# Patent

## Title
Compositions and methods for cancer and cancer stem cell detection and elimination

## Abstract

In alternative embodiments, the invention provides compositions and methods for inhibiting or ablating cancer stem cells. In alternative embodiments, the invention provides compositions and methods for inhibiting the action of double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes. In alternative embodiments, the invention provides compositions and methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, e.g., leukemia or Chronic Myeloid Leukemia (CML). In alternative embodiments, the invention provides compositions and methods for inhibiting a Sonic Hedgehog (Shh) pathway, e.g., by using a Smoothened (SMO) protein inhibitor. In alternative embodiments, the invention provides compositions and methods for measuring or determining, or predicting, chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance. In alternative embodiments, the invention provides compositions and methods for determining or measuring the effectiveness of a treatment, a drug, a therapy or a diet for eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This United States utility patent application is the §371 national phase of Patent Convention Treaty (PCT) International Application no. PCT/US2012/054307 having an international filing date of Sep. 7, 2012, which claims benefit of priority to U.S. Provisional Patent Application Ser. No. 61/532,417, filed Sep. 8, 2011; U.S. Ser. No. 61/537,157, filed Sep. 21, 2011; U.S. Ser. No. 61/537,161, filed Sep. 21, 2011; and U.S. Ser. No. 61/537,185, filed Sep. 21, 2011. The aforementioned applications are expressly incorporated herein by reference in their entirety and for all purposes.


TECHNICAL FIELD
This invention relates to oncology, cellular and developmental biology and drug discovery. In alternative embodiments, the invention provides compositions and methods for inhibiting or ablating cancer stem cells. In alternative embodiments, the invention provides compositions and methods for inhibiting the action of double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes. In alternative embodiments, the invention provides compositions and methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, e.g., leukemia or Chronic Myeloid Leukemia (CML).
In alternative embodiments, the invention provides compositions and methods for inhibiting a Sonic Hedgehog (Shh) pathway, e.g., by using a Smoothened (SMO) protein inhibitor. In alternative embodiments, the invention provides compositions and methods for inhibiting a Sonic Hedgehog (Shh) pathway, e.g., by using a Smoothened (SMO) protein inhibitor; to force, stimulate or initiate a dormant cell or a cancer stem cell (e.g., a Chronic Myelogenous Leukemia (CML) stem cell) to cycle so they the cell can more effectively targeted by a chemotherapy, a radiation therapy or a targeted tyrosine kinase inhibitor; as a biomarker of response to chemotherapy; and/or, as a target for drug development.
In alternative embodiments, the invention provides compositions and methods for measuring or determining, or predicting, chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance comprising measuring or determining, individually or together, levels or amounts of GLI2 transcript and/or protein (increasing) and/or GLI3 transcript and/or protein (decreasing) as prognostic biomarkers of chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance. In alternative embodiments, the invention provides compositions and methods for measuring or determining, or predicting, a response to an inhibitor or inhibitors of a Sonic Hedgehog (Shh) pathway, or a targeted Shh inhibition, or a selective Shh inhibition, comprising measuring or determining, individually or together, levels or amounts of GLI1 and/or GLI2 transcript and/or protein.
In alternative embodiments, the invention provides compositions and methods for determining or measuring the effectiveness of a treatment, a drug, a therapy or a diet for eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells. In alternative embodiments, the invention provides compositions and methods for determining or predicting a positive response or monitoring a response (predicting a negative or positive response) to a selective JAK2 inhibition therapy, drug or treatment.
BACKGROUND
RNA editing is a post-transcriptional processing mechanism that results in an RNA sequence that is different from that encoded by the genomic DNA and thereby diversifies the gene product and function. The type of RNA editing that is most prevalent in higher eukaryotes converts adenosine residues into inosine (A-to-I editing) in double-stranded RNA (dsRNA) through the action of double-stranded RNA-specific adenosine deaminases, or ADAR, enzymes.
ADAR is an enzyme that in humans is encoded by the ADAR gene (ADAR1 is an acronym for “adenosine deaminase acting on RNA 1”). ADAR1 RNA edits by site-specific deamination of adenosines. The ADAR1 enzyme destabilizes double stranded RNA through conversion of adenosine to inosine. The ADAR1 enzyme modifies cellular and viral RNAs, including coding and noncoding RNAs. ADAR1 is an RNA editing enzyme, required for hematopoiesis. ADAR1+/− chimeric embryos die before embryonic day 14 with defects in the hematopoietic system. Regulated levels of ADAR1 expression are critical for embryonic erythropoiesis in the liver. Mutations in the ADAR gene have been associated with dyschromatosis symmetrica hereditaria. Alternate transcriptional splice variants, encoding different isoforms, have been characterized.
