
C17orf39
−2.16
−3.69
0.646
0.42
1.53


79002
NM_024038
C19orf43
−2.54
−2.67
0.587
0.59
0.99


79098
NM_023938
C1orf116
−1.83
−1.56
0.695
0.75
0.92


26099
NM_015609
C1orf144
−2.02
−1.72
0.680
0.71
0.95


149466
NM_182517
C1orf210
−1.51
−1.60
0.731
0.76
0.96


54823
NM_017673
C1orf26
−1.93
−1.87
0.677
0.74
0.91


79078
NM_024097
C1orf50
−1.59
−1.40
0.741
0.78
0.96


79871
NM_024813
C1orf82
−3.98
−3.04
0.313
0.53
0.59


140701
NM_080622
C20orf135
−1.83
−2.55
0.676
0.63
1.08


253143
NM_173566
C22orf30
−1.73
−0.57
0.728
0.90
0.81


56947
NM_020194
C2orf33
−1.84
−1.21
0.703
0.81
0.86


80304
NM_025203
C2orf44
−1.89
−1.33
0.665
0.75
0.88


57415
NM_020685
C3orf14
−1.71
−1.56
0.721
0.76
0.95


25871
NM_015412
C3orf17
−1.78
−1.18
0.721
0.82
0.88


131831
NM_152394
C3orf44
−1.58
−1.76
0.715
0.72
0.99


206412
XM_116497
C6orf163
−1.72
−1.54
0.728
0.73
1.00


253714
NM_198468
C6orf167
−2.12
−1.24
0.665
0.79
0.84


221261
XM_168053
C6orf184
−2.22
−1.47
0.650
0.75
0.86


221718
NM_152738
C6orf218
−1.84
−1.20
0.709
0.79
0.90


84302
NM_032342
C9orf125
−2.46
−2.37
0.528
0.61
0.87


138240
XM_059954
C9orf57
−1.71
−1.30
0.668
0.77
0.86


55684
NM_024718
C9orf86
−1.61
−1.92
0.654
0.71
0.92


203245
NM_197956
C9orf90
−1.59
−1.10
0.749
0.82
0.91


56344
NM_019855
CABP5
−1.81
−0.50
0.708
0.92
0.77


773
NM_000068
CACNA1A
−2.11
−1.83
0.607
0.68
0.90


775
NM_000719
CACNA1C
−1.67
−1.39
0.688
0.76
0.91


83698
NM_031468
CALN1
−1.61
−0.95
0.695
0.84
0.83


55450
NM_018584
CAMK2N1
−3.80
−1.54
0.626
0.72
0.87


824
NM_001748
CAPN2
−2.18
−2.55
0.651
0.61
1.08


84290
NM_032330
CAPNS2
−1.68
−1.12
0.681
0.81
0.84


84433
NM_032415
CARD11
−1.64
−0.70
0.732
0.88
0.83


64170
NM_022352
CARD9
−1.53
−0.81
0.744
0.87
0.86


57524
NM_020764
CASKIN1
−1.84
−0.86
0.701
0.87
0.81


9994
NM_012115
CASP8AP2
−2.05
−1.66
0.631
0.70
0.90


863
NM_005187
CBFA2T3
−2.46
−1.32
0.673
0.74
0.90


79872
NM_024814
CBLL1
−2.41
−3.24
0.585
0.50
1.17


56267
NM_019610
CCBL2
−1.88
−1.17
0.695
0.82
0.85


80125
NM_182791
CCDC33
−1.80
−0.89
0.675
0.84
0.80


120935
NM_182496
CCDC38
−2.30
−2.54
0.588
0.65
0.91


152185
NM_144718
CCDC52
−1.96
−1.89
0.699
0.70
0.99


29080
NM_014167
CCDC59
−1.68
−1.50
0.702
0.77
0.92


83987
NM_032040
CCDC8
−1.89
−1.82
0.643
0.70
0.91


283234
NM_032251
CCDC88B
−1.73
−0.89
0.638
0.83
0.77


54908
NM_017785
CCDC99
−2.46
−3.48
0.420
0.48
0.88


6361
NM_002987
CCL17
−2.11
−2.30
0.610
0.64
0.96


6367
NM_002990
CCL22
−2.48
−1.42
0.685
0.79
0.87


6370
NM_005624
CCL25
−1.69
−1.03
0.688
0.83
0.83


10344
NM_006072
CCL26
−2.37
−1.16
0.697
0.82
0.85


891
NM_031966
CCNB1
−1.92
−1.21
0.652
0.78
0.83


8812
NM_003858
CCNK
−3.07
−2.54
0.443
0.54
0.81


1235
NM_004367
CCR6
−1.77
−1.74
0.674
0.73
0.92


1237
NM_005201
CCR8
−1.61
−0.87
0.704
0.86
0.82


23552
NM_012119
CCRK
−1.75
−1.15
0.744
0.84
0.88


10575
NM_006430
CCT4
−2.55
−1.70
0.538
0.69
0.78


930
NM_001770
CD19
−2.12
−0.99
0.726
0.85
0.85


910
NM_001764
CD1B
−1.90
−1.02
0.657
0.82
0.80


4345
NM_005944
CD200
−1.53
−1.77
0.706
0.71
1.00


934
NM_013230
CD24
−2.59
−2.38
0.527
0.57
0.92


51744
NM_016382
CD244
−1.51
−1.13
0.725
0.80
0.91


10849
NM_012099
CD3EAP
−1.76
−0.24
0.746
0.96
0.78


962
NM_001778
CD48
−1.59
−1.42
0.657
0.80
0.83


55143
NM_018101
CDCA8
−2.19
−3.49
0.480
0.48
1.00


8558
NM_003674
CDK10
−1.81
−1.03
0.734
0.86
0.86


80279
NM_025197
CDK5RAP3
−3.10
−2.74
0.548
0.63
0.87


125931
NM_198444
CEACAM20
−3.05
−2.95
0.451
0.59
0.77


1951
NM_001407
CELSR3
−1.53
−0.80
0.718
0.87
0.83


1062
NM_001813
CENPE
−2.75
−2.61
0.590
0.58
1.03


2491
NM_006733
CENPI
−1.54
−1.59
0.692
0.76
0.92


79172
NM_024322
CENPO
−2.42
−2.22
0.597
0.65
0.92


22897
XM_374936
CEP164
−3.04
−2.08
0.565
0.66
0.86


1073
NM_021914
CFL2
−1.80
−1.17
0.614
0.81
0.75


114335
NM_033377
CGB1
−1.57
−1.01
0.716
0.82
0.87


79094
NM_024111
CHAC1
−2.41
−2.44
0.602
0.61
0.98


1108
NM_001273
CHD4
−2.00
−2.22
0.702
0.64
1.10


1111
NM_001274
CHEK1
−4.19
−3.53
0.387
0.52
0.74


27243
NM_014453
CHMP2A
−1.95
−3.58
0.655
0.45
1.47


91782
NM_152272
CHMP7
−1.70
−2.32
0.676
0.67
1.01


91851
NM_145234
CHRDL1
−1.64
−1.11
0.689
0.84
0.82


1131
NM_000740
CHRM3
−1.58
−2.09
0.709
0.68
1.05


8973
NM_004198
CHRNA6
−2.24
−0.51
0.722
0.92
0.78


1178
NM_001828
CLC
−1.52
−0.67
0.670
0.89
0.75


1186
NM_001287
CLCN7
−2.32
−1.62
0.716
0.73
0.98


137075
NM_194284
CLDN23
−1.81
−1.03
0.655
0.83
0.79


7123
NM_003278
CLEC3B
−2.02
−1.63
0.712
0.76
0.93


25932
NM_013943
CLIC4
−2.44
−1.65
0.699
0.73
0.96


55907
NM_018686
CMAS
−1.63
−1.53
0.637
0.78
0.82


1259
NM_000087
CNGA1
−2.20
−1.70
0.604
0.70
0.87


10256
NM_006314
CNKSR1
−2.74
−3.15
0.599
0.57
1.05


23019
NM_016284
CNOT1
−2.74
−2.06
0.571
0.67
0.85


1271
NM_001842
CNTFR
−2.00
−0.63
0.722
0.90
0.80


53942
NM_014361
CNTN5
−1.72
−1.95
0.689
0.65
1.06


1297
NM_001851
COL9A1
−3.17
−2.16
0.605
0.66
0.91


150684
NM_152516
COMMD1
−2.28
−0.95
0.648
0.85
0.76


9276
NM_004766
COPB2
−4.48
−3.75
0.343
0.49
0.70


22820
NM_016128
COPG
−3.44
−2.50
0.506
0.59
0.86


8533
NM_003653
COPS3
−1.63
−0.62
0.705
0.89
0.79


10987
NM_006837
COPS5
−1.51
−1.19
0.727
0.79
0.92


10980
NM_006833
COPS6
−2.32
−1.14
0.666
0.81
0.82


22818
NM_016057
COPZ1
−3.63
−2.53
0.479
0.58
0.82


1325
NM_001302
CORT
−1.63
−2.84
0.667
0.55
1.22


1325
NM_001302
CORT
−2.15
−3.10
0.726
0.53
1.36


64506
NM_030594
CPEB1
−1.60
−1.93
0.743
0.70
1.06


1369
NM_001308
CPN1
−1.87
−0.86
0.701
0.87
0.81


1370
J05158
CPN2
−1.61
−1.29
0.705
0.77
0.92


58487
NM_021212
CREBZF
−2.50
−1.40
0.668
0.73
0.92


9282
NM_004229
CRSP2
−2.48
−1.88
0.690
0.71
0.98


9441
NM_004831
CRSP7
−3.43
−2.36
0.570
0.63
0.90


1436
NM_005211
CSF1R
−1.85
−2.13
0.730
0.71
1.03


1446
NM_001890
CSN1S1
−1.61
−2.03
0.646
0.68
0.95


1459
NM_001896
CSNK2A2
−2.56
−2.32
0.627
0.68
0.92


80777
NM_030579
CYB5B
−1.68
−1.91
0.691
0.66
1.05


1545
NM_000104
CYP1B1
−2.01
−0.30
0.724
0.95
0.76


1595
NM_000786
CYP51A1
−1.66
−2.44
0.651
0.61
1.06


153090
NM_032552
DAB2IP
−3.29
−2.88
0.491
0.55
0.89


23142
NM_015115
DCUN1D4
−1.71
−0.53
0.726
0.92
0.79


245932
NM_153289
DEFB119
−1.78
−1.33
0.721
0.77
0.93


140850
NM_139074
DEFB127
−2.20
−1.40
0.609
0.79
0.77


1676
NM_004401
DFFA
−2.36
−1.36
0.620
0.79
0.78


10901
NM_021004
DHRS4
−2.40
−1.05
0.653
0.83
0.79


79665
NM_024612
DHX40
−3.28
−3.35
0.433
0.48
0.90


27122
NM_013253
DKK3
−2.46
−2.37
0.565
0.63
0.89


1750
XM_376652
DLX6
−2.60
−2.26
0.655
0.56
1.17


29958
NM_013391
DMGDH
−3.45
−2.47
0.661
0.55
1.20


1763
XM_166103
DNA2L
−2.54
−2.55
0.463
0.59
0.78


1788
NM_022552
DNMT3A
−1.70
−2.24
0.728
0.65
1.11


116092
NM_052951
DNTTIP1
−1.71
−1.22
0.692
0.78
0.89


220164
NM_152721
DOK6
−2.81
−1.66
0.556
0.72
0.78


29980
NM_145794
DONSON
−4.14
−3.46
0.268
0.46
0.58


1802
NM_001384
DPH2
−1.91
−1.63
0.589
0.74
0.80


10231
NM_005822
DSCR1L1
−1.52
−1.97
0.632
0.68
0.93


1837
NM_001390
DTNA
−1.52
−1.54
0.667
0.77
0.87


151636
NM_138287
DTX3L
−1.89
−1.78
0.707
0.72
0.98


64118
NM_022156
DUS1L
−2.02
−0.85
0.662
0.86
0.77


1852
NM_001395
DUSP9
−2.13
−1.70
0.685
0.72
0.95


143241
NM_138812
DYDC1
−1.82
−1.12
0.676
0.83
0.82


84332
NM_032372
DYDC2
−1.66
−0.96
0.687
0.84
0.81


1781
NM_001378
DYNC1I2
−2.06
−1.72
0.732
0.74
0.98


1894
NM_018098
ECT2
−2.13
−0.93
0.733
0.85
0.86


1915
NM_001402
EEF1A1
−2.11
−1.76
0.620
0.69
0.90


29904
NM_013302
EEF2K
−2.93
−2.83
0.570
0.61
0.93


60678
NM_021937
EEFSEC
−1.98
−0.93
0.666
0.84
0.79


283229
NM_173584
EFCAB4A
−2.38
−2.66
0.494
0.47
1.05


65989
NM_023932
EGFL9
−1.74
−0.45
0.715
0.93
0.77


112399
NM_022073
EGLN3
−1.61
−1.82
0.678
0.72
0.94


8668
NM_003757
EIF3S2
−2.76
−1.95
0.729
0.65
1.13


63036
NM_033440
ELA2A
−1.51
−0.80
0.745
0.87
0.86


1996
NM_021952
ELAVL4
−2.31
−1.77
0.583
0.68
0.85


1999
NM_004433
ELF3
−1.90
−1.53
0.749
0.70
1.07


256364
NM_153265
EML3
−1.86
−2.27
0.621
0.54
1.16


2047
NM_004441
EPHB1
−2.11
−1.45
0.688
0.80
0.86


2066
NM_005235
ERBB4
−1.74
−2.36
0.743
0.68
1.10


2077
NM_006494
ERF
−2.70
−1.18
0.641
0.77
0.83


90332
NM_138568
EXOC3L2
−2.04
−1.45
0.591
0.79
0.75


5394
NM_002685
EXOSC10
−2.43
−2.01
0.518
0.67
0.77


3995
NM_021727
FADS3
−1.73
−1.79
0.653
0.73
0.90


11170
NM_007177
FAM107A
−2.39
−1.05
0.660
0.83
0.80


26071
NM_015582
FAM127B
−2.09
−2.47
0.670
0.60
1.11


80097
NM_025029
FAM128B
−1.75
−1.31
0.680
0.77
0.88


9715
NM_014690
FAM131B
−1.82
−3.96
0.599
0.28
2.17


83982
NM_032036
FAM14A
−1.53
−0.98
0.712
0.83
0.86


54801
NM_017645
FAM29A
−2.20
−1.54
0.633
0.79
0.80


26240
NM_012135
FAM50B
−2.43
−1.62
0.618
0.73
0.84


286336
NM_033387
FAM78A
−1.82
−1.22
0.668
0.82
0.81


84985
NM_032899
FAM83A
−3.11
−2.64
0.490
0.52
0.94


2176
NM_000136
FANCC
−1.61
−1.61
0.709
0.71
1.00


2178
NM_021922
FANCE
−2.19
−2.26
0.600
0.59
1.01


2197
NM_001997
FAU
−3.01
−1.72
0.641
0.72
0.89


54850
NM_017703
FBXL12
−1.60
−0.42
0.748
0.94
0.80


283807
NM_203373
FBXL22
−1.74
−0.84
0.640
0.82
0.78


93611
NM_033182
FBXO44
−2.30
−2.46
0.570
0.65
0.88


26270
NM_018438
FBXO6
−2.11
−1.31
0.688
0.79
0.87


6468
NM_022039
FBXW4
−2.27
−0.91
0.661
0.85
0.78


2213
NM_004001
FCGR2B
−2.14
−1.74
0.724
0.74
0.98


2222
NM_004462
FDFT1
−2.11
−1.52
0.663
0.77
0.86


2865
NM_005304
FFAR3
−1.56
−0.97
0.714
0.85
0.84


2268
NM_005248
FGR
−2.67
−2.20
0.610
0.70
0.87


84929
NM_032843
FIBCD1
−2.44
−1.47
0.598
0.73
0.82


80099
NM_025031
FLJ21075
−2.44
−2.10
0.553
0.63
0.88


80154
NM_025084
FLJ22795
−2.79
−2.57
0.493
0.54
0.91


220042
NM_145018
FLJ25416
−2.57
−2.05
0.594
0.65
0.91


254048
XM_376679
FLJ25778
−2.90
−2.67
0.543
0.55
0.99


152756
NM_153027