Traditional CML treatment, such as hydroxyurea and imatinib, is a great financial burden on patients. Moreover, they are not efficient at eradicate leukemia cancer stem cells, which often leads to disease progression and relapse. New drugs that target at cancer stem cells are urgently needed for patient care.
Studies suggested that leukemia stem cells (LSC) promote therapeutic resistance, relapse and disease progression, the leading causes of leukemia mortality, as a result of enhanced survival and self-renewal combined with a propensity to become dormant in supportive microenvironments. Therapies capable of breaking LSC quiescence while sparing normal hematopoietic stem cell (HSC) function have remained elusive. In chronic myeloid leukemia (CML) mouse models, Sonic hedgehog (Shh) pathway activation promotes LSC maintenance. However, the comparative role of Shh signaling in human normal HSC and LSC quiescence induction and self-renewal had not been determined.
Signal transducer and activator of transcription 5A (STAT5a) is a protein that in humans is encoded by the STAT5A gene. The protein encoded by this gene is a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein is activated by, and mediates the responses of many cell ligands, such as IL2, IL3, IL7 GM-CSF, erythropoietin, thrombopoietin, and different growth hormones. Activation of this protein in myeloma and lymphoma associated with a TEL/JAK2 gene fusion is independent of cell stimulus and has been shown to be essential for the tumorigenesis.
Janus kinase 2 (JAK2) is a human protein that has been implicated in signaling by members of the type II cytokine receptor family (e.g. interferon receptors), the GM-CSF receptor family, and the gp130 receptor family (e.g., IL-6R), and the single chain receptors such as Epo-R. JAK2 gene fusions with the TEL(ETV6) and PCMI genes have been found in leukemia patients. Mutations in JAK2 have been implicated in myeloproliferative disorders.
SUMMARY
RNA Editing as a Novel Cancer Stem Cell Target
In alternative embodiments, the invention provides methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation and/or self-renewal (or self-renewal capacity) of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, comprising,
(a) providing a composition that inhibits or slows the expression of or the activity of: and ADAR1 gene (adenosine deaminase acting on RNA 1), and ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 polypeptide; and
(b) administering a sufficient amount of the composition to an individual in need thereof, wherein a sufficient amount comprises the inhibition or slowing of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells.
In alternative embodiments of the methods, the hematopoietic stem cell or cancer stem cell comprises a cancer stem cell, or a leukemia cell, or a Chronic Myeloid Leukemia (CML) cell, a leukemia stem cell, or a Chronic Myeloid Leukemia (CML) stem cell.
In alternative embodiments of the methods, the composition that inhibits or slows the expression of an ADAR1 gene, an ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 polypeptide comprises:
(a) an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript;
(b) a polypeptide, peptide or an antibody inhibitory to the expression of the ADAR1 gene or ADAR1 gene transcript, or activity or expression of the ADAR1 enzyme;
(c) the method of (a), wherein the inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript comprises: an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA); or
(d) the method of (a) or (c), wherein inhibitory nucleic acid molecule comprises a ribozyme.
In alternative embodiments of the methods, the inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript comprises a single or doublestranded and/or sense or antisense sequence or subsequence comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
In alternative embodiments of the methods, the antibody inhibitory to the expression of the ADAR1 gene or ADAR1 gene transcript, or activity or expression of the ADAR1 enzyme comprises an antibody or antigen-binding fragment thereof that specifically binds to a protein as set forth in SEQ ID NO:3.
In alternative embodiments of the methods, the polypeptide or peptide inhibitory to the expression of the ADAR1 gene or ADAR1 gene transcript, or activity or expression of the ADAR1 enzyme comprises a peptide aptamer or an ADAR1-binding polypeptide or peptide.