FLJ31659
−2.26
−1.93
0.652
0.70
0.93


201283
NM_153032
FLJ32065
−2.17
−1.88
0.657
0.69
0.96


400629
NM_207459
FLJ35767
−2.91
−2.91
0.458
0.49
0.94


168455
NM_175884
FLJ36031
−2.00
−1.95
0.653
0.69
0.94


151258
NM_173512
FLJ39822
−1.66
−0.46
0.744
0.93
0.80


147699
NM_178494
FLJ40125
−1.65
−1.34
0.708
0.80
0.88


206338
NM_173800
FLJ90650
−1.90
−0.72
0.702
0.87
0.81


2324
NM_002020
FLT4
−1.85
−0.86
0.729
0.88
0.83


8061
NM_005438
FOSL1
−2.05
−0.85
0.727
0.83
0.87


2300
NM_005250
FOXL1
−2.14
−1.86
0.716
0.63
1.13


116113
NM_138457
FOXP4
−1.63
−1.72
0.693
0.75
0.92


53826
NM_022003
FXYD6
−1.58
−2.08
0.742
0.71
1.04


2585
NM_002044
GALK2
−2.75
−2.39
0.598
0.67
0.89


253959
XM_210022
GARNL1
−1.67
−1.34
0.737
0.77
0.95


2618
NM_000819
GART
−2.28
−1.56
0.713
0.69
1.03


2633
NM_002053
GBP1
−2.12
−3.02
0.711
0.52
1.36


2657
NM_001492
GDF1
−1.98
−2.39
0.748
0.64
1.17


124975
NM_153338
GGT6
−1.99
−2.23
0.647
0.69
0.94


168537
NM_153236
GIMAP7
−2.20
−1.13
0.620
0.82
0.76


256356
NM_152776
GK5
−2.15
−1.51
0.546
0.69
0.79


83468
NM_031302
GLT8D2
−2.03
−1.69
0.616
0.72
0.85


10691
NM_006582
GMEB1
−2.09
−1.57
0.723
0.69
1.04


94235
NM_033258
GNG8
−1.55
−1.78
0.689
0.73
0.94


27238
NM_015698
GPKOW
−1.68
−0.44
0.700
0.93
0.75


283554
XM_290615
GPR137C
−2.18
−1.55
0.513
0.68
0.75


390212
NM_206997
GPR152
−1.97
−1.51
0.627
0.73
0.86


2846
NM_005296
GPR23
−1.73
−1.57
0.682
0.75
0.91


2852
NM_001505
GPR30
−1.85
−1.10
0.661
0.82
0.80


2853
NM_005299
GPR31
−1.71
−1.04
0.687
0.84
0.82


2854
NM_001506
GPR32
−1.77
−2.67
0.675
0.59
1.14


2866
NM_005305
GPR42
−1.69
−0.86
0.690
0.87
0.80


10149
NM_005756
GPR64
−1.60
−1.69
0.705
0.74
0.95


53831
NM_020370
GPR84
−2.09
−1.84
0.615
0.71
0.86


53836
NM_023915
GPR87
−1.90
−2.10
0.653
0.67
0.98


285513
NM_198281
GPRIN3
−1.62
−2.88
0.705
0.58
1.21


80852
XM_042936
GRIP2
−2.15
−2.41
0.610
0.57
1.08


2926
NM_002092
GRSF1
−1.83
−1.95
0.604
0.71
0.85


84163
NM_173537
GTF2IRD2
−1.55
−1.00
0.669
0.85
0.79


26164
NM_015666
GTPBP5
−2.03
−1.11
0.681
0.82
0.83


2978
NM_000409
GUCA1A
−1.51
−1.53
0.704
0.75
0.94


3000
NM_000180
GUCY2D
−2.59
−1.81
0.620
0.75
0.82


60484
NM_021817
HAPLN2
−1.85
−1.02
0.687
0.83
0.83


253018
NM_181717
HCG27
−2.47
−1.40
0.611
0.76
0.80


57493
XM_087386
HEG1
−2.41
−1.43
0.610
0.78
0.78


3280
NM_005524
HES1
−1.52
−1.64
0.673
0.75
0.90


57801
NM_021170
HES4
−2.53
−2.94
0.455
0.57
0.80


135114
NM_138571
HINT3
−1.60
−1.56
0.675
0.74
0.91


3093
NM_005339
HIP2
−2.23
−2.50
0.711
0.63
1.13


8479
NM_003609
HIRIP3
−1.76
−1.71
0.665
0.72
0.92


23262
NM_015216
HISPPD1
−2.01
−0.62
0.744
0.88
0.85


121504
NM_175054
HIST4H4
−2.04
−2.20
0.640
0.69
0.93


3099
NM_000189
HK2
−2.19
−1.49
0.678
0.80
0.85


3146
NM_002128
HMGB1
−1.91
−1.64
0.722
0.77
0.94


3156
NM_000859
HMGCR
−2.02
−1.24
0.743
0.75
0.99


3150
NM_004965
HMGN1
−1.80
−1.16
0.654
0.81
0.81


220988
NM_194247
HNRPA3
−2.18
−2.51
0.656
0.58
1.14


50863
NM_016522
HNT
−1.81
−2.15
0.611
0.69
0.89


84525
NM_032495
HOP
−1.53
−0.26
0.748
0.96
0.78


3238
NM_021193
HOXD12
−2.65
−2.00
0.675
0.62
1.09


154791
NM_197964
HSPC268
−3.61
−2.71
0.441
0.57
0.78


23553
NM_012269
HYAL4
−3.54
−2.99
0.444
0.52
0.86


3382
NM_004968
ICA1
−1.70
−1.41
0.659
0.78
0.84


23463
NM_012405
ICMT
−1.81
−0.73
0.708
0.88
0.81


3467
NM_002177
IFNW1
−2.83
−1.85
0.636
0.72
0.88


3543
NM_020070
IGLL1
−1.72
−0.92
0.666
0.85
0.78


3550
NM_006083
IK
−4.13
−3.31
0.469
0.50
0.93


3596
NM_002188
IL13
−1.72
−1.81
0.666
0.71
0.94


3601
NM_002189
IL15RA
−2.13
−2.03
0.725
0.69
1.05


3553
NM_000576
IL1B
−2.98
−2.49
0.617
0.62
0.99


3611
NM_004517
ILK
−2.18
−1.69
0.568
0.73
0.77


55364
NM_018439
IMPACT
−1.68
−2.16
0.634
0.68
0.93


3619
NM_020238
INCENP
−3.05
−3.25
0.386
0.51
0.76


26173
XM_291222
INTS1
−1.88
−1.39
0.704
0.77
0.91


57508
NM_020748
INTS2
−2.14
−0.99
0.654
0.85
0.77


79711
NM_024658
IPO4
−1.53
−1.42
0.732
0.78
0.94


79192
XM_380171
IRX1
−2.16
−1.71
0.628
0.74
0.85


122961
NM_194279
ISCA2
−1.58
−1.79
0.719
0.75
0.95


23479
NM_014301
ISCU
−2.62
−1.60
0.589
0.74
0.79


3713
NM_005547
IVL
−1.73
−1.61
0.653
0.75
0.87


126306
NM_144616
JSRP1
−1.72
−2.33
0.690
0.67
1.02


353219
NM_181337
KAAG1
−1.55
−1.04
0.709
0.81
0.87


126823
NM_152366
KARCA1
−1.57
−0.86
0.718
0.88
0.81


3780
NM_002248
KCNN1
−1.64
−0.72
0.668
0.89
0.75


3785
NM_004518
KCNQ2
−2.29
−1.30
0.734
0.79
0.93


9132
NM_004700
KCNQ4
−2.43
−1.89
0.709
0.69
1.03


283518
NM_173605
KCNRG
−1.82
−1.50
0.621
0.70
0.88


57582
XM_029962
KCNT1
−2.28
−2.42
0.726
0.60
1.21


202559
NM_152688
KHDRBS2
−2.54
−2.08
0.601
0.64
0.93


23334
NM_015284
KIAA0467
−1.60
−0.88
0.740
0.86
0.86


23354
XM_049237
KIAA0841
−2.55
−1.54
0.595
0.75
0.79


23383
XM_048457
KIAA0892
−1.62
−0.89
0.739
0.86
0.86


23379
XM_029101
KIAA0947
−2.87
−2.25
0.545
0.64
0.85


23325
NM_015275
KIAA1033
−1.95
−0.59
0.690
0.91
0.76


57179
NM_020444
KIAA1191
−1.99
−1.20
0.676
0.82
0.83


57535
NM_020775
KIAA1324
−2.62
−1.57
0.574
0.76
0.76


57650
NM_020890
KIAA1524
−1.65
−0.95
0.722
0.84
0.86


80256
NM_025182
KIAA1539
−1.78
−1.41
0.677
0.75
0.90


57703
XM_034594
KIAA1604
−2.57
−2.89
0.566
0.51
1.12


165215
NM_177454
KIAA1946
−1.61
−1.30
0.719
0.80
0.90


3832
NM_004523
KIF11
−4.90
−3.56
0.267
0.42
0.64


113220
NM_138424
KIF12
−3.83
−2.76
0.429
0.55
0.78


81930
NM_031217
KIF18A
−3.12
−2.18
0.532
0.64
0.82


9493
NM_004856
KIF23
−3.15
−2.68
0.530
0.57
0.94


57565
NM_020805
KLHL14
−1.71
−1.11
0.710
0.81
0.88


57563
NM_020803
KLHL8
−2.16
−1.73
0.636
0.71
0.90


25818
NM_012427
KLK5
−2.11
−2.03
0.669
0.68
0.99


11202
NM_007196
KLK8
−2.18
−0.72
0.689
0.88
0.79


3837
NM_002265
KPNB1
−3.32
−2.97
0.384
0.47
0.81


3887
NM_002281
KRT81
−2.14
−2.40
0.587
0.61
0.96


3857
NM_000226
KRT9
−1.68
−1.55
0.672
0.75
0.90


81851
NM_030967
KRTAP1-1
−1.73
−1.62
0.683
0.71
0.96


337959
NM_181621
KRTAP13-2
−2.33
−2.54
0.615
0.63
0.98


3916
NM_005561
LAMP1
−1.60
−1.47
0.678
0.78
0.87


353139
NM_178428
LCE2A
−1.82
−1.26
0.659
0.77
0.86


79143
NM_024298
LENG4
−2.71
−2.73
0.475
0.56
0.85


114783
XM_055866
LMTK3
−1.79
−0.97
0.646
0.84
0.77


146325
NM_145270
LOC146325
−1.84
−1.93
0.673
0.71
0.94


147646
XM_085833
LOC147646
−1.50
−1.24
0.734
0.82
0.89


150223
XM_097886
LOC150223
−2.43
−1.33
0.624
0.79
0.79


161247
NM_203402
LOC161247
−2.02
−1.04
0.688
0.84
0.82


196264
NM_198275
LOC196264
−1.62
−2.03
0.720
0.68
1.06


283152
XM_378314
LOC283152
−1.83
−2.30
0.591
0.53
1.11


283547
XM_378454
LOC283547
−2.22
−1.59
0.507
0.67
0.76


283871
XM_208887
LOC283871
−1.52
−1.01
0.666
0.79
0.84


339745
NM_001001664
LOC339745
−1.63
−3.03
0.726
0.55
1.31


374973
XM_371248
LOC374973
−2.30
−1.67
0.572
0.70
0.82


399947
NM_207645
LOC399947
−1.73
−2.10
0.676
0.63
1.07


400451
NM_207446
LOC400451
−1.79
−2.58
0.665
0.55
1.22


90120
XM_379680
LOC90120
−1.53
−0.65
0.693
0.91
0.76


4023
NM_000237
LPL
−1.88
−2.27
0.742
0.64
1.15


84918
NM_032832
LRP11
−1.57
−1.14
0.703
0.80
0.88


10128
NM_133259
LRPPRC
−1.84
−1.10
0.734
0.85
0.87


9684
NM_014665
LRRC14
−1.59
−1.60
0.694
0.74
0.94


55631
NM_017768
LRRC40
−1.85
−1.37
0.642
0.78
0.82


115353
NM_052940
LRRC42
−1.82
−0.93
0.661
0.87
0.76


116064
XM_057296
LRRC58
−2.54
−3.12
0.516
0.55
0.93


65999
NM_023942
LRRC61
−1.88
−1.97
0.690
0.69
1.00


51599
NM_015925
LSR
−2.61
−2.86
0.501
0.53
0.94


55692
NM_018032
LUC7L
−1.61
−1.34
0.644
0.81
0.80


66004
NM_177477
LYNX1
−2.42
−2.71
0.597
0.58
1.04


116068
XM_371760
LYSMD3
−2.17
−1.99
0.583
0.71
0.82


4101
NM_005361
MAGEA2
−2.02
−2.23
0.593
0.66
0.90


9175
NM_004721
MAP3K13
−1.50
−0.83
0.697
0.87
0.80


5594
NM_002745
MAPK1
−1.57
−0.52
0.683
0.91
0.75


225689
NM_139021
MAPK15
−1.74
−0.67
0.744
0.91
0.82


23031
XM_038150
MAST3
−1.53
−1.52
0.705
0.75
0.93


4147
NM_002380
MATN2
−1.79
−2.39
0.639
0.64
1.00


4149
NM_002382
MAX
−1.53
−1.29
0.700
0.79
0.88


4152
NM_002384
MBD1
−2.24
−0.88
0.725
0.83
0.87


125997
NM_144614
MBD3L2
−1.88
−1.67
0.664
0.77
0.86


4199
NM_002395
ME1
−2.62
−1.81
0.669
0.64
1.05


4200
NM_002396
ME2
−1.52
−0.99
0.694
0.85
0.82


4201
NM_014623
MEA1
−1.59
−1.92
0.679
0.71
0.96


84246
NM_032286
MED10
−1.54
−1.68
0.708
0.73
0.97


80306
NM_025205
MED28
−2.77
−2.63
0.497
0.53
0.94


10001
NM_005466
MED6
−2.01
−1.99
0.748
0.69
1.08


112950
NM_052877
MED8
−2.06
−2.20
0.602
0.64
0.93


1953
XM_031401
MEGF6
−2.16
−1.92
0.528
0.69
0.76


4225
NM_005925
MEP1B
−2.09
−1.54
0.600
0.75
0.80


59274
NM_022566
MESDC1
−1.81
−0.93
0.653
0.85
0.77


23184
XM_370880
MESDC2
−2.08
−1.53
0.602
0.75
0.81


4236
NM_005926
MFAP1
−2.75
−3.08
0.447
0.53
0.84


84310
NM_032350
MGC11257
−1.65
−1.37
0.688
0.77
0.89


94107
NM_053045
MGC14327
−2.77
−3.16
0.471
0.55
0.86


84848
NM_032762
MGC16121
−1.64
−1.11
0.732
0.80
0.92


93624
XM_291105
MGC21874
−1.95
−2.84
0.628
0.59
1.07


200424
XM_371501
MGC22014
−1.61
−1.03
0.721
0.83
0.86


84730
NM_032644
MGC2452
−1.56
−1.67
0.746
0.70
1.07


169166
NM_152628
MGC39715
−1.68
−1.66
0.712
0.74
0.96


84752
NM_033309
MGC4655
−2.75
−1.72
0.548
0.69
0.80


202915
NM_152689
MGC9712
−2.69
−1.98
0.575
0.67
0.86


2872
NM_017572
MKNK2
−1.89
−1.03
0.635
0.84
0.76


64976
NM_003776
MRPL40
−1.77
−0.98
0.711
0.84
0.84


122704
NM_181307
MRPL52
−2.03
−1.61
0.636
0.78
0.82


65005
NM_031420
MRPL9
−1.50
−0.13
0.747
0.97
0.77


64951
NM_032014
MRPS24
−1.72
−0.67
0.712
0.89
0.80


10903
NM_181873
MTMR11
−2.10
−1.54
0.693
0.75
0.93


4609
NM_002467
MYC
−2.13
−1.70
0.740
0.67
1.11


4624
NM_002471
MYH6
−1.77
−0.79
0.731
0.89
0.82


4635
NM_002476
MYL4
−2.43
−1.77
0.708
0.71
1.00


85366
NM_033118