In alternative embodiments of the methods, the composition that inhibits or slows the expression of or the activity of: an ADAR1 gene (adenosine deaminase acting on RNA 1), an ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 polypeptide is administered in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides compositions, pharmaceutical compositions or formulations, or equivalents, comprising a composition that inhibits or slows the expression of or the activity of: an ADAR1 gene (adenosine deaminase acting on RNA 1), and ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 polypeptide, wherein optionally the composition or formulation is formulated for administration in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
Compositions and Methods for Dormant Cancer Stem Cell Detection and Elimination
In alternative embodiments, the invention provides compositions and methods to detect dormant cancer stem cells, e.g., Chronic Myelogenous Leukemia (CML) stem cells. In alternative embodiments, the invention provides compositions and methods for use therapeutically to force dormant cancer stem cells, e.g., Chronic Myelogenous Leukemia (CML) stem cells, into cycle so they can be targeted by a therapeutic agent or procedure, e.g., chemotherapy, radiation therapy or targeted tyrosine kinase inhibitors.
In alternative embodiments, the invention provides methods for activating, stimulating or initiating in a cancer stem cell a transition from G0 to G1 of the cell cycle, or initiating cell cycling in a cancer stem cell, or breaking dormancy in a cancer stem cell, or inducing in a stem cell susceptibility to BCR-ABL inhibition, comprising:
(a) providing a composition that inhibits a Sonic Hedgehog (Shh), or providing a Smoothened (SMO) protein inhibitor (Smoothened (SMO) is an integral membrane protein mediator, a of Hedgehog signaling); and
(b) administering an effective amount of the Sonic Hedgehog (Shh) inhibitor or the Smoothened (SMO) protein inhibitor to the cancer stem cell, thereby activating, stimulating or initiating in the cancer stem cell a transition from G0 to G1 of the cell cycle, or initiating cell cycling in the cancer stem cell, inducing in the stem cell susceptibility to BCR-ABL inhibition, or breaking dormancy in the stem cell.
In alternative embodiments, the invention provides methods for radiosensitization of a cancer stem cell, or sensitizing a cancer stem cell to a treatment or protocol that targets dividing cells, or sensitizing a cancer stem cell to a chemotherapy, a radiation therapy or a targeted tyrosine kinase inhibitor, comprising,
(a) providing a composition that inhibits a Sonic Hedgehog (Shh), or providing a Smoothened (SMO) protein inhibitor; and
(b) administering an effective amount of the Sonic Hedgehog (Shh) inhibitor or the Smoothened (SMO) protein inhibitor to the cancer stem cell, thereby radiosensitizing the cancer stem cell, or sensitizing the cancer stem cell to a treatment or protocol that targets dividing cells, or sensitizing the cancer stem cell to a chemotherapy, a radiation therapy or a targeted tyrosine kinase inhibitor.
In alternative embodiments of the methods, the cancer stem cell is a hematopoietic cancer stem cell, or the cancer stem cell is a leukemia stem cell, or a Chronic Myeloid Leukemia (CML) stem cell or a Chronic Myeloid Leukemia (CML) stem cell.
In alternative embodiments of the methods, the composition that inhibits or slows the expression of an Shh gene, an Shh gene product, an Shh transcript, and/or an Shh polypeptide comprises:
(a) an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of a Shh gene or Shh gene transcript;
(b) a polypeptide, peptide or an antibody inhibitory to the expression of the Shh gene or Shh gene transcript, or activity or expression of the Shh polypeptide;
(c) the method of (a), wherein the inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the Shh gene or Shh gene transcript comprises: an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA); or
(d) the method of (a) or (c), wherein inhibitory nucleic acid molecule comprises a ribozyme.
In alternative embodiments of the methods, the composition that inhibits or slows the expression of a Shh gene, a Shh gene product, a Shh transcript, and/or a Shh polypeptide comprises a PF-04449913 (structure illustrated in FIG. 17a), or an equivalent thereof, or a bioisostere thereof.
In alternative embodiments of the methods, the antibody inhibitory to the expression of a Shh gene, a Shh gene product or a Shh transcript, or activity or expression of the Shh polypeptide, comprises an antibody or antigen-binding fragment thereof that specifically binds to a Shh protein.
In alternative embodiments of the methods, the polypeptide or peptide inhibitory to the expression of a Shh gene, a Shh gene product or a Shh transcript, or activity or expression of the Shh protein comprises a peptide aptamer or a Shh protein-binding polypeptide or peptide.