MYLK2
−1.59
−0.91
0.698
0.86
0.81


23026
XM_028522
MYO16
−1.91
−1.26
0.700
0.80
0.88


4666
NM_005594
NACA
−1.55
−1.48
0.740
0.78
0.95


23148
XM_166571
NACAD
−2.13
−1.70
0.662
0.73
0.91


54187
NM_018946
NANS
−1.77
−1.28
0.694
0.82
0.84


23397
NM_015341
NCAPH
−1.94
−1.74
0.690
0.72
0.96


23154
NM_014284
NCDN
−2.18
−1.76
0.655
0.72
0.91


56926
NM_020170
NCLN
−4.19
−3.59
0.320
0.44
0.72


4536
NM_173709
ND2
−1.53
−1.67
0.694
0.74
0.93


54820
NM_017668
NDE1
−3.86
−3.41
0.260
0.44
0.60


81565
NM_030808
NDEL1
−1.84
−1.52
0.649
0.75
0.87


27158
NM_014434
NDOR1
−1.79
−0.76
0.680
0.88
0.77


10397
NM_006096
NDRG1
−1.87
−1.21
0.641
0.80
0.80


55967
NM_018838
NDUFA12
−1.51
−1.77
0.669
0.73
0.91


4700
NM_002490
NDUFA6
−1.84
−0.86
0.691
0.87
0.79


4707
NM_004545
NDUFB1
−2.24
−1.69
0.623
0.75
0.83


4734
NM_006154
NEDD4
−1.85
−1.64
0.726
0.73
0.99


79858
NM_024800
NEK11
−1.58
−0.52
0.695
0.92
0.75


284086
NM_178170
NEK8
−2.10
−1.77
0.587
0.73
0.80


349565
NM_178177
NMNAT3
−1.93
−1.14
0.687
0.83
0.82


283820
NM_173614
NOMO2
−1.62
−1.17
0.664
0.76
0.87


9520
NM_006310
NPEPPS
−2.82
−2.20
0.613
0.65
0.94


23467
NM_014293
NPTXR
−1.90
−1.63
0.732
0.73
1.00


2494
NM_003822
NR5A2
−2.24
−2.51
0.720
0.61
1.18


80023
NM_024958
NRSN2
−1.60
−0.46
0.716
0.92
0.78


25936
NM_015471
NSL1
−1.66
−0.13
0.739
0.98
0.76


4923
NM_002531
NTSR1
−1.93
−2.23
0.633
0.63
1.00


170685
NM_153183
NUDT10
−1.73
−1.57
0.700
0.75
0.93


11051
NM_007006
NUDT21
−2.43
−1.19
0.652
0.80
0.81


23279
XM_113678
NUP160
−2.11
−1.27
0.666
0.80
0.84


4928
NM_005387
NUP98
−2.55
−2.58
0.694
0.58
1.20


55916
NM_018698
NXT2
−2.18
−2.94
0.517
0.57
0.91


11054
NM_007346
OGFR
−2.00
−1.99
0.749
0.69
1.08


611
NM_001708
OPN1SW
−2.05
−2.27
0.605
0.63
0.96


10133
NM_021980
OPTN
−1.65
−2.10
0.683
0.65
1.05


4991
NM_002548
OR1D2
−1.53
−1.59
0.706
0.74
0.96


8387
NM_003553
OR1E1
−1.97
−2.21
0.621
0.64
0.97


26211
NM_012369
OR2F1
−1.86
−2.11
0.653
0.66
0.99


114881
NM_017731
OSBPL7
−1.55
−1.46
0.688
0.78
0.88


64172
NM_022353
OSGEPL1
−1.75
−0.39
0.709
0.94
0.75


150677
NM_148961
OTOS
−2.80
−1.96
0.567
0.68
0.83


27199
NM_080818
OXGR1
−1.87
−1.27
0.657
0.80
0.82


27334
NM_014499
P2RY10
−1.61
−1.34
0.695
0.78
0.89


8106
NM_004643
PABPN1
−1.54
−1.07
0.705
0.82
0.86


85315
NM_133367
PAQR8
−2.37
−1.44
0.612
0.74
0.83


344838
NM_198504
PAQR9
−2.02
−3.47
0.667
0.49
1.35


5092
NM_000281
PCBD1
−2.10
−1.88
0.596
0.69
0.87


57060
NM_020418
PCBP4
−2.01
−1.02
0.700
0.86
0.82


5116
NM_006031
PCNT
−2.14
−2.64
0.639
0.61
1.05


9141
NM_004708
PDCD5
−3.14
−3.53
0.398
0.42
0.96


5145
NM_000440
PDE6A
−1.80
−1.28
0.655
0.79
0.83


8622
NM_003719
PDE8B
−2.35
−0.89
0.693
0.87
0.80


5159
NM_002609
PDGFRB
−1.80
−1.58
0.642
0.74
0.87


204474
NM_174924
PDILT
−2.98
−1.71
0.530
0.70
0.75


10630
NM_006474
PDPN
−1.50
−1.56
0.640
0.74
0.86


23037
NM_015022
PDZD2
−1.83
−0.91
0.720
0.86
0.84


10455
NM_006117
PECI
−1.63
−1.10
0.688
0.82
0.84


27043
NM_014389
PELP1
−2.08
−1.98
0.602
0.67
0.90


64065
NM_022121
PERP
−1.73
−2.24
0.668
0.63
1.06


92960
NM_080662
PEX11G
−2.18
−4.07
0.585
0.41
1.42


5207
NM_002625
PFKFB1
−2.30
−2.34
0.546
0.62
0.88


192111
NM_138575
PGAM5
−1.59
−1.00
0.720
0.83
0.86


80055
NM_024989
PGAP1
−1.74
−1.11
0.684
0.81
0.85


5232
NM_138733
PGK2
−2.53
−2.32
0.501
0.63
0.80


221692
XM_166420
PHACTR1
−1.86
−1.34
0.704
0.76
0.92


112885
NM_138415
PHF21B
−1.51
−2.39
0.697
0.64
1.09


84844
NM_032758
PHF5A
−2.27
−1.75
0.627
0.67
0.93


51588
NM_015897
PIAS4
−2.04
−1.34
0.748
0.74
1.00


5291
NM_006219
PIK3CB
−1.87
−1.64
0.640
0.73
0.88


26034
NM_015553
PIP3-E
−1.57
−0.68
0.751
0.88
0.85


55124
NM_018068
PIWIL2
−1.75
−1.21
0.665
0.80
0.83


168507
NM_138295
PKD1L1
−2.07
−2.06
0.644
0.68
0.95


9033
NM_016112
PKD2L1
−1.85
−1.87
0.657
0.67
0.98


5570
NM_032471
PKIB
−2.34
−1.95
0.538
0.69
0.78


123745
NM_198442
PLA2G4E
−1.99
−2.92
0.642
0.59
1.09


22874
NM_014935
PLEKHA6
−2.09
−1.04
0.697
0.83
0.84


58473
NM_021200
PLEKHB1
−1.54
−0.79
0.741
0.86
0.86


10979
NM_006832
PLEKHC1
−1.69
−1.32
0.676
0.78
0.86


57480
XM_027307
PLEKHG1
−1.60
−0.94
0.742
0.85
0.87


5356
NM_002669
PLRG1
−2.11
−1.42
0.738
0.78
0.95


5359
NM_021105
PLSCR1
−1.81
−1.73
0.665
0.69
0.96


57048
NM_020360
PLSCR3
−1.83
−1.14
0.660
0.80
0.82


5373
NM_000303
PMM2
−3.21
−2.03
0.594
0.60
1.00


5395
NM_000535
PMS2
−1.83
−1.79
0.735
0.74
0.99


25953
NM_015488
PNKD
−1.72
−1.63
0.669
0.73
0.92


11284
NM_007254
PNKP
−1.56
−1.68
0.688
0.73
0.95


5430
NM_000937
POLR2A
−5.91
−5.55
0.188
0.13
1.46


5432
NM_002694
POLR2C
−2.85
−2.66
0.523
0.61
0.86


5433
NM_004805
POLR2D
−5.28
−2.62
0.332
0.48
0.69


5438
NM_006233
POLR2I
−2.04
−1.29
0.656
0.81
0.81


5439
NM_006234
POLR2J
−2.71
−3.22
0.544
0.52
1.04


246721
NM_032958
POLR2J2
−2.01
−2.41
0.684
0.60
1.14


5441
NM_021128
POLR2L
−1.95
−1.19
0.672
0.83
0.81


171568
NM_138338
POLR3H
−2.16
−2.14
0.624
0.66
0.95


10940
NM_015029
POP1
−4.24
−3.02
0.390
0.50
0.78


5456
NM_000307
POU3F4
−2.32
−1.82
0.713
0.65
1.10


5498
NM_000309
PPOX
−2.04
−1.50
0.741
0.70
1.06


23645
NM_014330
PPP1R15A
−2.20
−1.48
0.698
0.77
0.91


5534
NM_000945
PPP3R1
−2.89
−1.67
0.603
0.74
0.82


343070
XM_291638
PRAMEF9
−2.37
−1.86
0.613
0.73
0.84


59336
NM_021620
PRDM13
−2.48
−2.63
0.524
0.57
0.93


56978
NM_020226
PRDM8
−1.63
−1.79
0.687
0.70
0.98


5550
NM_002726
PREP
−1.97
−1.68
0.668
0.75
0.89


5553
NM_002728
PRG2
−2.27
−2.72
0.617
0.60
1.03


166336
NM_198859
PRICKLE2
−2.13
−2.68
0.632
0.58
1.09


5564
NM_006253
PRKAB1
−2.19
−2.65
0.580
0.56
1.03


5565
NM_005399
PRKAB2
−1.80
−1.41
0.656
0.77
0.86


5567
NM_002731
PRKACB
−1.80
−0.95
0.639
0.84
0.76


53632
NM_017431
PRKAG3
−1.52
−1.29
0.702
0.79
0.89


5617
NM_000948
PRL
−2.06
−1.25
0.741
0.80
0.92


23627
NM_012409
PRND
−2.02
−2.26
0.612
0.63
0.98


5625
NM_016335
PRODH
−2.35
−2.40
0.702
0.52
1.34


58510
NM_021232
PRODH2
−3.37
−1.97
0.574
0.61
0.94


8559
NM_003675
PRPF18
−2.48
−1.27
0.652
0.82
0.80


27339
NM_014502
PRPF19
−1.79
−2.09
0.682
0.68
1.01


26121
NM_015629
PRPF31
−1.88
−1.33
0.639
0.78
0.82


5698
NM_002800
PSMB9
−2.94
−1.80
0.627
0.64
0.98


5714
NM_002812
PSMD8
−4.26
−4.07
0.182
0.33
0.55


5740
NM_000961
PTGIS
−2.23
−0.75
0.717
0.85
0.84


5757
NM_002823
PTMA
−2.62
−2.15
0.571
0.63
0.91


5805
NM_000317
PTS
−1.50
−0.92
0.738
0.84
0.88


9543
NM_004884
PUNC
−2.25
−0.91
0.710
0.86
0.82


84074
NM_032134
QRICH2
−1.67
−2.79
0.686
0.55
1.26


5768
NM_002826
QSCN6
−2.59
−2.10
0.570
0.63
0.90


26056
NM_015470
RAB11FIP5
−1.64
−1.48
0.741
0.76
0.97


9609
NM_004914
RAB36
−1.95
−1.94
0.747
0.71
1.05


338382
NM_177403
RAB7B
−1.84
−1.17
0.699
0.83
0.84


4218
NM_005370
RAB8A
−1.62
−1.88
0.689
0.69
1.00


5876
NM_004582
RABGGTB
−2.34
−1.78
0.704
0.65
1.09


5885
NM_006265
RAD21
−1.83
−1.09
0.744
0.85
0.88


5888
NM_002875
RAD51
−1.87
−2.50
0.722
0.59
1.22


10411
NM_006105
RAPGEF3
−2.07
−1.79
0.696
0.76
0.92


9462
NM_004841
RASAL2
−2.61
−2.66
0.661
0.60
1.09


10235
NM_005825
RASGRP2
−4.38
−2.56
0.419
0.50
0.84


8045
NM_003475
RASSF7
−1.74
−2.48
0.722
0.62
1.16


10741
NM_006606
RBBP9
−4.88
−3.74
0.190
0.35
0.54


79171
NM_024321
RBM42
−1.73
−1.83
0.711
0.71
1.00


27303
NM_014483
RBMS3
−1.55
−1.60
0.733
0.72
1.02


5950
NM_006744
RBP4
−2.25
−1.67
0.627
0.71
0.88


9978
NM_014248
RBX1
−1.94
−1.42
0.715
0.77
0.93


9986
NM_005133
RCE1
−2.36
−1.98
0.700
0.61
1.16


57333
NM_020650
RCN3
−2.49
−3.01
0.596
0.53
1.12


25807
NM_012265
RHBDD3
−1.74
−1.10
0.726
0.82
0.89


57127
NM_020407
RHBG
−1.83
−1.13
0.699
0.83
0.85


391
NM_001665
RHOG
−1.61
−0.56
0.738
0.91
0.81


171177
NM_133639
RHOV
−1.52
−1.86
0.716
0.67
1.07


83732
NM_031480
RIOK1
−2.26
−1.70
0.575
0.76
0.76


84659
NM_032572
RNASE7
−1.87
−1.61
0.691
0.71
0.97


140432
NM_178861
RNF113B
−1.76
−1.42
0.643
0.78
0.83


79845
NM_024787
RNF122
−2.03
−1.15
0.652
0.82
0.79


158763
NM_144967
RP13-102H20.1
−2.11
−3.99
0.673
0.37
1.84


6134
NM_006013
RPL10
−1.58
−2.08
0.736
0.64
1.15


4736
NM_007104
RPL10A
−1.51
−2.41
0.745
0.65
1.15


11224
NM_007209
RPL35
−1.83
−1.69
0.701
0.73
0.96


6125
NM_000969
RPL5
−1.62
−2.14
0.734
0.63
1.17


6217
NM_001020
RPS16
−1.63
−1.69
0.731
0.71
1.03


6187
NM_002952
RPS2
−2.72
−1.81
0.676
0.70
0.96


6227
NM_001024
RPS21
−3.01
−1.89
0.641
0.69
0.93


6188
NM_001005
RPS3
−2.44
−1.62
0.710
0.73
0.97


6193
NM_001009
RPS5
−2.80
−2.12
0.666
0.65
1.02


6196
NM_021135
RPS6KA2
−1.59
−1.25
0.720
0.82
0.87


3921
NM_002295
RPSA
−2.47
−2.35
0.654
0.62
1.06


64121
NM_022157
RRAGC
−1.79
−1.16
0.701
0.80
0.87


6240
NM_001033
RRM1
−5.53
−3.82
0.299
0.24
1.24


6241
NM_001034
RRM2
−6.12
−4.07
0.226
0.19
1.18


54700
NM_018427
RRN3
−2.99
−1.54
0.705
0.72
0.98


23212
NM_015169
RRS1
−1.77
−2.08
0.720
0.67
1.08


349667
NM_178570
RTN4RL2
−2.87
−1.66
0.596
0.73
0.82


860
NM_004348
RUNX2
−2.50
−1.01
0.694
0.81
0.86


113174
NM_138421
SAAL1
−1.86
−1.48
0.623
0.78
0.80


6299
NM_002968
SALL1
−2.70
−2.24
0.626
0.68
0.93


9092
NM_005146
SART1
−1.70
−0.64
0.708
0.89
0.80


6307
NM_006745
SC4MOL
−2.57
−1.79
0.674
0.64
1.05


113178
NM_079834
SCAMP4
−2.18
−1.71
0.559
0.74
0.75


92344
NM_152281
SCYL1BP1
−1.65
−1.53
0.688
0.78
0.88


6392
NM_003002
SDHD
−2.06
−1.21
0.642
0.79
0.81


284904
NM_174977
SEC14L4
−1.54
−1.82
0.719
0.74
0.97


81929
NM_031216
SEH1L
−1.77
−1.12
0.673
0.80
0.84


22929
NM_012247
SEPHS1
−1.51
−0.68
0.731
0.90
0.81


871
NM_001235
SERPINH1
−2.63
−0.82
0.651
0.84
0.78


5274
NM_005025
SERPINI1
−2.31
−2.56
0.702
0.62
1.13


54093
NM_017438
SETD4
−2.82
−2.84
0.517
0.61
0.85


387893
NM_020382
SETD8
−1.57
−1.54
0.708
0.73
0.97


387893
NM_020382
SETD8
−2.15
−1.49
0.728
0.70
1.03


124925
NM_178860
SEZ6
−2.75
−2.58
0.507
0.63
0.80


10291
NM_005877
SF3A1
−2.45
−2.31
0.657
0.67
0.99


10946
NM_006802
SF3A3
−3.21
−2.27
0.551
0.67
0.82


23451
NM_012433
SF3B1
−2.80
−3.80
0.561
0.39
1.43


10992
XM_290506
SF3B2
−2.97
−3.05
0.572
0.50
1.15


23450
NM_012426
SF3B3
−2.68
−3.02
0.580
0.52
1.13


83443
NM_031287
SF3B5
−2.85
−2.42
0.481
0.56
0.85


6421
NM_005066
SFPQ
−3.20
−2.15
0.554
0.69
0.80


6428
NM_003017
SFRS3
−2.44
−1.02
0.657
0.85
0.77


6433
NM_152235
SFRS8
−2.02
−0.27
0.716
0.95
0.75


118980
NM_178858
SFXN2
−2.36
−2.38
0.577
0.67
0.86


151648
NM_138484
SGOL1
−2.79
−3.45
0.567
0.46
1.24


10044
NM_005489
SH2D3C
−3.26
−3.20
0.579
0.52
1.10


79628
NM_024577
SH3TC2
−1.87
−1.78
0.673
0.73
0.93


25942
NM_015477
SIN3A
−2.22
−1.74
0.679
0.75
0.91


140885
NM_080792
SIRPA
−1.58
−0.36
0.737
0.94
0.79


284759
XM_209363
SIRPB2
−2.09
−2.32
0.620
0.67
0.93


51804
NM_017420
SIX4
−2.08
−1.56
0.746
0.70
1.07


147912
NM_175875
SIX5
−1.60
−1.10
0.716
0.84
0.85


4990
NM_007374
SIX6
−2.27
−1.42
0.722
0.72
1.00


7884
NM_006527
SLBP
−3.33
−2.00
0.536
0.71
0.75


10723
NM_006598
SLC12A7
−3.67
−2.99
0.473
0.51
0.93


117247
NM_018593
SLC16A10
−1.99
−3.01
0.642
0.58
1.11


123041
NM_153646
SLC24A4
−2.17
−2.11
0.615
0.71
0.87


10166
NM_014252
SLC25A15
−1.52
−2.07
0.719
0.63
1.14


115286
NM_173471
SLC25A26
−2.52
−2.99
0.522
0.57
0.91


64924
NM_022902
SLC30A5
−1.56
−1.73
0.739
0.72
1.02


10463
NM_006345
SLC30A9
−1.93
−2.12
0.536
0.65
0.82


285641
NM_181774
SLC36A3
−1.92
−1.43
0.653
0.79
0.82


29986
NM_014579
SLC39A2
−1.68
−1.73
0.710
0.73
0.97


283375
NM_173596
SLC39A5
−2.26
−1.71
0.506
0.65
0.78


23446
NM_022109
SLC44A1
−2.56
−2.65
0.599
0.58
1.04


85414
NM_033102
SLC45A3
−1.99
−2.12
0.674
0.61
1.10


11309
NM_007256
SLCO2B1
−1.99
−1.67
0.714
0.73
0.98


6603
NM_003077
SMARCD2
−2.09
−0.28
0.740
0.94
0.79


23049
NM_014006
SMG1
−1.75
−2.13
0.685
0.70
0.98


6606
NM_000344
SMN1
−2.54
−1.74
0.662
0.66
1.01


6607
NM_017411
SMN2
−1.65
−1.57
0.732
0.73
1.01


55234
NM_018225
SMU1
−3.35
−3.96
0.208
0.40
0.52


80725
NM_025248
SNIP
−1.64
−1.09
0.699
0.81
0.87


51429
NM_016224
SNX9
−2.56
−1.33
0.646
0.78
0.83


8435
NM_003578
SOAT2
−2.47
−2.23
0.688
0.56
1.23


6651
NM_003103
SON
−5.33
−3.44
0.253
0.51
0.50


10615
NM_006461
SPAG5
−1.81
−1.73
0.572
0.72
0.80


83893
NM_031955
SPATA16
−1.96
−1.47
0.627
0.76
0.82


219938
NM_174927
SPATA19
−1.85
−1.75
0.708
0.70
1.01


84651
NM_032566
SPINK7
−1.65
−1.58
0.729
0.71
1.02


81848
NM_030964
SPRY4
−1.68
−1.63
0.690
0.70
0.99


136853
NM_080744
SRCRB4D
−2.36
−2.26
0.695
0.66
1.05


253017
XM_171068
SRD5A2L2
−2.28
−1.31
0.639
0.78
0.82


6756
NM_005635
SSX1
−2.26
−0.88
0.723
0.83
0.87


57559
NM_020799
STAMBPL1
−1.78
−0.89
0.670
0.84
0.80


134429
NM_139164
STARD4
−2.01
−1.44
0.597
0.78
0.77


80765
NM_030574
STARD5
−1.69
−1.50
0.686
0.74
0.93


10273
NM_005861
STUB1
−2.00
−0.64
0.699
0.90
0.78


6804
NM_004603
STX1A
−2.18
−0.72
0.732
0.88
0.83


56670
NM_033050
SUCNR1
−1.51
−1.94
0.724
0.70
1.03


6829
NM_003169
SUPT5H
−2.87
−2.40
0.527
0.58
0.90


6830
NM_003170
SUPT6H
−2.94
−2.08
0.619
0.69
0.90


9412
NM_004264
SURB7
−2.22
−2.07
0.714
0.69
1.03


136306
NM_174959
SVOPL
−2.43
−2.66
0.522
0.58
0.89


25949
NM_015484
SYF2
−2.96
−3.00
0.535
0.52
1.04


54843
NM_032379
SYTL2
−2.23
−1.98
0.634
0.73
0.87


10460
NM_006342
TACC3
−2.06
−0.78
0.663
0.86
0.77


11138
NM_007063
TBC1D8
−2.10
−1.87
0.698
0.69
1.01


90665
NM_033284
TBL1Y
−2.12
−2.38
0.571
0.65
0.87


6911
NM_004608
TBX6
−2.44
−1.22
0.697
0.77
0.91


8557
NM_003673
TCAP
−3.41
−1.87
0.591
0.69
0.85


200132
NM_152665
TCTEX1D1
−1.88
−1.83
0.673
0.72
0.94


7011
NM_007110
TEP1
−1.83
−1.75
0.749
0.72
1.03


10227
NM_001120
TETRAN
−1.55
−1.25
0.626
0.80
0.78


56159
NM_031276
TEX11
−1.72
−1.31
0.721
0.80
0.91


7018
NM_001063
TP
−1.90
−2.37
0.730
0.66
1.11


7069
NM_003251
THRSP
−1.60
−0.53
0.744
0.92
0.81


10333
NM_006068
TLR6
−1.68
−1.28
0.653
0.82
0.80


117532
NM_080751
TMC2
−1.55
−1.34
0.714
0.81
0.88


23671
NM_016192
TMEFF2
−1.70
−1.50
0.733
0.76
0.97


200728
NM_198276
TMEM17
−2.02
−1.07
0.646
0.82
0.79


80775
NM_030577
TMEM177
−2.49
−1.63
0.542
0.72
0.75


199964
NM_182532
TMEM61
−1.97
−1.35
0.660
0.78
0.84


137835
NM_144649
TMEM71
−1.80
−1.16
0.650
0.83
0.78


388730
NM_203376
TMEM81
−1.91
−1.25
0.565
0.74
0.76


388364
NM_206832
TMIGD1
−2.12
−2.28
0.610
0.60
1.01


28983
NM_014058
TMPRSS11E
−1.87
−0.86
0.659
0.84
0.78


23495
NM_012452
TNFRSF13B
−2.22
−1.33
0.688
0.78
0.88


8711
NM_003985
TNK1
−1.64
−1.81
0.682
0.74
0.92


85456
NM_033396
TNKS1BP1
−2.17
−1.33
0.643
0.75
0.86


7134
NM_003280
TNNC1
−2.35
−1.45
0.688
0.72
0.96


30000
NM_013433
TNPO2
−3.20
−3.50
0.489
0.46
1.06


26058
NM_015575
TNRC15
−1.63
−1.57
0.744
0.76
0.98


57690
NM_018996
TNRC6C
−2.03
−1.25
0.656
0.78
0.84


7153
NM_001067
TOP2A
−2.28
−3.06
0.635
0.53
1.20


7163
NM_005079
TPD52
−2.30
−1.61
0.629
0.75
0.84


93492
NM_130785
TPTE2
−1.68
−1.09
0.741
0.82
0.90


22974
NM_012112
TPX2
−5.55
−3.99
0.202
0.34
0.59


11139
NM_007064
TRAD
−1.84
−1.79
0.632
0.74
0.85


7185
NM_005658
TRAF1
−1.80
−1.64
0.713
0.75
0.95


84231
NM_032271
TRAF7
−1.97
−1.44
0.624
0.76
0.82


58485
NM_021210
TRAPPC1
−1.60
−1.48
0.734
0.75
0.98


54209
NM_018965
TREM2
−2.49
−3.33
0.653
0.46
1.43


80128
NM_025058
TRIM46
−1.58
−1.15
0.709
0.79
0.89


84676
NM_032588
TRIM63
−1.87
−1.65
0.693
0.70
0.99


81786
NM_033342
TRIM7
−1.64
−1.82
0.696
0.67
1.04


54802
NM_017646
TRIT1
−1.75
−0.52
0.714
0.93
0.77


54822
NM_017672
TRPM7
−1.63
−0.38
0.711
0.94
0.75


29122
NM_013270
TSP50
−1.94
−1.42
0.658
0.78
0.84


26262
NM_130465
TSPAN17
−2.78
−2.50
0.558
0.62
0.90


84630
XM_166453
TTBK1
−1.86
−1.66
0.669
0.76
0.87


54902
NM_017775
TTC19
−1.85
−1.20
0.746
0.81
0.92


83538
NM_031421
TTC25
−1.65
−1.82
0.690
0.70
0.98


7272
NM_003318
TTK
−1.78
−2.34
0.670
0.67
1.00


26140
NM_015644
TTLL3
−2.13
−2.96
0.663
0.52
1.27


57348
NM_020659
TTYH1
−1.58
−1.89
0.742
0.71
1.05


10376
NM_006082
TUBA1B
−2.55
−1.42
0.650
0.78
0.84


84790
NM_032704
TUBA1C
−2.60
−1.83
0.575
0.67
0.86


7283
NM_001070
TUBG1
−2.08
−0.55
0.714
0.91
0.78


11338
NM_007279
U2AF2
−2.96
−1.88
0.576
0.69
0.83


7311
NM_003333
UBA52
−2.72
−2.01
0.675
0.67
1.01


51271
NM_016525
UBAP1
−1.66
−0.46
0.733
0.93
0.79


9040
NM_003969
UBE2M
−1.80
−1.42
0.709
0.78
0.91


54658
NM_000463
UGT1A1
−1.63
−1.77
0.739
0.72
1.02


54579
NM_019078
UGT1A5
−1.59
−1.19
0.716
0.84
0.86


51720
NM_016290
UIMC1
−1.98
−1.19
0.725
0.80
0.90


90249
XM_030300
UNC5A
−2.03
−2.14
0.590
0.68
0.86


23353
NM_025154
UNC84A
−2.07
−1.88
0.676
0.70
0.97


85451
XM_036115
UNK
−1.55
−0.56
0.745
0.89
0.83


388325
NM_207103
UNQ5783
−1.95
−1.26
0.636
0.78
0.82


139596
NM_145052
UPRT
−1.56
−1.92
0.696
0.71
0.98


27089
NM_014402
UQCRQ
−2.12
−2.02
0.624
0.69
0.91


9097
NM_005151
USP14
−1.83
−0.67
0.719
0.89
0.80


373856
XM_036729
USP41
−1.58
−2.22
0.710
0.60
1.18


9712
NM_014688
USP6NL
−1.99
−3.76
0.557
0.31
1.79


10208
NM_005800
USPL1
−2.20
−1.42
0.672
0.77
0.88


9686
NM_014667
VGLL4
−1.69
−2.30
0.617
0.57
1.08


7431
NM_003380
VIM
−2.39
−1.95
0.713
0.68
1.05


8876
NM_004666
VNN1
−1.67
−0.42
0.732
0.94
0.78


155382
XM_376631
VPS37D
−1.84
−0.95
0.717
0.84
0.85


11193
NM_007187
WBP4
−1.91
−0.83
0.724
0.86
0.84


23001
NM_014991
WDFY3
−2.12
−2.04
0.660
0.67
0.98


11169
NM_007086
WDHD1
−2.17
−2.19
0.691
0.64
1.08


91833
NM_144574
WDR20
−1.59
−1.08
0.696
0.85
0.82


79819
NM_024763
WDR78
−2.50
−2.83
0.564
0.56
1.00


7465
NM_003390
WEE1
−3.71
−5.41
0.316
0.23
1.37


164237
NM_172005
WFDC13
−1.53
−0.56
0.735
0.91
0.81


140686
NM_181522
WFDC3
−1.56
−1.00
0.684
0.83
0.82


90199
NM_130896
WFDC8
−2.52
−2.56
0.494
0.62
0.79


147179
NM_133264
WIPF2
−2.23
−2.43
0.604
0.64
0.94


7478
NM_031933
WNT8A
−2.09
−2.53
0.708
0.59
1.20


56949
NM_020196
XAB2
−4.01
−3.62
0.349
0.44
0.80


170626
NM_130776
XAGE3
−2.48
−2.76
0.570
0.56
1.01


7512
NM_003399
XPNPEP2
−2.25
−1.68
0.692
0.74
0.94


2547
NM_001469
XRCC6
−2.21
−1.88
0.697
0.71
0.99


26137
NM_015642
ZBTB20
−2.12
−1.74
0.665
0.72
0.92


360023
NM_194314
ZBTB41
−1.55
−1.79
0.719
0.71
1.02


84186
NM_032226
ZCCHC7
−1.91
−1.27
0.640
0.79
0.81


55146
NM_018106
ZDHHC4
−1.61
−1.49
0.630
0.77
0.82


60685
NM_021943
ZFAND3
−1.81
−0.95
0.695
0.84
0.83


153527
NM_144723
ZMAT2
−1.79
−0.40
0.724
0.94
0.77


118490
NM_178451
ZMYND17
−1.55
−0.50
0.717
0.93
0.77


10778
NM_006629
ZNF271
−1.79
−1.50
0.744
0.75
0.99


92822
NM_152287
ZNF276
−2.13
−2.77
0.598
0.60
1.00


91975
NM_052860
ZNF300
−1.81
−2.22
0.650
0.68
0.95


57343
NM_020657
ZNF304
−2.46
−2.14
0.600
0.67
0.90


162967
XM_371190
ZNF320
−1.81
−1.18
0.684
0.81
0.85


79893
NM_024835
ZNF403
−1.66
−2.55
0.713
0.61
1.18


126070
NM_152357
ZNF440
−1.64
−2.02
0.704
0.72
0.98


168544
XM_095168
ZNF467
−1.61
−2.10
0.722
0.67
1.08


197407
NM_152652
ZNF553
−1.75
−1.73
0.693
0.72
0.96


147837
NM_145276
ZNF563
−1.55
−0.93
0.724
0.86
0.84


201514
NM_173548
ZNF584
−2.15
−1.61
0.659
0.74
0.90


169270
NM_173539
ZNF596
−1.51
−0.60
0.737
0.89
0.82


80095
NM_025027
ZNF606
−1.85
−1.12
0.656
0.80
0.82


23352
NM_020765
ZUBR1
−1.93
−0.63
0.692
0.90
0.77
















TABLE 2A







Highly Selective siRNAs (SEQ ID NOs: 1-4, 6, 7, 13, 14, 23, 29, 33-35, 37, 38, 66, 72,


77, 78, 84, 91, 93, and 151)


















Entrez










Cher-


Gene

Gene

MAD
MAD
Fold




ry


ID
Accession #
Symbol
Catalog #
BPLER
HMLER
BPLER
Fold_HMLER
BPLER_RSD
HMLER_RSD
Ratio
Score





