In alternative embodiments of the methods, the composition that inhibits or slows the expression of or the activity of: a Shh gene, a Shh gene product or a Shh transcript, and/or an Shh polypeptide is administered in vitro, ex vivo or in vivo.
The invention provides compositions, pharmaceutical compositions or formulations comprising a composition that inhibits or slows the expression of or the activity of: a Shh gene, a Shh gene product or a Shh transcript, and/or an Shh polypeptide, wherein optionally the composition or formulation is formulated for administration in vitro, ex vivo or in vivo. In alternative embodiments, the composition that inhibits or slows the expression of or the activity of: a Shh gene, a Shh gene product or a Shh transcript, and/or an Shh polypeptide comprises any composition used to practice a method of the invention, e.g., a PF-04449913 (structure illustrated in FIG. 17a), or an equivalent thereof, or a bioisostere thereof.
The invention provides arrays or kits comprising a cancer stem cell splice isoform and/or proteome detection platform, wherein the array or kit comprises a sufficient plurality of nucleic acids and/or proteins to detect a dormant cancer stem cell from a non-dormant stem cell or a cancer stem cell transitioning from G0 to G1 of the cell cycle.
The invention provides methods for determining the effectiveness of a test compound for: activating, stimulating or initiating in a cancer stem cell a transition from G0 to G1 of the cell cycle; or initiating cell cycling in a cancer stem cell; or breaking dormancy in a cancer stem cell; or radiosensitizing of a cancer stem cell; or sensitizing a cancer stem cell to a treatment or protocol that targets dividing cells; inducing in a stem cell susceptibility to BCR-ABL inhibition; or sensitizing a cancer stem cell to a chemotherapy, a radiation therapy or a targeted tyrosine kinase inhibitor, comprising
analyzing the cancer stem cell transcript (RNA) splice isoform pattern and/or the proteome before and after contacting the test compound to the stem cell, wherein a change of the cancer stem cell to a non-dormant transcript (RNA) splice isoform pattern and/or the proteome pattern indicates that the test compound is effective for: activating, stimulating or initiating in a cancer stem cell a transition from G0 to G1 of the cell cycle; or initiating cell cycling in a cancer stem cell; or breaking dormancy in a cancer stem cell; or radiosensitizing of a cancer stem cell; or sensitizing a cancer stem cell to a treatment or protocol that targets dividing cells; inducing in a stem susceptibility to BCR-ABL inhibition; or sensitizing a cancer stem cell to a chemotherapy, a radiation therapy or a targeted tyrosine kinase inhibitor, wherein optionally the analyzing comprises use of an array or a kit of the invention.
The invention provides kits comprising: a composition used to practice a method of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
Sonic Hedgehog Targets as Biomarkers of Prognosis and Response for Human Chronic Myelogenous Leukemia
In alternative embodiments, the invention provides compositions and methods to detect dormant cancer stem cells, e.g., Chronic Myelogenous Leukemia (CML) stem cells. In alternative embodiments, the invention provides compositions and methods for use therapeutically to force dormant cancer stem cells, e.g., Chronic Myelogenous Leukemia (CML) stem cells, into cycle so they can be targeted by a therapeutic agent or procedure, e.g., chemotherapy, radiation therapy or targeted tyrosine kinase inhibitors.
In alternative embodiments, the invention provides compositions and methods for measuring or determining, or predicting, chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance, comprising: measuring or determining, individually or together, levels or amounts of GLI2 transcript and/or protein (increasing) and/or GLI3 transcript and/or protein (decreasing) as prognostic biomarkers of chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance, wherein increased or increasing levels of GLI2 transcript and/or protein and/or decreasing levels of GLI3 transcript and/or protein indicate and/or predict chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance.
In alternative embodiments, the invention provides compositions and methods for measuring or determining, or predicting, a response to an inhibitor or inhibitors of a Sonic Hedgehog (Shh) pathway, or a targeted Shh inhibition, or a selective Shh inhibition, comprising:
measuring or determining, individually or together, levels or amounts of GLI1 and/or GLI2 transcript and/or protein,
wherein the presence of one or both GLI1 and/or GLI2 transcript and/or protein, and/or increased or increasing levels of one or both GLI1 and/or GLI2 transcript and/or protein indicates a response to an inhibitor or inhibitors of a Sonic Hedgehog (Shh) pathway, or a targeted Shh inhibition, or a selective Shh inhibition.
In alternative embodiments, the invention provides compositions and methods for measuring or determining, or predicting, whether a cancer stem cell has or will transition from G0 to G1 of the cell cycle, or has or will initiate cell cycling, or has or will break dormancy, or has been induced to have a susceptibility to BCR-ABL inhibition, comprising:
measuring or determining, individually or together, levels or amounts of GLI1 and/or GLI2 transcript and/or protein,
wherein the presence of one or both GLI1 and/or GLI2 transcript and/or protein, and/or increased or increasing levels of one or both GLI1 and/or GLI2 transcript and/or protein, indicates a response to an inhibitor or inhibitors of a Sonic Hedgehog (Shh) pathway, or a targeted Shh inhibition, or a selective Shh inhibition, thereby also indicating or predicting whether a cancer stem cell has or will transition from G0 to G1 of the cell cycle, or has or will initiate cell cycling, or has or will break dormancy, or has been induced to have a susceptibility to BCR-ABL inhibition.
In alternative embodiments, for methods of the invention, the presence, absence and/or amount of a GLI1, GLI2 and/or GLI3 transcript and/or protein is measured using an array, an immunoassay, an immunoprecipitation, a kit, a polymerase chain reaction (PCR), a qRT-PCR, a nanofluidic assay or device, a nanofluidic proteome assay, a chromatography, a nanoproteomics quantification, an isoelectric focusing assay, or a combination thereof.
In alternative embodiments, the invention provides compositions and methods for assessing the eradication of self-renewing cancer stem cells, or Leukemic Stem Cells (LSCs), comprising:
measuring or determining, individually or together, levels or amounts of GLI1 and/or GLI2 transcript and/or protein,
wherein the absence of one or both GLI1 and/or GLI2 transcript and/or protein, and/or decreasing levels of one or both GLI1 and/or GLI2 transcript and/or protein, indicates a response to an inhibitor or inhibitors of a Sonic Hedgehog (Shh) pathway, or a targeted Shh inhibition, or a selective Shh inhibition, thereby also indicating or predicting an eradication or diminishment of self-renewing cancer stem cells, or Leukemic Stem Cells (LSCs).
In alternative embodiments, the invention provides arrays, immunoassays, kits and the like comprising nucleic acids, proteins or antibodies capable of determining or measuring the presence, absence and/or amount of a GLI1, GLI2 and/or GLI3 transcript and/or protein. In alternative embodiments, the arrays or kits comprise a composition used to practice a method of the invention, or a composition, and optionally comprising instructions for use thereof.
Spliced Isoform Biomarkers to Assess Responses to Cancer Stem Cell Targeted Therapies
In alternative embodiments, the invention provides compositions and methods for determining or measuring the effectiveness of a treatment, a drug, a therapy or a diet for eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells, comprising:
(a) determining or measuring the amount of an alternatively spliced phosphoStat5a and/or a phospho-JAK2;
(b) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2; or
(c) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2 transcript or message;
wherein a decrease in the amount of the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, determines or predicts that the treatment, drug, therapy or diet will be effective for the treatment, prevention or amelioration of a leukemic stem cell (LSC) or cells, or eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells.
In alternative embodiments, the invention provides compositions and methods for selecting a diet, a treatment, a drug or a therapy; to treat or ameliorate a leukemic stem cell (LSC), or, for eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells, comprising:
(a) applying, contacting or administering a diet, a treatment, a drug or a therapy to a LSC cell or a cell population or subpopulation, and
(b)(i) determining or measuring the amount of an alternatively spliced phosphoStat5a and/or a phospho-JAK2;
(ii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2; or
(iii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2 transcript or message;
wherein a decrease in the amount of the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, determines or predicts that the treatment, drug, therapy or diet will be effective for the treatment, prevention or amelioration of a leukemic stem cell (LSC) or cells, or eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells.
In alternative embodiments, the invention provides compositions and methods for determining or predicting a positive response or monitoring a response (predicting a negative or positive response) to a selective JAK2 inhibition therapy, drug or treatment, comprising
(a) applying, contacting or administering a diet, a treatment, a drug or a therapy to a LSC cell or a cell population or subpopulation, and
(b)(i) determining or measuring the amount of an alternatively spliced phosphoStat5a and/or a phospho-JAK2;
(ii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2; or
(iii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2 transcript or message;
wherein a decrease in the amount of the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, determines or predicts that the selective JAK2 inhibition therapy, drug or treatment will be (or is) effective for the treatment, prevention or amelioration of a leukemic stem cell (LSC) or cells, or eliminating, killing or reducing the amounts of a leukemic stem cell (LSC) or cells.