5692
NM_002796
PSMB4
M-011362-00
−6.73
−2.41
0.147
0.52
0.07
0.13
0.28
4


5682
NM_002786
PSMA1
M-010123-01
−5.53
−2.21
0.240
0.65
0.06
0.07
0.37
4


29127
NM_013277
RACGAP1
M-008650-00
−3.61
−1.46
0.361
0.77
0.14
0.08
0.47
4


5702
NM_002804
PSMC3
M-008738-01
−1.77
−1.02
0.399
0.81
0.10
0.07
0.49
4


1659
NM_004941
DHX8
M-010506-01
−4.27
−1.24
0.413
0.81
0.05
0.05
0.51
4


5684
NM_002788
PSMA3
M-011758-00
−1.72
−0.54
0.434
0.90
0.17
0.09
0.48
4


11269
NM_007242
DDX19B
M-013471-00
−4.84
−2.09
0.304
0.66
0.07
0.06
0.46
3


22938
NM_012245
SNW1
M-012446-00
−5.45
−1.31
0.335
0.75
0.10
0.08
0.45
3


79441
NM_024511
C4orf15
M-018131-00
−2.52
0.55
0.560
1.09
0.09
0.03
0.52
3


5683
NM_002787
PSMA2
M-011757-00
−4.14
−2.79
0.205
0.54
0.15
0.01
0.38
2


10594
NM_006445
PRPF8
M-012252-01
−5.34
−2.64
0.246
0.62
0.22
0.04
0.40
2


5713
NM_002811
PSMD7
M-009621-01
−3.88
−2.86
0.256
0.53
0.08
0.02
0.49
2


5901
NM_006325
RAN
M-010353-00
−5.67
−2.89
0.266
0.57
0.06
0.10
0.47
2


59286
NM_024292
UBL5
M-014320-00
−4.14
−2.33
0.337
0.64
0.11
0.01
0.53
2


5515
NM_002715
PPP2CA
M-003598-00
−2.77
−0.87
0.457
0.86
0.07
0.09
0.53
2


5700
NM_002802
PSMC1
M-009578-00
−1.50
−0.45
0.476
0.91
0.19
0.04
0.52
2


57474
NM_020714
ZNF490
M-013937-00
−4.40
−2.66
0.285
0.59
0.08
0.03
0.49
1


147015
NM_144683
DHRS13
M-008777-00
−3.66
−1.69
0.350
0.75
0.04
0.07
0.47
1


64763
NM_022752
ZNF574
M-007054-00
−3.52
−1.41
0.425
0.79
0.15
0.11
0.54
1


91869
NM_052859
RFT1
M-018174-00
−2.82
0.04
0.468
1.01
0.12
0.05
0.47
1


65243
NM_023070
ZNF643
M-007056-00
−3.05
−0.52
0.493
0.92
0.14
0.03
0.54
1


84922
NM_032836
FIZ1
M-015014-00
−2.97
−0.32
0.514
0.94
0.07
0.05
0.55
1


23474
NM_014297
ETHE1
M-012508-00
−2.19
1.03
0.641
1.17
0.25
0.05
0.55
1


80863
NM_030651
PRRT1
M-016655-00
−3.57
−2.00
0.348
0.64
0.18
0.10
0.54
0


2850
NM_018971
GPR27
M-005562-01
−3.35
−1.23
0.385
0.81
0.05
0.15
0.47
0


9503
NM_020411
XAGE1
M-013187-00
−2.09
0.53
0.541
1.11
0.09
0.17
0.49
0
















TABLE 2B





Moderately Selective siRNAs





















Entrez

Gene

MAD
MAD



Gene ID
Accession #
Symbol
Catalog #
BPLER
HMLER
Fold_BPLER





84950
NM_032864
PRPF38A
M-014833-00
−3.64
−1.70
0.403


5431
NM_000938
POLR2B
M-011187-00
−4.09
−1.97
0.438


115106
NM_138443
CCDC5
M-015491-00
−2.18
−0.02
0.583


11157
NM_007080
LSM6
M-019754-00
−2.83
−0.41
0.593


5708
NM_002808
PSMD2
M-017212-01
−4.64
−2.67
0.343


10213
NM_005805
PSMD14
M-006024-00
−3.37
−2.28
0.352


10403
NM_006101
NDC80
M-004106-00
−3.24
−2.06
0.377


79680
NM_024627
C22orf29
M-016338-00
−3.47
−2.46
0.400


24148
NM_012469
PRPF6
M-012821-00
−3.13
−1.23
0.508


57819
NM_021177
LSM2
M-017813-00
−2.90
−0.95
0.512


9861
NM_014814
PSMD6
M-021249-00
−2.05
−0.79
0.516


91746
NM_133370
YTHDC1
M-015332-00
−2.31
−0.26
0.567


57461
NM_020701
ISY1
M-013894-00
−4.27
−2.95
0.306


55696
NM_018047
RBM22
M-021186-00
−2.37
−1.53
0.477


151903
NM_144716
CCDC12
M-015455-00
−3.22
−1.34
0.502


83540
NM_031423
NUF2
M-005289-01
−2.68
−0.90
0.515


26036
NM_015555
ZNF451
M-013935-00
−3.07
−0.77
0.516


1213
NM_004859
CLTC
M-004001-00
−2.02
0.05
0.570


2597
NM_002046
GAPDH
M-004253-01
−4.30
−0.19
0.576


5211
NM_002626
PFKL
M-006822-00
−1.85
−0.10
0.630


55735
NM_018198
DNAJC11
M-021205-00
−1.52
0.75
0.656


57835
NM_021196
SLC4A5
M-007585-00
−1.94
0.63
0.672


388531
NM_207391
RGS9BP
M-032069-00
−1.58
0.53
0.707


154007
NM_152551
C6orf151
M-018855-00
−1.75
1.64
0.730


1315
NM_016451
COPB1
M-017940-00
−3.13
−2.91
0.326


3190
NM_002140
HNRPK
M-011692-00
−3.35
−2.71
0.358


10114
NM_005734
HIPK3
M-004810-00
−3.97
−2.34
0.418


11325
NM_007372
DDX42
M-012393-00
−4.00
−1.78
0.425


90407
NM_080652
TMEM41A
M-015245-00
−2.70
−1.83
0.457


9821
NM_014781
RB1CC1
M-021117-00
−3.21
−1.50
0.466


51477
NM_016368
ISYNA1
M-009669-00
−1.95
−0.82
0.489


286826
NM_173083
LIN9
M-018918-00
−2.90
−0.72
0.525


84975
NM_032889
MFSD5
M-018634-00
−2.81
−0.38
0.540


79622
NM_024571
C16orf33
M-014370-00
−2.67
−0.58
0.540


6457
NM_003027
SH3GL3
M-015728-00
−2.67
−0.55
0.544


5128
NM_002595
PCTK2
M-004835-01
−2.29
−0.79
0.552


29945
NM_013367
ANAPC4
M-013642-00
−2.96
−0.79
0.557


64236
NM_021630
PDLIM2
M-010731-00
−2.65
−0.34
0.563


27316
NM_002139
RBMX
M-011691-00
−2.43
−0.57
0.569


56931
NM_020175
DUS3L
M-031942-00
−2.65
−0.71
0.571


51473
NM_016356
DCDC2
M-020868-00
−1.61
−0.02
0.571


55957
NM_019104
LIN37
M-013311-00
−1.92
−0.19
0.571


4247
NM_002408
MGAT2
M-011333-00
−2.06
−0.19
0.586


115361
NM_052941
GBP4
M-018177-00
−2.09
−0.14
0.608


8291
NM_003494
DYSF
M-003652-01
−2.13
0.64
0.614


57456
NM_020696
KIAA1143
M-013876-00
−2.27
0.86
0.631


255104
NM_181719
TMCO4
M-018952-00
−1.74
−0.01
0.638


3763
NM_002240
KCNJ6
M-006251-00
−1.88
0.12
0.654


8621
NM_003718
CDC2L5
M-004688-00
−2.34
0.95
0.656


89777
NM_080474
SERPINB12
M-008758-00
−1.66
0.63
0.668


26994
NM_014372
RNF11
M-006971-00
−1.86
1.11
0.671


91978
NM_033513
C19orf20
M-023892-00
−1.69
0.87
0.684


192134
NM_138706
B3GNT6
M-016388-00
−1.80
0.45
0.691


154075
NM_152552
SAMD3
M-015449-00
−1.99
0.68
0.692


2838
NM_005290
GPR15
M-005550-00
−1.58
0.72
0.708


79443
NM_024513
FYCO1
M-014350-00
−1.67
0.86
0.714


150946
XM_097977
FAM59B
M-022647-00
−1.85
0.68
0.714


59269
NM_024503
HIVEP3
M-014345-00
−5.23
−2.35
0.306


554
NM_000054
AVPR2
M-005432-00
−3.26
−2.16
0.401


57621
NM_020861
ZBTB2
M-014129-00
−3.50
−1.59
0.412


57701
XM_035497
KIAA1602
M-026953-00
−3.29
−1.54
0.446


148930
NM_182516
L5
M-018573-00
−2.89
−1.47
0.486


285676
NM_182594
ZNF454
M-018860-00
−2.78
−0.94
0.494


1633
NM_000788
DCK
M-006710-00
−3.39
−1.32
0.502


8335
NM_003513
HIST1H2AB
M-017596-00
−1.78
−1.23
0.504


84458
XM_050988
LCOR
M-026303-00
−2.98
−0.81
0.511


11281
NM_007252
POU6F2
M-019645-00
−3.27
−0.75
0.529


8653
NM_004660
DDX3Y
M-011904-00
−1.66
−1.17
0.537


1175
NM_004069
AP2S1
M-011833-00
−1.99
−0.34
0.562


83787
NM_031905
ARMC10
M-018188-00
−2.13
0.45
0.596


9085
NM_004680
CDY1
M-008916-00
−1.86
−0.49
0.599


285888
XM_376727
CNPY1
M-028750-00
−2.15
0.40
0.612


117178
NM_014021
SSX2IP
M-020361-00
−2.15
−0.09
0.615


1769
NM_001371
DNAH8
M-010075-00
−1.78
−0.24
0.621


149954
NM_182519
C20orf186
M-009125-00
−1.71
1.00
0.695


167838
XM_371849
TXLNB
M-024703-00
−1.58
1.06
0.724

















Entrez




Cherry



Gene ID
Fold_HMLER
BPLER_RSD
HMLER_RSD
Ratio
Score







84950
0.69
0.05
0.02
0.58
4



 5431
0.69
0.04
0.01
0.63
4



115106 
1.00
0.21
0.02
0.59
4



11157
0.93
0.02
0.08
0.64
4



 5708
0.61
0.16
0.04
0.56
3



10213
0.62
0.06
0.10
0.56
3



10403
0.66
0.07
0.02
0.57
3



79680
0.62
0.05
0.02
0.64
3



24148
0.81
0.20
0.13
0.62
3



57819
0.84
0.09
0.05
0.61
3



 9861
0.87
0.05
0.08
0.59
3



91746
0.96
0.19
0.06
0.59
3



57461
0.54
0.07
0.06
0.56
2



55696
0.78
0.08
0.03
0.61
2



151903 
0.78
0.14
0.15
0.64
2



83540
0.84
0.06
0.05
0.62
2



26036
0.86
0.19
0.17
0.60
2



 1213
1.01
0.05
0.00
0.57
2



 2597
0.97
0.11
0.09
0.60
2



 5211
0.98
0.17
0.02
0.64
2



55735
1.11
0.16
0.03
0.59
2



57835
1.11
0.21
0.06
0.61
2



388531 
1.10
0.07
0.06
0.64
2



154007 
1.26
0.10
0.02
0.58
2



 1315
0.54
0.24
0.09
0.61
1



 3190
0.55
0.12
0.08
0.65
1



10114
0.68
0.07
0.02
0.61
1



11325
0.71
0.04
0.04
0.60
1



90407
0.73
0.08
0.04
0.63
1



 9821
0.74
0.18
0.04
0.63
1



51477
0.87
0.18
0.07
0.57
1



286826 
0.90
0.08
0.07
0.59
1



84975
0.93
0.10
0.05
0.58
1



79622
0.91
0.07
0.03
0.59
1



 6457
0.90
0.15
0.03
0.60
1



 5128
0.87
0.05
0.06
0.64
1



29945
0.87
0.06
0.04
0.64
1



64236
0.95
0.10
0.03
0.60
1



27316
0.91
0.16
0.03
0.62
1



56931
0.89
0.08
0.02
0.64
1



51473
1.00
0.19
0.19
0.57
1



55957
0.97
0.21
0.02
0.59
1



 4247
0.97
0.02
0.07
0.60
1



115361 
0.99
0.15
0.15
0.62
1



 8291
1.12
0.11
0.08
0.55
1



57456
1.14
0.14
0.06
0.56
1



255104 
0.99
0.13
0.04
0.64
1



 3763
1.04
0.16
0.14
0.63
1



 8621
1.13
0.03
0.08
0.58
1



89777
1.09
0.07
0.04
0.61
1



26994
1.17
0.01
0.07
0.57
1



91978
1.13
0.09
0.04
0.61
1



192134 
1.07
0.17
0.07
0.65
1



154075 
1.11
0.09
0.06
0.62
1



 2838
1.11
0.09
0.03
0.64
1



79443
1.13
0.05
0.01
0.63
1



150946 
1.10
0.20
0.07
0.65
1



59269
0.54
0.10
0.05
0.57
0



 554
0.66
0.10
0.14
0.60
0



57621
0.73
0.09
0.02
0.56
0



57701
0.74
0.11
0.04
0.60
0



148930 
0.78
0.04
0.08
0.62
0



285676 
0.87
0.03
0.21
0.57
0



 1633
0.82
0.05
0.01
0.61
0



 8335
0.83
0.07
0.01
0.61
0



84458
0.85
0.10
0.03
0.60
0



11281
0.88
0.16
0.08
0.60
0



 8653
0.83
0.08
0.05
0.64
0



 1175
0.94
0.19
0.09
0.60
0



83787
1.07
0.12
0.06
0.56
0



 9085
0.93
0.04
0.12
0.64
0



285888 
1.06
0.05
0.08
0.58
0



117178 
0.98
0.01
0.04
0.63
0



 1769
0.96
0.14
0.01
0.65
0



149954 
1.15
0.11
0.02
0.61
0



167838 
1.16
0.14
0.13
0.62
0

















TABLE 2C





Modestly Selective siRNAs






















Gene





Accession #
Catalog #
Symbol
MAD_BPLER
MAD_HMLER
Fold_BPLER





NM_002696
M-011357-00
POLR2G
−3.59
−1.68
0.507


NM_018097
M-021161-00
CEP27
−1.99
−1.76
0.522


XM_058073
M-010646-00
NUP205
−2.88
−1.57
0.544


XM_375557
M-031204-00
C19orf29
−2.24
−0.36
0.625


NM_001253
M-011237-00
CDC5L
−1.60
−0.87
0.654


NM_006292
M-003549-01
TSG101
−2.45
0.23
0.684


NM_016937
M-020856-00
POLA1
−2.01
−0.17
0.724


NM_032916
M-015046-00
FAM86B1
−2.54
−2.05
0.491


NM_001237
M-003205-02
CCNA2
−2.79
−1.39
0.499


NM_016047
M-020260-00
SF3B14
−2.97
−2.06
0.500


NM_007364
M-008051-00
TMED3
−2.93
−1.28
0.537


NM_014347
M-006964-00
ZNF324
−2.91
−1.05
0.543


NM_007263
M-017632-00
COPE
−3.15
−1.43
0.547


NM_005063
M-005061-01
SCD
−3.24
−1.58
0.555


NM_032425
M-022058-00
KIAA1822
−2.67
−1.27
0.563


NM_020860
M-013166-00
STIM2
−2.39
−0.99
0.597


NM_018242
M-013347-00
SLC47A1
−1.62
−0.71
0.613


NM_004336
M-004102-00
BUB1
−2.62
−0.62
0.617


NM_152655
M-016432-00
ZNF585A
−2.06
−0.70
0.642


NM_182640
M-019184-00
MRPS9
−2.00
−0.45
0.669


NM_022830
M-014221-00
TUT1
−1.91
−0.07
0.680


XM_370928
M-022880-00
TBC1D24
−1.97
−0.32
0.680


NM_152374
M-016893-00
FLJ38984
−1.50
−0.23
0.693


NM_006590
M-006087-00
USP39
−2.13
−0.04
0.695


NM_016200
M-017030-00
LSM8
−1.76
−0.26
0.700


NM_003981
M-019491-00
PRC1
−3.08
−2.40
0.409


NM_020882
M-024094-00
COL20A1
−3.47
−2.58
0.416


NM_021974
M-004723-00
POLR2F
−3.46
−2.98
0.417


NM_018271
M-020368-00
FLJ10916
−2.39
−2.48
0.440


XM_378175
M-003993-01
LOC124446
−3.04
−2.49
0.458


NM_014456
M-004438-02
PDCD4
−2.81
−2.25
0.462


NM_152496
M-016951-00
MANEAL
−3.02
−2.41
0.464


NM_174931
M-017855-00
CCDC75
−3.33
−2.06
0.474


XM_371354
M-022129-00
KIF26B
−1.95
−1.15
0.536


NM_004739
M-008482-00
MTA2
−3.71
−1.07
0.545


NM_198080
M-021432-00
MSRB3
−2.72
−0.86
0.571


NM_000792
M-011170-00
DIO1
−1.96
−1.39
0.573


NM_013366
M-003200-02
ANAPC2
−2.80
−1.31
0.580


NM_001746
M-003636-02
CANX
−1.92
−1.38
0.587


NM_148571
M-013182-00
MRPL27
−1.57
−1.17
0.587


NM_002255
M-018983-00
KIR2DL4
−2.02
−0.98
0.593


NM_024762
M-014438-00
ZNF552
−2.35
−1.30
0.595


NM_052946
M-015170-00
NOSTRIN
−2.06
−0.69
0.604


NM_002636
M-011353-00
PHF1
−3.21
−0.72
0.606


NM_015046
M-021420-00
SETX
−2.40
−0.72
0.617


NM_006204
M-007653-00
PDE6C
−1.98
−0.33
0.621


NM_001225
M-004404-00
CASP4
−2.36
−0.92
0.623


NM_000172
M-009827-01
GNAT1
−2.03
−0.36
0.627


NM_002270
M-011308-00
TNPO1
−1.99
−0.59
0.630


NM_030969
M-015288-00
TMEM14B
−1.99
−0.56
0.636


NM_020689
M-007466-00
SLC24A3
−2.23
−0.75
0.637


NM_020395
M-015624-00
INTS12
−2.22
−0.74
0.639


NM_021640
M-013747-00
C12orf10
−2.11
−0.54
0.644


NM_014750
M-016846-00
DLG7
−1.93
−0.56
0.646


NM_194324
M-019295-00
MGC39900
−2.13
−0.87
0.647


NM_014420
M-020520-00
DKK4
−1.90
−0.72
0.657


NM_012202
M-012804-00
GNG3
−1.86
−0.62
0.658


NM_001797
M-013493-00
CDH11
−1.56
−0.69
0.663


NM_002513
M-006753-00
NME3
−1.71
0.12
0.668


NM_005954
M-012728-00
MT3
−1.77
−0.16
0.672


NM_000322
M-011102-00
PRPH2
−1.69
−0.56
0.675


NM_021960
M-004501-04
MCL1
−1.74
−0.13
0.679


NM_000131
M-005871-00
F7
−2.01
−0.06
0.679


NM_020380
M-015673-00
CASC5
−1.86
−0.56
0.686


NM_022304
M-005630-01
HRH2
−1.68
−0.39
0.693


NM_005003
M-019897-00
NDUFAB1
−1.82
−0.20
0.694


NM_032024
M-014765-00
C10orf11
−1.58
−0.06
0.702


NM_134444
M-015351-00
NLRP4
−1.68
0.11
0.702


XM_371074
M-030921-00
TANC2
−1.86
0.40
0.709


NM_080730
M-012988-00
HOM-TES-
−1.84
−0.09
0.711




103


NM_005713
M-012101-00
COL4A3BP
−1.96
0.03
0.715


NM_152905
M-008306-00
NEDD1
−1.56
0.59
0.721


NM_020528
M-013199-00
PCBP3
−1.69
0.15
0.727


NM_006156
M-020081-00
NEDD8
−1.79
0.25
0.736


NM_012141
M-012417-00
INTS6
−3.48
−2.65
0.369


NM_173626
M-007490-00
SLC26A11
−3.21
−2.50
0.415


NM_003301
M-005747-00
TRHR
−3.02
−2.31
0.423


NM_020809
M-026514-00
ARHGAP20
−3.36
−2.17
0.434


NM_004277
M-007483-00
SLC25A27
−2.81
−1.80
0.476


NM_016082
M-013297-01
CDK5RAP1
−3.57
−2.01
0.477


NM_030962
M-014684-00
SBF2
−2.84
−1.47
0.481


NM_001538
M-011295-00
HSF4
−4.01
−1.43
0.512


NM_173556
M-018459-00
CCDC83
−3.06
−1.36
0.518


XM_016548
M-025053-00
CDY2B
−3.04
−1.73
0.521


XM_056282
M-024572-00
LRRC62
−2.35
−1.74
0.528


NM_182703
M-019312-00
ANKDD1A
−2.87
−1.76
0.532


NM_005831
M-010637-00
CALCOCO2
−2.43
−1.58
0.533


NM_003526
M-011446-00
HIST1H2BC
−1.64
−1.61
0.541


NM_005911
M-008818-00
MAT2A
−2.25
−1.21
0.547


NM_022567
M-014165-00
NYX
−2.34
−0.97
0.550


NM_018360
M-020815-00
CXorf15
−2.01
−1.59
0.555


NM_182558
M-018912-00
C12orf36
−1.99
−0.99
0.556


NM_016388
M-020821-00
TRAT1
−2.24
−1.24
0.557


NM_006413
M-015336-00
RPP30
−1.84
−1.38
0.558


NM_024619
M-006817-00
FN3KRP
−2.98
−1.53
0.562


NM_001937
M-011641-00
DPT
−2.02
−1.01
0.565


XM_375187
M-022211-00
NUT
−1.98
−0.97
0.574


NM_178168
M-008750-00
OR10A5
−2.31
−1.21
0.576


NM_020320
M-013767-00
RARS2
−2.61
−1.37
0.576


NM_003685
M-009490-00
KHSRP
−1.93
−1.15
0.582


NM_152603
M-016451-00
ZNF567
−2.39
−1.09
0.585


XM_114430
M-024410-00
LOC202051
−2.59
−0.79
0.592


NM_198494
M-027243-00
ZNF642
−2.47
−0.63
0.594


NM_152465
M-016422-00
PROCA1
−2.24
−1.20
0.602


NM_152647
M-016412-00
C15orf33
−2.27
−0.50
0.604


NM_138451
M-015615-00
IQCD
−2.04
−1.20
0.605


NM_024011
M-004026-01
CDC2L2
−2.70
−0.55
0.605


NM_024647
M-018906-00
NUP43
−2.40
−1.30
0.605


NM_152515
M-018844-00
CKAP2L
−2.56
−1.17
0.605


NM_207401
M-032078-00
FLJ45717
−1.68
−0.82
0.611


NM_144702
M-015707-00
C1orf92
−2.17
−0.90
0.615


XM_371575
M-027984-00
PRPF40A
−1.75
−0.96
0.623


XM_209941
M-026255-00
C9orf117
−2.01
−0.84
0.635


NM_015446
M-013961-00
AHCTF1
−2.29
−0.69
0.635


NM_019067
M-015743-00
GNL3L
−2.24
−0.43
0.636


NM_018922
M-013292-00
PCDHGB1
−1.67
−0.81
0.637


NM_032866
M-019002-00
CGNL1
−2.22
−0.57
0.637


NM_213604
M-023895-00
ADAMTSL5
−2.21
−0.83
0.640


NM_018129
M-009715-00
PNPO
−1.55
−0.72
0.641


NM_002510
M-011741-00
GPNMB
−1.52
−0.76
0.641


NM_004783
M-004171-03
TAOK2
−1.82
−0.32
0.648


XM_089747
M-026312-00
C10orf80
−2.25
−0.73
0.653


NM_173821
M-018155-00
FLJ33590
−1.90
−0.56
0.656


NM_014891
M-017675-00
PDAP1
−1.78
−0.08
0.658


NM_005301
M-005567-00
GPR35
−1.85
−0.49
0.661


NM_021049
M-010800-00
MAGEA5
−1.68
−0.36
0.662


NM_002218
M-017838-00
ITIH4
−1.65
−0.33
0.668


NM_000188
M-006820-01
HK1
−2.21
−0.05
0.678


NM_015338
M-012856-00
ASXL1
−1.86
−0.39
0.680


NM_019070
M-017975-00
DDX49
−1.92
−0.33
0.683


NM_173853
M-017757-00
KRTCAP3
−1.83
−0.34
0.683


XM_373109
M-027171-00
RP11-
−1.93
0.07
0.686




145H9.1


NM_005544
M-003015-01
IRS1
−1.57
−0.47
0.695


NM_007205
M-032280-00
TREX2
−2.22
−0.39
0.697


NM_007358
M-012796-00
MTF2
−2.11
−0.40
0.699


NM_173497
M-007198-00
HECTD2
−1.66
−0.39
0.705


NM_003247
M-019745-00
THBS2
−2.48
−0.31
0.705


NM_173846
M-017754-00
TPPP2
−1.65
0.12
0.707


XM_030729
M-026260-00
FAM22F
−1.72
−0.23
0.714


NM_015690
M-005039-01
STK36
−1.55
0.46
0.718


NM_002082
M-004627-01
GRK6
−1.90
0.29
0.722


NM_004673
M-007804-01
ANGPTL1
−1.88
0.62
0.738


NM_182755
M-019384-00
ZNF438
−1.62
0.10
0.742






















Cherry



Accession #
Fold_HMLER
BPLER_RSD
HMLER_RSD
Ratio
Score







NM_002696
0.74
0.07
0.05
0.69
3



NM_018097
0.74
0.10
0.09
0.70
3



XM_058073
0.75
0.02
0.05
0.73
3



XM_375557
0.94
0.12
0.01
0.67
3



NM_001253
0.87
0.05
0.12
0.75
3



NM_006292
1.04
0.09
0.04
0.66
3



NM_016937
0.97
0.05
0.04
0.74
3



NM_032916
0.70
0.15
0.05
0.70
2



NM_001237
0.75
0.07
0.05
0.67
2



NM_016047
0.72
0.09
0.03
0.70
2



NM_007364
0.80
0.16
0.04
0.68
2



NM_014347
0.84
0.19
0.07
0.65
2



NM_007263
0.77
0.06
0.03
0.71
2



NM_005063
0.75
0.04
0.04
0.74
2



NM_032425
0.77
0.02
0.08
0.73
2



NM_020860
0.83
0.05
0.04
0.72
2



NM_018242
0.90
0.04
0.12
0.68
2



NM_004336
0.91
0.08
0.03
0.67
2



NM_152655
0.89
0.04
0.05
0.72
2



NM_182640
0.92
0.06
0.07
0.72
2



NM_022830
0.99
0.11
0.00
0.69
2



XM_370928
0.95
0.08
0.03
0.71
2



NM_152374
0.97
0.17
0.01
0.72
2



NM_006590
0.99
0.05
0.05
0.70
2



NM_016200
0.96
0.10
0.04
0.73
2



NM_003981
0.60
0.18
0.19
0.68
1



NM_020882
0.56
0.04
0.04
0.74
1



NM_021974
0.56
0.08
0.08
0.74
1



NM_018271
0.62
0.16
0.12
0.71
1



XM_378175
0.66
0.12
0.13
0.70
1



NM_014456
0.63
0.16
0.04
0.73
1



NM_152496
0.64
0.08
0.05
0.72
1



NM_174931
0.65
0.20
0.03
0.72
1



XM_371354
0.82
0.13
0.07
0.65
1



NM_004739
0.79
0.16
0.12
0.69
1



NM_198080
0.85
0.09
0.08
0.67
1



NM_000792
0.78
0.22
0.07
0.74
1



NM_013366
0.79
0.07
0.03
0.74
1



NM_001746
0.80
0.07
0.08
0.74
1



NM_148571
0.81
0.20
0.04
0.73
1



NM_002255
0.85
0.01
0.08
0.69
1



NM_024762
0.80
0.03
0.05
0.75
1



NM_052946
0.90
0.13
0.03
0.67
1



NM_002636
0.86
0.10
0.12
0.71
1



NM_015046
0.89
0.08
0.17
0.70
1



NM_006204
0.95
0.08
0.07
0.66
1



NM_001225
0.86
0.15
0.05
0.73
1



NM_000172
0.95
0.07
0.06
0.66
1



NM_002270
0.90
0.09
0.06
0.70
1



NM_030969
0.90
0.11
0.02
0.71
1



NM_020689
0.88
0.08
0.03
0.72
1



NM_020395
0.88
0.13
0.03
0.72
1



NM_021640
0.90
0.04
0.13
0.71
1



NM_014750
0.90
0.15
0.05
0.72
1



NM_194324
0.87
0.22
0.11
0.74
1



NM_014420
0.89
0.23
0.04
0.74
1



NM_012202
0.90
0.10
0.05
0.73
1



NM_001797
0.90
0.05
0.11
0.73
1



NM_002513
1.02
0.17
0.05
0.65
1



NM_005954
0.97
0.04
0.08
0.69
1



NM_000322
0.91
0.03
0.15
0.74
1



NM_021960
0.98
0.07
0.01
0.69
1



NM_000131
0.99
0.13
0.05
0.69
1



NM_020380
0.92
0.07
0.05
0.75
1



NM_022304
0.93
0.07
0.07
0.74
1



NM_005003
0.97
0.01
0.04
0.72
1



NM_032024
0.97
0.12
0.22
0.72
1



NM_134444
1.01
0.17
0.04
0.69
1



XM_371074
1.05
0.20
0.09
0.67
1



NM_080730
0.99
0.03
0.02
0.72
1



NM_005713
1.00
0.13
0.06
0.71
1



NM_152905
1.08
0.06
0.02
0.67
1



NM_020528
1.02
0.12
0.05
0.71
1



NM_006156
1.04
0.12
0.08
0.71
1



NM_012141
0.53
0.16
0.09
0.70
0



NM_173626
0.64
0.08
0.03
0.65
0



NM_003301
0.62
0.05
0.05
0.68
0



NM_020809
0.63
0.05
0.02
0.69
0



NM_004277
0.68
0.22
0.15
0.70
0



NM_016082
0.73
0.09
0.09
0.66
0



NM_030962
0.74
0.06
0.02
0.65
0



NM_001538
0.72
0.02
0.06
0.71
0



NM_173556
0.77
0.02
0.08
0.68
0



XM_016548
0.71
0.10
0.03
0.74
0



XM_056282
0.74
0.08
0.13
0.71
0



NM_182703
0.74
0.17
0.13
0.72
0



NM_005831
0.74
0.07
0.03
0.72
0



NM_003526
0.77
0.12
0.06
0.70
0



NM_005911
0.81
0.02
0.04
0.67
0



NM_022567
0.84
0.12
0.02
0.66
0



NM_018360
0.77
0.14
0.11
0.72
0



NM_182558
0.79
0.13
0.21
0.71
0



NM_016388
0.83
0.20
0.06
0.67
0



NM_006413
0.78
0.15
0.09
0.71
0



NM_024619
0.79
0.18
0.05
0.71
0



NM_001937
0.84
0.18
0.02
0.67
0



XM_375187
0.80
0.05
0.05
0.72
0



NM_178168
0.81
0.03
0.08
0.71
0



NM_020320
0.79
0.11
0.02
0.73
0



NM_003685
0.82
0.15
0.13
0.71
0



NM_152603
0.83
0.10
0.06
0.71
0



XM_114430
0.85
0.14
0.19
0.69
0



NM_198494
0.91
0.08
0.10
0.65
0



NM_152465
0.82
0.06
0.08
0.73
0



NM_152647
0.91
0.12
0.13
0.66
0



NM_138451
0.83
0.14
0.10
0.73
0



NM_024011
0.93
0.03
0.02
0.65
0



NM_024647
0.81
0.09
0.03
0.75
0



NM_152515
0.81
0.22
0.12
0.75
0



NM_207401
0.83
0.21
0.05
0.73
0



NM_144702
0.87
0.10
0.12
0.71
0



XM_371575
0.86
0.10
0.01
0.73
0



XM_209941
0.88
0.13
0.18
0.72
0



NM_015446
0.90
0.11
0.06
0.71
0



NM_019067
0.94
0.15
0.02
0.68
0



NM_018922
0.89
0.05
0.18
0.71
0



NM_032866
0.90
0.23
0.11
0.71
0



NM_213604
0.88
0.09
0.08
0.73
0



NM_018129
0.90
0.13
0.05
0.72
0



NM_002510
0.87
0.05
0.06
0.73
0



NM_004783
0.95
0.09
0.04
0.68
0



XM_089747
0.89
0.09
0.12
0.74
0



NM_173821
0.92
0.12
0.13
0.72
0



NM_014891
0.99
0.11
0.06
0.67
0



NM_005301
0.92
0.19
0.18
0.72
0



NM_021049
0.95
0.03
0.07
0.70
0



NM_002218
0.95
0.01
0.06
0.70
0



NM_000188
0.99
0.12
0.02
0.68
0



NM_015338
0.93
0.20
0.16
0.73
0



NM_019070
0.96
0.11
0.01
0.71
0



NM_173853
0.93
0.09
0.19
0.73
0



XM_373109
1.01
0.21
0.12
0.68
0



NM_005544
0.93
0.06
0.05
0.75
0



NM_007205
0.94
0.18
0.07
0.74
0



NM_007358
0.94
0.07
0.06
0.74
0



NM_173497
0.94
0.08
0.03
0.75
0



NM_003247
0.95
0.11
0.22
0.75
0



NM_173846
1.01
0.10
0.11
0.70
0



XM_030729
0.97
0.03
0.05
0.74
0



NM_015690
1.07
0.18
0.01
0.67
0



NM_002082
1.04
0.05
0.01
0.69
0



NM_004673
1.11
0.04
0.18
0.67
0



NM_182755
1.02
0.23
0.16
0.73
0

















TABLE 3







Triple Negative Breast Cancer Gene Signature (TGS) Genes or Malignancy-Associated


Response Signature (MARS) Genes
















Gene
SEQ ID
Entrez

MAD
MAD
Fold
Fold
Ratio
Cherry


Symbol
NO:
Gene ID
Accession #
BPLER
HMLER
BPLER
HMLER
BPLER/HMLER
Score



















PSMB4
1
5692
NM_002796
−6.73
−2.41
0.147
0.52
0.28
4


PSMA1
2
5682
NM_002786
−5.53
−2.21
0.240
0.65
0.37
4


RACGAP1
3
29127
NM_013277
−3.61
−1.46
0.361
0.77
0.47
4


PSMC3
4
5702
NM_002804
−1.77
−1.02
0.399
0.81
0.49
4


PRPF38A
5
84950
NM_032864
−3.64
−1.70
0.403
0.69
0.58
4


DHX8
6
1659
NM_004941
−4.27
−1.24
0.413
0.81
0.51
4


PSMA3
7
5684
NM_002788
−1.72
−0.54
0.434
0.90
0.48
4


POLR2B
8
5431
NM_000938
−4.09
−1.97
0.438
0.69
0.63
4


CCDC5
9
115106
NM_138443
−2.18
−0.02
0.583
1.00
0.59
4


LSM6
10
11157
NM_007080
−2.83
−0.41
0.593
0.93
0.64
4


PRPF6
11
24148
NM_012469
−3.13
−1.23
0.508
0.81
0.62
3


NUP205
12
23165
XM_058073
−2.88
−1.57
0.544
0.75
0.73
3


DDX19B
13
11269
NM_007242
−4.84
−2.09
0.304
0.66
0.46
3


SNW1
14
22938
NM_012245
−5.45
−1.31
0.335
0.75
0.45
3


PSMD2
15
5708
NM_002808
−4.64
−2.67
0.343
0.61
0.56
3


PSMD14
16
10213
NM_005805
−3.37
−2.28
0.352
0.62
0.56
3


NDC80
17
10403
NM_006101
−3.24
−2.06
0.377
0.66
0.57
3


C22orf29
18
79680
NM_024627
−3.47
−2.46
0.400
0.62
0.64
3


POLR2G
19
5436
NM_002696
−3.59
−1.68
0.507
0.74
0.69
3


LSM2
20
57819
NM_021177
−2.90
−0.95
0.512
0.84
0.61
3


PSMD6
21
9861
NM_014814
−2.05
−0.79
0.516
0.87
0.59
3


CEP27
22
55142
NM_018097
−1.99
−1.76
0.522
0.74
0.70
3


C4orf15
23
79441
NM_024511
−2.52
0.55
0.560
1.09
0.52
3


YTHDC1
24
91746
NM_133370
−2.31
−0.26
0.567
0.96
0.59
3


C19orf29
25
58509
XM_375557
−2.24
−0.36
0.625
0.94
0.67
3


CDC5L
26
988
NM_001253
−1.60
−0.87
0.654
0.87
0.75
3


TSG101
27
7251
NM_006292
−2.45
0.23
0.684
1.04
0.66
3


POLA1
28
5422
NM_016937
−2.01
−0.17
0.724
0.97
0.74
3


UBL5
29
59286
NM_024292
−4.14
−2.33
0.337
0.64
0.53
2


CCNA2
30
890
NM_001237
−2.79
−1.39
0.499
0.75
0.67
2


NUF2
31
83540
NM_031423
−2.68
−0.90
0.515
0.84
0.62
2


COPE
32
11316
NM_007263
−3.15
−1.43
0.547
0.77
0.71
2


PSMA2
33
5683
NM_002787
−4.14
−2.79
0.205
0.54
0.38
2


PSMD7
34
5713
NM_002811
−3.88
−2.86
0.256
0.53
0.49
2


RAN
35
5901
NM_006325
−5.67
−2.89
0.266
0.57
0.47
2


ISY1
36
57461
NM_020701
−4.27
−2.95
0.306
0.54
0.56
2


PPP2CA
37
5515
NM_002715
−2.77
−0.87
0.457
0.86
0.53
2


PSMC1
38
5700
NM_002802
−1.50
−0.45
0.476
0.91
0.52
2


RBM22
39
55696
NM_018047
−2.37
−1.53
0.477
0.78
0.61
2


FAM86B1
40
85002
NR_003494
−2.54
−2.05
0.491
0.70
0.70
2





NM_032916





(SEQ ID





NO: 181)


SF3B14
41
51639
NM_016047
−2.97
−2.06
0.500
0.72
0.70
2


CCDC12
42
151903
NM_144716
−3.22
−1.34
0.502
0.78
0.64
2


ZNF451
43
26036
NM_015555
−3.07
−0.77
0.516
0.86
0.60
2


TMED3
44
23423
NM_007364
−2.93
−1.28
0.537
0.80
0.68
2


ZNF324
45
25799
NM_014347
−2.91
−1.05
0.543
0.84
0.65
2


SCD
46
6319
NM_005063
−3.24
−1.58
0.555
0.75
0.74
2


KIAA1822
47
84439
NM_032425
−2.67
−1.27
0.563
0.77
0.73
2


CLTC
48
1213
NM_004859
−2.02
0.05
0.570
1.01
0.57
2


GAPDH
49
2597
NM_002046
−4.30
−0.19
0.576
0.97
0.60
2


STIM2
50
57620
NM_020860
−2.39
−0.99
0.597
0.83
0.72
2


SLC47A1
51
55244
NM_018242
−1.62
−0.71
0.613
0.90
0.68
2


BUB1
52
699
NM_004336
−2.62
−0.62
0.617
0.91
0.67
2


ZNF585A
53
199704
NM_152655
−2.06
−0.70
0.642
0.89
0.72
2


DNAJC11
54
55735
NM_018198
−1.52
0.75
0.656
1.11
0.59
2


MRPS9
55
64965
NM_182640
−2.00
−0.45
0.669
0.92
0.72
2


SLC4A5
56
57835
NM_021196
−1.94
0.63
0.672
1.11
0.61
2


TUT1
57
64852
NM_022830
−1.91
−0.07
0.680
0.99
0.69
2


TBC1D24
58
57465
NM_020705
−1.97
−0.32
0.680
0.95
0.71
2





XM_370928





(SEQ ID





NO: 182)


FLJ38984
59
127703
NM_152374
−1.50
−0.23
0.693
0.97
0.72
2


USP39
60
10713
NM_006590
−2.13
−0.04
0.695
0.99
0.70
2


LSM8
61
51691
NM_016200
−1.76
−0.26
0.700
0.96
0.73
2


RGS9BP
62
388531
NM_207391
−1.58
0.53
0.707
1.10
0.64
2


C6orf151
63
154007
NM_152551
−1.75
1.64
0.730
1.26
0.58
2


COPB1
64
1315
NM_016451
−3.13
−2.91
0.326
0.54
0.61
1


ISYNA1
65
51477
NM_016368
−1.95
−0.82
0.489
0.87
0.57
1


FIZ1
66
84922
NM_032836
−2.97
−0.32
0.514
0.94
0.55
1


PDLIM2
67
64236
NM_021630
−2.65
−0.34
0.563
0.95
0.60
1


ANAPC2
68
29882
NM_013366
−2.80
−1.31
0.580
0.79
0.74
1


MGAT2
69
4247
NM_002408
−2.06
−0.19
0.586
0.97
0.60
1


MRPL27
70
51264
NM_148571
−1.57
−1.17
0.587
0.81
0.73
1


KIR2DL4
71
3805
NM_002255
−2.02
−0.98
0.593
0.85
0.69
1


ETHE1
72
23474
NM_014297
−2.19
1.03
0.641
1.17
0.55
1


C12orf10
73
60314
NM_021640
−2.11
−0.54
0.644
0.90
0.71
1


RNF11
74
26994
NM_014372
−1.86
1.11
0.671
1.17
0.57
1


FAM59B
75
150946
XM_097977
−1.85
0.68
0.714
1.10
0.65
1


NEDD8
76
4738
NM_006156
−1.79
0.25
0.736
1.04
0.71
1


ZNF490
77
57474
NM_020714
−4.40
−2.66
0.285
0.59
0.49
1


DHRS13
78
147015
NM_144683
−3.66
−1.69
0.350
0.75
0.47
1


HNRPK
79
3190
NM_002140
−3.35
−2.71
0.358
0.55
0.65
1


PRC1
80
9055
NM_003981
−3.08
−2.40
0.409
0.60
0.68
1


COL20A1
81
57642
NM_020882
−3.47
−2.58
0.416
0.56
0.74
1


POLR2F
82
5435
NM_021974
−3.46
−2.98
0.417
0.56
0.74
1


DDX42
83
11325
NM_007372
−4.00
−1.78
0.425
0.71
0.60
1


ZNF574
84
64763
NM_022752
−3.52
−1.41
0.425
0.79
0.54
1


FLJ10916
85
55258
NM_018271
−2.39
−2.48
0.440
0.62
0.71
1


TMEM41A
86
90407
NM_080652
−2.70
−1.83
0.457
0.73
0.63
1


LOC124446
87
124446
XM_378175
−3.04
−2.49
0.458
0.66
0.70
1


PDCD4
88
27250
NM_014456
−2.81
−2.25
0.462
0.63
0.73
1


MANEAL
89
149175
NM_152496
−3.02
−2.41
0.464
0.64
0.72
1


RB1CC1
90
9821
NM_014781
−3.21
−1.50
0.466
0.74
0.63
1


RFT1
91
91869
NM_052859
−2.82
0.04
0.468
1.01
0.47
1


CCDC75
92
253635
NM_174931
−3.33
−2.06
0.474
0.65
0.72
1


ZNF643
93
65243
NM_023070
−3.05
−0.52
0.493
0.92
0.54
1


LIN9
94
286826
NM_173083
−2.90
−0.72
0.525
0.90
0.59
1


KIF26B
95
55083
NM-
−1.95
−1.15
0.536
0.82
0.65
1





018012





XM_371354





(SEQ ID





NO: 183)


MFSD5
96
84975
NM_032889
−2.81
−0.38
0.540
0.93
0.58
1


C16orf33
97
79622
NM_024571
−2.67
−0.58
0.540
0.91
0.59
1


SH3GL3
98
6457
NM_003027
−2.67
−0.55
0.544
0.90
0.60
1


MTA2
99
9219
NM_004739
−3.71
−1.07
0.545
0.79
0.69
1


PCTK2
100
5128
NM_002595
−2.29
−0.79
0.552
0.87
0.64
1


ANAPC4
101
29945
NM_013367
−2.96
−0.79
0.557
0.87
0.64
1


RBMX
102
27316
NM_002139
−2.43
−0.57
0.569
0.91
0.62
1


DUS3L
103
56931
NM_020175
−2.65
−0.71
0.571
0.89
0.64
1


MSRB3
104
253827
NM_198080
−2.72
−0.86
0.571
0.85
0.67
1


DCDC2
105
51473
NM_016356
−1.61
−0.02
0.571
1.00
0.57
1


LIN37
106
55957
NM_019104
−1.92
−0.19
0.571
0.97
0.59
1


DIO1
107
1733
NM_000792
−1.96
−1.39
0.573
0.78
0.74
1


CANX
108
821
NM_001746
−1.92
−1.38
0.587
0.80
0.74
1


ZNF552
109
79818
NM_024762
−2.35
−1.30
0.595
0.80
0.75
1


NOSTRIN
110
115677
NM_052946
−2.06
−0.69
0.604
0.90
0.67
1


PHF1
111
5252
NM_002636
−3.21
−0.72
0.606
0.86
0.71
1


GBP4
112
115361
NM_052941
−2.09
−0.14
0.608
0.99
0.62
1


DYSF
113
8291
NM_003494
−2.13
0.64
0.614
1.12
0.55
1


SETX
114
23064
NM_015046
−2.40
−0.72
0.617
0.89
0.70
1


PDE6C
115
5146
NM_006204
−1.98
−0.33
0.621
0.95
0.66
1


CASP4
116
837
NM_001225
−2.36
−0.92
0.623
0.86
0.73
1


GNAT1
117
2779
NM_000172
−2.03
−0.36
0.627
0.95
0.66
1


TNPO1
118
3842
NM_002270
−1.99
−0.59
0.630
0.90
0.70
1


KIAA1143
119
57456
NM_020696
−2.27
0.86
0.631
1.14
0.56
1


TMEM14B
120
81853
NM_030969
−1.99
−0.56
0.636
0.90
0.71
1


SLC24A3
121
57419
NM_020689
−2.23
−0.75
0.637
0.88
0.72
1


TMCO4
122
255104
NM_181719
−1.74
−0.01
0.638
0.99
0.64
1


INTS12
123
57117
NM_020395
−2.22
−0.74
0.639
0.88
0.72
1


DLG7
124
9787
NM_014750
−1.93
−0.56
0.646
0.90
0.72
1


MGC39900
125
286527
NM_194324
−2.13
−0.87
0.647
0.87
0.74
1


KCNJ6
126
3763
NM_002240
−1.88
0.12
0.654
1.04
0.63
1


CDC2L5
127
8621
NM_003718
−2.34
0.95
0.656
1.13
0.58
1


DKK4
128
27121
NM_014420
−1.90
−0.72
0.657
0.89
0.74
1


CDH11
129
1009
NM_001797
−1.56
−0.69
0.663
0.90
0.73
1


NME3
130
4832
NM_002513
−1.71
0.12
0.668
1.02
0.65
1


SERPINB12
131
89777
NM_080474
−1.66
0.63
0.668
1.09
0.61
1


MT3
132
4504
NM_005954
−1.77
−0.16
0.672
0.97
0.69
1


PRPH2
133
5961
NM_000322
−1.69
−0.56
0.675
0.91
0.74
1


MCL1
134
4170
NM_021960
−1.74
−0.13
0.679
0.98
0.69
1


F7
135
2155
NM_000131
−2.01
−0.06
0.679
0.99
0.69
1


C19orf20
136
91978
NM_033513
−1.69
0.87
0.684
1.13
0.61
1


CASC5
137
570820
NM_020380
−1.86
−0.56
0.686
0.92
0.75
1


B3GNT6
138
192134
NM_138706
−1.80
0.45
0.691
1.07
0.65
1


SAMD3
139
154075
NM_152552
−1.99
0.68
0.692
1.11
0.62
1


HRH2
140
3274
NM_022304
−1.68
−0.39
0.693
0.93
0.74
1


NDUFAB1
141
4706
NM_005003
−1.82
−0.20
0.694
0.97
0.72
1


C10orf11
142
83938
NM_032024
−1.58
−0.06
0.702
0.97
0.72
1


NLRP4
143
147945
NM_134444
−1.68
0.11
0.702
1.01
0.69
1


GPR15
144
2838
NM_005290
−1.58
0.72
0.708
1.11
0.64
1


TANC2
145
26115
NM_025185
−1.86
0.40
0.709
1.05
0.67
1





XM_371074





(SEQ ID





NO: 184)


HOM-TES-
146
25900
NM_080730
−1.84
−0.09
0.711
0.99
0.72
1


103


FYCO1
147
794433
NM_024513
−1.67
0.86
0.714
1.13
0.63
1


COL4A3BP
148
10087
NM_005713
−1.96
0.03
0.715
1.00
0.71
1


NEDD1
149
121441
NM_152905
−1.56
0.59
0.721
1.08
0.67
1


PCBP3
150
54039
NM_020528
−1.69
0.15
0.727
1.02
0.71
1


PRPF8
151
10594
NM_006445
−5.34
−2.64
0.246
0.62
0.40
1


HIPK3
152
10114
NM_005734
−3.97
−2.34
0.418
0.68
0.61
1


PFKL
153
5211
NM_002626
−1.85
−0.10
0.630
0.98
0.64
1


GNG3
154
2785
NM_012202
−1.86
−0.62
0.658
0.90
0.73
1
















TABLE 4A





Triple-negative Gene Signature





















Gene
Entrez





Gene function
Symbol
Gene ID
Accession #
MAD_BPLER
MAD_HMLER





Apoptosis
CASP4
837
NM_001225
−2.36
−0.92


Apoptosis
MCL1
4170
NM_021960
−1.74
−0.13


Apoptosis
PDCD4
27250
NM_014456
−2.81
−2.25


Cell Adhesion
CDH11
1009
NM_001797
−1.56
−0.69


Cell Adhesion
COL20A1
57642
NM_020882
−3.47
−2.58


Cell Adhesion
COL4A3BP
10087
NM_005713
−1.96
0.03


Cell Adhesion
PDLIM2
64236
NM_021630
−2.65
−0.34


Cell Signaling
DKK4
27121
NM_014420
−1.90
−0.72


Cell Signaling
MTA2
9219
NM_004739
−3.71
−1.07


Cell Signaling
PRPH2
5961
NM_000322
−1.69
−0.56


Cell Signaling
RACGAP1
29127
NM_013277
−3.61
−1.46


Cell Signaling
RGS9BP
388531
NM_207391
−1.58
0.53


Cell Signaling
TBC1D24
57465
XM_370928
−1.97
−0.32


DNA binding
FIZ1
84922
NM_032836
−2.97
−0.32


DNA binding
PHF1
5252
NM_002636
−3.21
−0.72


DNA binding
ZNF324
25799
NM_014347
−2.91
−1.05


DNA binding
ZNF451
26036
NM_015555
−3.07
−0.77


DNA binding
ZNF490
57474
NM_020714
−4.40
−2.66


DNA binding
ZNF552
79818
NM_024762
−2.35
−1.30


DNA binding
ZNF574
64763
NM_022752
−3.52
−1.41


DNA binding
ZNF585A
199704
NM_152655
−2.06
−0.70


DNA binding
ZNF643
65243
NM_023070
−3.05
−0.52


DNA repair
GNAT1
2779
NM_000172
−2.03
−0.36


DNA repair
GNG3
2785
NM_012202
−1.86
−0.62


DNA repair
SETX
23064
NM_015046
−2.40
−0.72


DNA repair
TSG101
7251
NM_006292
−2.45
0.23


G1/S transition
CCNA2
890
NM_001237
−2.79
−1.39


G1/S transition
LIN37
55957
NM_019104
−1.92
−0.19


G1/S transition
LIN9
286826
NM_173083
−2.90
−0.72


G1/S transition
POLA1
5422
NM_016937
−2.01
−0.17


G1/S transition
RB1CC1
9821
NM_014781
−3.21
−1.50


Gene Expression
C16orf33
79622
NM_024571
−2.67
−0.58


Gene Expression
C6orf151
154007
NM_152551
−1.75
1.64


Gene Expression
CCDC12
151903
NM_144716
−3.22
−1.34


Gene Expression
DDX42
11325
NM_007372
−4.00
−1.78


Gene Expression
DHX8
1659
NM_004941
−4.27
−1.24


Gene Expression
HIPK3
10114
NM_005734
−3.97
−2.34


Gene Expression
HNRPK
3190
NM_002140
−3.35
−2.71


Gene Expression
MRPL27
51264
NM_148571
−1.57
−1.17


Gene Expression
MRPS9
64965
NM_182640
−2.00
−0.45


Gene Expression
POLR2B
5431
NM_000938
−4.09
−1.97


Gene Expression
POLR2F
5435
NM_021974
−3.46
−2.98


Gene Expression
POLR2G
5436
NM_002696
−3.59
−1.68


Gene Expression
RBM22
55696
NM_018047
−2.37
−1.53


Gene Expression
RBMX
27316
NM_002139
−2.43
−0.57


Gene Expression
SNW1
22938
NM_012245
−5.45
−1.31


Inflammation
HRH2
3274
NM_022304
−1.68
−0.39


Inflammation
KIR2DL4
3805
NM_002255
−2.02
−0.98


Inflammation
NLRP4
147945
NM_134444
−1.68
0.11


Inflammation
NOSTRIN
115677
NM_052946
−2.06
−0.69


Metabolism
B3GNT6
192134
NM_138706
−1.80
0.45


Metabolism
DHRS13
147015
NM_144683
−3.66
−1.69


Metabolism
DIO1
1733
NM_000792
−1.96
−1.39


Metabolism
ETHE1
23474
NM_014297
−2.19
1.03


Metabolism
GAPDH
2597
NM_002046
−4.30
−0.19


Metabolism
GBP4
115361
NM_052941
−2.09
−0.14


Metabolism
ISYNA1
51477
NM_016368
−1.95
−0.82


Metabolism
KCNJ6
3763
NM_002240
−1.88
0.12


Metabolism
MGAT2
4247
NM_002408
−2.06
−0.19


Metabolism
MSRB3
253827
NM_198080
−2.72
−0.86


Metabolism
NDUFAB1
4706
NM_005003
−1.82
−0.20


Metabolism
PFKL
5211
NM_002626
−1.85
−0.10


Metabolism
SCD
6319
NM_005063
−3.24
−1.58


Mitosis
ANAPC2
29882
NM_013366
−2.80
−1.31


Mitosis
ANAPC4
29945
NM_013367
−2.96
−0.79


Mitosis
BUB1
699
NM_004336
−2.62
−0.62


Mitosis
C4orf15
79441
NM_024511
−2.52
0.55


Mitosis
CASC5
57082
NM_020380
−1.86
−0.56


Mitosis
CCDC5
115106
NM_138443
−2.18
−0.02


Mitosis
CEP27
55142
NM_018097
−1.99
−1.76


Mitosis
DLG7
9787
NM_014750
−1.93
−0.56


Mitosis
NDC80
10403
NM_006101
−3.24
−2.06


Mitosis
NME3
4832
NM_002513
−1.71
0.12


Mitosis
NUF2
83540
NM_031423
−2.68
−0.90


Mitosis
PPP2CA
5515
NM_002715
−2.77
−0.87


Mitosis
PRC1
9055
NM_003981
−3.08
−2.40


Mitosis
RAN
5901
NM_006325
−5.67
−2.89


Molecular
CANX
821
NM_001746
−1.92
−1.38


Transport


Molecular
CLTC
1213
NM_004859
−2.02
0.05


Transport


Molecular
COPB1
1315
NM_016451
−3.13
−2.91


Transport


Molecular
COPE
11316
NM_007263
−3.15
−1.43


Transport


Molecular
DDX19B
11269
NM_007242
−4.84
−2.09


Transport


Molecular
FYCO1
79443
NM_024513
−1.67
0.86


Transport


Molecular
MFSD5
84975
NM_032889
−2.81
−0.38


Transport


Molecular
NUP205
23165
XM_058073
−2.88
−1.57


Transport


Molecular
RFT1
91869
NM_052859
−2.82
0.04


Transport


Molecular
SH3GL3
6457
NM_003027
−2.67
−0.55


Transport


Molecular
SLC24A3
57419
NM_020689
−2.23
−0.75


Transport


Molecular
SLC47A1
55244
NM_018242
−1.62
−0.71


Transport


Molecular
SLC4A5
57835
NM_021196
−1.94
0.63


Transport


Molecular
TNPO1
3842
NM_002270
−1.99
−0.59


Transport


Proteasome
NEDD1
121441
NM_152905
−1.56
0.59


Degradation


Proteasome
NEDD8
4738
NM_006156
−1.79
0.25


Degradation


Proteasome
PSMA1
5682
NM_002786
−5.53
−2.21


Degradation


Proteasome
PSMA2
5683
NM_002787
−4.14
−2.79


Degradation


Proteasome
PSMA3
5684
NM_002788
−1.72
−0.54


Degradation


Proteasome
PSMB4
5692
NM_002796
−6.73
−2.41


Degradation


Proteasome
PSMC1
5700
NM_002802
−1.50
−0.45


Degradation


Proteasome
PSMC3
5702
NM_002804
−1.77
−1.02


Degradation


Proteasome
PSMD14
10213
NM_005805
−3.37
−2.28


Degradation


Proteasome
PSMD2
5708
NM_002808
−4.64
−2.67


Degradation


Proteasome
PSMD6
9861
NM_014814
−2.05
−0.79


Degradation


Proteasome
PSMD7
5713
NM_002811
−3.88
−2.86


Degradation


Proteasome
RNF11
26994
NM_014372
−1.86
1.11


Degradation


Proteasome
UBL5
59286
NM_024292
−4.14
−2.33


Degradation


Proteasome
USP39
10713
NM_006590
−2.13
−0.04


Degradation


RNA splicing
C22orf29
79680
NM_024627
−3.47
−2.46


RNA splicing
CDC2L5
8621
NM_003718
−2.34
0.95


RNA splicing
CDC5L
988
NM_001253
−1.60
−0.87


RNA splicing
INTS12
57117
NM_020395
−2.22
−0.74


RNA splicing
ISY1
57461
NM_020701
−4.27
−2.95


RNA splicing
LSM2
57819
NM_021177
−2.90
−0.95


RNA splicing
LSM6
11157
NM_007080
−2.83
−0.41


RNA splicing
LSM8
51691
NM_016200
−1.76
−0.26


RNA splicing
PRPF38A
84950
NM_032864
−3.64
−1.70


RNA splicing
PRPF6
24148
NM_012469
−3.13
−1.23


RNA splicing
PRPF8
10594
NM_006445
−5.34
−2.64


RNA splicing
SF3B14
51639
NM_016047
−2.97
−2.06


RNA splicing
TUT1
64852
NM_022830
−1.91
−0.07


Unassigned
F7
2155
NM_000131
−2.01
−0.06


(Blood clotting


cascade/Hemostasis/


Protein


metabolism)


Unassigned
SERPINB12
89777
NM_080474
−1.66
0.63


(coagulation)


Unassigned
KIF26B
55083
XM_371354
−1.95
−1.15


(development)


Unassigned
GPR15
2838
NM_005290
−1.58
0.72


(GPCRs, Class


A Rhodopsin-


like)


Unassigned
PCBP3
54039
NM_020528
−1.69
0.15


(RNA binding)


Unassigned
MT3
4504
NM_005954
−1.77
−0.16


(Target of


EGFR1/TGF


beta/TNF-alpha


signaling, metal)


Unassigned
DYSF
8291
NM_003494
−2.13
0.64


(Target of IL-1


singlaing)


Unassigned
PCTK2
5128
NM_002595
−2.29
−0.79


(Target of TGF


beta signaling)


Unassigned
C10orf11
83938
NM_032024
−1.58
−0.06


Unassigned
C12orf10
60314
NM_021640
−2.11
−0.54


Unassigned
C19orf20
91978
NM_033513
−1.69
0.87


Unassigned
C19orf29
58509
XM_375557
−2.24
−0.36


Unassigned
CCDC75
253635
NM_174931
−3.33
−2.06


Unassigned
DCDC2
51473
NM_016356
−1.61
−0.02


Unassigned
DNAJC11
55735
NM_018198
−1.52
0.75


Unassigned
DUS3L
56931
NM_020175
−2.65
−0.71


Unassigned
FAM59B
150946
XM_097977
−1.85
0.68


Unassigned
FAM86B1
85002
NM_032916
−2.54
−2.05


Unassigned
FLJ10916
55258
NM_018271
−2.39
−2.48


Unassigned
FLJ38984
127703
NM_152374
−1.50
−0.23


Unassigned
HOM-TES-
25900
NM_080730
−1.84
−0.09



103


Unassigned
KIAA1143
57456
NM_020696
−2.27
0.86


Unassigned
KIAA1822
84439
NM_032425
−2.67
−1.27


Unassigned
MANEAL
149175
NM_152496
−3.02
−2.41


Unassigned
MGC39900
286527
NM_194324
−2.13
−0.87


Unassigned
PDE6C
5146
NM_006204
−1.98
−0.33


Unassigned
SAMD3
154075
NM_152552
−1.99
0.68


Unassigned
STIM2
57620
NM_020860
−2.39
−0.99


Unassigned
TANC2
26115
XM_371074
−1.86
0.40


Unassigned
TMCO4
255104
NM_181719
−1.74
−0.01


Unassigned
TMED3
23423
NM_007364
−2.93
−1.28


Unassigned
TMEM14B
81853
NM_030969
−1.99
−0.56


Unassigned
TMEM219
124446
XM_378175
−3.04
−2.49


Unassigned
TMEM41A
90407
NM_080652
−2.70
−1.83


Unassigned
YTHDC1
91746
NM_133370
−2.31
−0.26



















Ratio




Gene function
Fold_BPLER
Fold_HMLER
BPLER/HMLER
Cherry_Score







Apoptosis
0.623
0.86
0.73
1



Apoptosis
0.679
0.98
0.69
1



Apoptosis
0.462
0.63
0.73
1



Cell Adhesion
0.663
0.90
0.73
1



Cell Adhesion
0.416
0.56
0.74
1



Cell Adhesion
0.715
1.00
0.71
1



Cell Adhesion
0.563
0.95
0.60
1



Cell Signaling
0.657
0.89
0.74
1



Cell Signaling
0.545
0.79
0.69
1



Cell Signaling
0.675
0.91
0.74
1



Cell Signaling
0.361
0.77
0.47
4



Cell Signaling
0.707
1.10
0.64
2



Cell Signaling
0.680
0.95
0.71
2



DNA binding
0.514
0.94
0.55
1



DNA binding
0.606
0.86
0.71
1



DNA binding
0.543
0.84
0.65
2



DNA binding
0.516
0.86
0.60
2



DNA binding
0.285
0.59
0.49
1



DNA binding
0.595
0.80
0.75
1



DNA binding
0.425
0.79
0.54
1



DNA binding
0.642
0.89
0.72
2



DNA binding
0.493
0.92
0.54
1



DNA repair
0.627
0.95
0.66
1



DNA repair
0.658
0.90
0.73
1



DNA repair
0.617
0.89
0.70
1



DNA repair
0.684
1.04
0.66
3



G1/S transition
0.499
0.75
0.67
2



G1/S transition
0.571
0.97
0.59
1



G1/S transition
0.525
0.90
0.59
1



G1/S transition
0.724
0.97
0.74
3



G1/S transition
0.466
0.74
0.63
1



Gene Expression
0.540
0.91
0.59
1



Gene Expression
0.730
1.26
0.58
2



Gene Expression
0.502
0.78
0.64
2



Gene Expression
0.425
0.71
0.60
1



Gene Expression
0.413
0.81
0.51
4



Gene Expression
0.418
0.68
0.61
1



Gene Expression
0.358
0.55
0.65
1



Gene Expression
0.587
0.81
0.73
1



Gene Expression
0.669
0.92
0.72
2



Gene Expression
0.438
0.69
0.63
4



Gene Expression
0.417
0.56
0.74
1



Gene Expression
0.507
0.74
0.69
3



Gene Expression
0.477
0.78
0.61
2



Gene Expression
0.569
0.91
0.62
1



Gene Expression
0.335
0.75
0.45
3



Inflammation
0.693
0.93
0.74
1



Inflammation
0.593
0.85
0.69
1



Inflammation
0.702
1.01
0.69
1



Inflammation
0.604
0.90
0.67
1



Metabolism
0.691
1.07
0.65
1



Metabolism
0.350
0.75
0.47
1



Metabolism
0.573
0.78
0.74
1



Metabolism
0.641
1.17
0.55
1



Metabolism
0.576
0.97
0.60
2



Metabolism
0.608
0.99
0.62
1



Metabolism
0.489
0.87
0.57
1



Metabolism
0.654
1.04
0.63
1



Metabolism
0.586
0.97
0.60
1



Metabolism
0.571
0.85
0.67
1



Metabolism
0.694
0.97
0.72
1



Metabolism
0.630
0.98
0.64
1



Metabolism
0.555
0.75
0.74
2



Mitosis
0.580
0.79
0.74
1



Mitosis
0.557
0.87
0.64
1



Mitosis
0.617
0.91
0.67
2



Mitosis
0.560
1.09
0.52
3



Mitosis
0.686
0.92
0.75
1



Mitosis
0.583
1.00
0.59
4



Mitosis
0.522
0.74
0.70
3



Mitosis
0.646
0.90
0.72
1



Mitosis
0.377
0.66
0.57
3



Mitosis
0.668
1.02
0.65
1



Mitosis
0.515
0.84
0.62
2



Mitosis
0.457
0.86
0.53
2



Mitosis
0.409
0.60
0.68
1



Mitosis
0.266
0.57
0.47
2



Molecular
0.587
0.80
0.74
1



Transport



Molecular
0.570
1.01
0.57
2



Transport



Molecular
0.326
0.54
0.61
1



Transport



Molecular
0.547
0.77
0.71
2



Transport



Molecular
0.304
0.66
0.46
3



Transport



Molecular
0.714
1.13
0.63
1



Transport



Molecular
0.540
0.93
0.58
1



Transport



Molecular
0.544
0.75
0.73
3



Transport



Molecular
0.468
1.01
0.47
1



Transport



Molecular
0.544
0.90
0.60
1



Transport



Molecular
0.637
0.88
0.72
1



Transport



Molecular
0.613
0.90
0.68
2



Transport



Molecular
0.672
1.11
0.61
2



Transport



Molecular
0.630
0.90
0.70
1



Transport



Proteasome
0.721
1.08
0.67
1



Degradation



Proteasome
0.736
1.04
0.71
1



Degradation



Proteasome
0.240
0.65
0.37
4



Degradation



Proteasome
0.205
0.54
0.38
2



Degradation



Proteasome
0.434
0.90
0.48
4



Degradation



Proteasome
0.147
0.52
0.28
4



Degradation



Proteasome
0.476
0.91
0.52
2



Degradation



Proteasome
0.399
0.81
0.49
4



Degradation



Proteasome
0.352
0.62
0.56
3



Degradation



Proteasome
0.343
0.61
0.56
3



Degradation



Proteasome
0.516
0.87
0.59
3



Degradation



Proteasome
0.256
0.53
0.49
2



Degradation



Proteasome
0.671
1.17
0.57
1



Degradation



Proteasome
0.337
0.64
0.53
2



Degradation



Proteasome
0.695
0.99
0.70
2



Degradation



RNA splicing
0.400
0.62
0.64
3



RNA splicing
0.656
1.13
0.58
1



RNA splicing
0.654
0.87
0.75
3



RNA splicing
0.639
0.88
0.72
1



RNA splicing
0.306
0.54
0.56
2



RNA splicing
0.512
0.84
0.61
3



RNA splicing
0.593
0.93
0.64
4



RNA splicing
0.700
0.96
0.73
2



RNA splicing
0.403
0.69
0.58
4



RNA splicing
0.508
0.81
0.62
3



RNA splicing
0.246
0.62
0.40
1



RNA splicing
0.500
0.72
0.70
2



RNA splicing
0.680
0.99
0.69
2



Unassigned
0.679
0.99
0.69
1



(Blood clotting



cascade/Hemostasis/



Protein



metabolism)



Unassigned
0.668
1.09
0.61
1



(coagulation)



Unassigned
0.536
0.82
0.65
1



(development)



Unassigned
0.708
1.11
0.64
1



(GPCRs, Class



A Rhodopsin-



like)



Unassigned
0.727
1.02
0.71
1



(RNA binding)



Unassigned
0.672
0.97
0.69
1



(Target of



EGFR1/TGF



beta/TNF-alpha



signaling, metal)



Unassigned
0.614
1.12
0.55
1



(Target of IL-1



singlaing)



Unassigned
0.552
0.87
0.64
1



(Target of TGF



beta signaling)



Unassigned
0.702
0.97
0.72
1



Unassigned
0.644
0.90
0.71
1



Unassigned
0.684
1.13
0.61
1



Unassigned
0.625
0.94
0.67
3



Unassigned
0.474
0.65
0.72
1



Unassigned
0.571
1.00
0.57
1



Unassigned
0.656
1.11
0.59
2



Unassigned
0.571
0.89
0.64
1



Unassigned
0.714
1.10
0.65
1



Unassigned
0.491
0.70
0.70
2



Unassigned
0.440
0.62
0.71
1



Unassigned
0.693
0.97
0.72
2



Unassigned
0.711
0.99
0.72
1



Unassigned
0.631
1.14
0.56
1



Unassigned
0.563
0.77
0.73
2



Unassigned
0.464
0.64
0.72
1



Unassigned
0.647
0.87
0.74
1



Unassigned
0.621
0.95
0.66
1



Unassigned
0.692
1.11
0.62
1



Unassigned
0.597
0.83
0.72
2



Unassigned
0.709
1.05
0.67
1



Unassigned
0.638
0.99
0.64
1



Unassigned
0.537
0.80
0.68
2



Unassigned
0.636
0.90
0.71
1



Unassigned
0.458
0.66
0.70
1



Unassigned
0.457
0.73
0.63
1



Unassigned
0.567
0.96
0.59
3

















TABLE 4B





Classification of Genes in TGS Gene Signature






















Cell
Cell
DNA

G1/S
Gene


Apoptosis
Adhesion
Signaling
binding
DNA repair
transition
Expression





CASP4
CDH11
DKK4
FIZ1
GNAT1
CCNA2
C16orf33


MCL1
COL20A1
MTA2
PHF1
GNG3
LIN37
C6orf151


PDCD4
COL4A3BP
PRPH2
ZNF324
SETX
LIN9
CCDC12



PDLIM2
RACGAP1
ZNF451
TSG101
POLA1
DDX42




RGS9BP
ZNF490

RB1CC1
DHX8




TBC1D24
ZNF552


HIPK3





ZNF574


HNRPK





ZNF585A


MRPL27





ZNF643


MRPS9








POLR2B








POLR2F








POLR2G








RBM22








RBMX








SNW1








Molecular
Proteasome
RNA


Inflammation
Metabolism
Mitosis
Transport
Degradation
splicing
Unassigned





HRH2
B3GNT6
ANAPC2
CANX
NEDD1
C22orf29
F7


KIR2DL4
DHRS13
ANAPC4
CLTC
NEDD8
CDC2L5
SERPINB12


NLRP4
DIO1
BUB1
COPB1
PSMA1
CDC5L
KIF26B


NOSTRIN
ETHE1
C4orf15
COPE
PSMA2
INTS12
GPR15



GAPDH
CASC5
DDX19B
PSMA3
ISY1
PCBP3



GBP4
CCDC5
FYCO1
PSMB4
LSM2
MT3



ISYNA1
CEP27
MFSD5
PSMC1
LSM6
DYSF



KCNJ6
DLG7
NUP205
PSMC3
LSM8
PCTK2



MGAT2
NDC80
RFT1
PSMD14
PRPF38A
C10orf11



MSRB3
NME3
SH3GL3
PSMD2
PRPF6
C12orf10



NDUFAB1
NUF2
SLC24A3
PSMD6
PRPF8
C19orf20



PFKL
PPP2CA
SLC47A1
PSMD7
SF3B14
C19orf29



SCD
PRC1
SLC4A5
RNF11
TUT1
CCDC75




RAN
TNPO1
UBL5

DCDC2






USP39

DNAJC11








DUS3L








FAM59B








FAM86B1








FLJ10916
















TABLE 5







Perturbagens inducing a TGS-related signature (CMAP)





















Percent


Rank
CMAP name
Mean
N
Enrichment
p-value
Specificity
non-null

















3
trichostatin A
0.381
182
0.439
0
0.4218
70


4
methylergometrine
0.604
4
0.896
0.0001
0
100


8
piribedil
0.581
4
0.83
0.00127
0.0089
100


11
deferoxamine
0.432
8
0.593
0.00309
0
87


13
sulfametoxydiazine
0.57
4
0.793
0.00354
0.0094
100


15
diltiazem
0.543
5
0.716
0.00433
0
80


16
nabumetone
0.526
4
0.78
0.0044
0.0065
100


18
meptazinol
0.548
4
0.764
0.00585
0.0079
100


19
remoxipride
0.53
4
0.759
0.00631
0.015
100


21
adrenosterone
0.48
4
0.735
0.00965
0.0217
100


24
canavanine
0.534
3
0.821
0.01148
0
100


25
phthalylsulfathiazole
0.504
5
0.662
0.01178
0.0974
80


26
butacaine
0.438
4
0.718
0.01281
0.0158
100


27
gabapentin
0.482
4
0.718
0.01307
0
100


37
vorinostat
0.339
12
0.42
0.0187
0.6181
66


40
sulfabenzamide
0.369
4
0.683
0.02174
0.0288
75


43
conessine
0.454
4
0.678
0.02335
0
75


45
adenosine
0.317
4
0.677
0.02381
0.0124
50



phosphate


46
loperamide
0.288
6
0.566
0.02459
0.1616
66


49
fludroxycortide
0.441
5
0.608
0.02784
0.0238
80


51
tacrine
0.339
4
0.664
0.02896
0.0126
75


52
flufenamic acid
0.428
6
0.554
0.02994
0.0365
83


54
practolol
0.435
4
0.657
0.03209
0.0259
75


56
4,5-
0.554
2
0.869
0.03493
0.0407
100



dianilinophthalimide


59
0316684-0000
0.298
4
0.644
0.03893
0.0192
50


60
resveratrol
0.332
9
0.439
0.04238
0.4363
55


62
STOCK1N-28457
0.461
3
0.717
0.04465
0.0174
100


65
pipemidic acid
0.387
3
0.715
0.04543
0.0238
66


66
hesperetin
0.486
5
0.571
0.04588
0.0325
80


67
vigabatrin
0.313
3
0.712
0.04649
0.1529
66


69
triamterene
0.374
5
0.569
0.04724
0.0403
60


70
biotin
0.352
3
0.711
0.04745
0.0074
66


72
amikacin
0.365
4
0.627
0.04828
0.0089
50


75
CP-690334-01
0.177
8
0.454
0.04924
0.2
50


76
methyldopa
0.391
5
0.566
0.04948
0.031
60


77
cetirizine
0.441
4
0.625
0.04957
0.0857
75


78
5252917
0.545
2
0.843
0.04982
0.0959
100
















TABLE 6







CMAP transcripts decreased in breast cancer cells after


treatment with Trichostatin A (Instance ID 6709)











Probe Set ID
Gene Symbol
Rank
Score
Amplitude














201267_s_at
PSMC3
22265
0.001
−1.66


200796_s_at
MCL1
22207
−0.002
−1.46


209055_s_at
CDC5L
21939
0.004
−1.19


AFFX-
GAPDH
21909
0
−1.17


HUMGAPDH/


M33197_5_at


215424_s_at
SNW1
21890
−0.005
−1.16


202731_at
PDCD4
21746
−0.005
−1.07


204162_at
NDC80
21731
−0.01
−1.07


201705_at
PSMD7
21564
−0.008
−1.01


211162_x_at
SCD
21329
−0.003
−0.92


207318_s_at
CDK13
21150
−0.001
−0.87


215509_s_at
BUB1
21032
−0.001
−0.84


203418_at
CCNA2
20991
−0.005
−0.83


211708_s_at
SCD
20983
−0.011
−0.83


208853_s_at
CANX
20890
−0.012
−0.81


207319_s_at
CDK13
20833
−0.015
−0.79


212861_at
MFSD5
20698
−0.015
−0.77


218134_s_at
RBM22
20612
−0.017
−0.75


203444_s_at
MTA2
20562
−0.032
−0.74


206474_at
CDK17
20587
−0.027
−0.74


210759_s_at
PSMA1
20591
−0.022
−0.74


202576_s_at
DDX19A ///
20554
−0.037
−0.73



DDX19B


221573_at
C7orf25 /// PSMA2
20330
−0.033
−0.7


209226_s_at
TNPO1
20264
−0.036
−0.68


201102_s_at
PFKL
20192
−0.044
−0.67


203101_s_at
MGAT2
20193
−0.038
−0.67


218009_s_at
PRC1
20166
−0.049
−0.67


221918_at
CDK17
20122
−0.052
−0.66


212247_at
NUP205
20070
−0.056
−0.65


211565_at
SH3GL3
20018
−0.059
−0.64


201532_at
PSMA3
19951
−0.062
−0.63


213226_at
CCNA2
19947
−0.068
−0.63


AFFX-
GAPDH
19837
−0.068
−0.62


HUMGAPDH/


M33197_M_at


204862_s_at
NME3
19691
−0.068
−0.6


213762_x_at
RBMX
19570
−0.068
−0.58


217829_s_at
USP39
19438
−0.068
−0.56


208852_s_at
CANX
19388
−0.071
−0.55


204219_s_at
PSMC1
19242
−0.07
−0.53


200749_at
—
18994
−0.065
−0.5


218204_s_at
FYCO1
18978
−0.07
−0.5


218220_at
C12orf10
18967
−0.075
−0.5


215954_s_at
C19orf29
18872
−0.077
−0.49


200750_s_at
RAN
18834
−0.087
−0.48


221452_s_at
TMEM14B
18856
−0.082
−0.48


215792_s_at
DNAJC11
18761
−0.089
−0.47


219119_at
NAA38
18734
−0.094
−0.47


210148_at
HIPK3
18651
−0.096
−0.46


203334_at
DHX8
18517
−0.101
−0.45


211746_x_at
PSMA1
18546
−0.097
−0.45


209449_at
LSM2
18476
−0.105
−0.44


202243_s_at
PSMB4
18410
−0.108
−0.43


209511_at
POLR2F
18356
−0.111
−0.43


218660_at
DYSF
18347
−0.116
−0.43


207657_x_at
TNPO1
18249
−0.124
−0.42


208837_at
TMED3
18268
−0.119
−0.42


201317_s_at
PSMA2
18181
−0.126
−0.41


201676_x_at
PSMA1
18061
−0.127
−0.4









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## Claims

1. A method of identifying a candidate therapeutic agent against a cancer initiating cell comprising the step of
a. exposing a human breast primary epithelial cell transformed derivative (BPLER) cell culture to a test agent, wherein said BPLER cell culture comprises human breast primary epithelial cells (BPE) transformed with at least one genetic element selected from hTERT, SV40 early region and h-RASV12;
b. measuring expression of at least ten malignancy associated response signature biomarkers wherein the at least 10 biomarkers are selected from the expression signature biomarker set consisting of SEQ ID NOs:1-154 in the BPLER cell culture to obtain a measured expression level,
c. comparing the measured expression level from step (b) to an expression signature reference from a BPLER cell culture that has not been exposed to the test agent, wherein a decrease of expression of at least 5 of the at least 10 biomarkers in the test culture compared to the expression signature reference indicates that the test agent is a candidate therapeutic agent against a cancer initiating cell.

2. The method of claim 1, wherein the cancer initiating cell is a basal-like breast tumor-initiating cell.