In alternative embodiments, the invention provides compositions and methods for assessing the resistance, or relative resistance, of a self-renewing leukemic stem cell (LSC) or cells to a selective JAK2 inhibition therapy, drug or treatment, comprising:
(i) determining or measuring the amount of an alternatively spliced phosphoStat5a and/or a phospho-JAK2;
(ii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2; or
(iii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2 transcript or message;
wherein an increased amount of, or the presence of, the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, determines or predicts that the self-renewing leukemic stem cell (LSC) or cells will be resistant, or relatively resistant, to a selective JAK2 inhibition therapy, drug or treatment, or
a decreased amount of, or lack of, the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, determines or predicts that the self-renewing leukemic stem cell (LSC) or cells will be sensitive or responsive to a selective JAK2 inhibition therapy, drug or treatment.
In alternative embodiments, the invention provides compositions and methods for distinguishing leukemic progenitors from their normal counterparts, comprising:
(i) determining or measuring the amount of an alternatively spliced phosphoStat5a and/or a phospho-JAK2;
(ii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2; or
(iii) determining or measuring the amount of an alternatively spliced Stat5a and/or a JAK2 transcript or message;
wherein the presence of the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, distinguishes the leukemic progenitors from their normal counterparts.
In alternative embodiments of methods of the invention, the method detects a cancer stem cell specific JAK/STAT signaling pathway splice isoforms by RNA sequencing qRT-PCR and/or nanoproteomics.
In alternative embodiments of methods of the invention, the LSC is a chronic myelogenous or myeloid leukemia (CML) stem cell, or a cancer stem cell in a primary or metastatic niche, or a cancer stem cell in a setting of inflammatory cytokines and interleukins as elaborated in a cancer.
In alternative embodiments of methods of the invention, the presence, absence and/or amount of the alternatively spliced phosphoStat5a and/or a phospho-JAK2, or alternatively spliced Stat5a and/or a JAK2 transcript or message, or alternatively spliced Stat5a and/or a JAK2 transcript or message, is measured by a procedure or device comprising (or comprising use of): a fluorescent activated cell sorter (FACS), an array, an immunoassay, an immunoprecipitation, a kit, a polymerase chain reaction (PCR), a qRT-PCR, a nanofluidic assay or device, a nanofluidic proteome assay, a chromatography, a nanoproteomics quantification, or an isoelectric focusing assay, or any combination thereof.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.



DESCRIPTION OF DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
RNA Editing as a Novel Cancer Stem Cell Target
FIG. 1A graphically illustrates data showing that the expression of ADAR1 p150 is increasing as CML progresses from CP to BC; the values were normalized to HPRT; a significant increase of ADAR1 p150 expression was observed in CML BC comparing to CML CP. FIG. 1B graphically illustrates data showing the ratio of ADAR1 isoforms (p150/p110) increases from normal cord blood; qRT-PCR of ADAR1 isoform mRNA in FACS sorted CD34+38+lin− cells from normal cord blood (n=8), CML CP (n=6) and CML BC (n=7); as described in detail in Example 1, below.
FIGS. 2A and 2B graphically illustrate data showing that an increase in ADAR1 expression is driven by Bcr-Abl in CML BC patients; Bcr-Abl and ADAR1 expression were analyzed in CML CP (n=6) and CML BC (n=4); a positive correlation is observed in only CML BC instead of CML CP; as described in detail in Example 1, below.
FIG. 3 graphically illustrates data showing that knock down of ADAR1 leads to increase of Bfu-E colonies and decrease of Macrophage (M) colonies in CML CP (n=3) and BC (n=1) but not in normal cord blood (n=3); sorted CD34+38+Lin− cells from normal cord blood, CML CP and BC patients were transduced with either shADAR1 or shControl lentivirus, *, p<0.05, **, p<0.005; ***, p<0.0001; for CML CP p=0.0037, p=0.0001, (n=3); for CML BC (n=1), p=0.0167, p=0.0030; as described in detail in Example 1, below.
FIG. 4 graphically illustrates data showing lentiviral overexpression of ADAR1 leads to decrease of Bfu-E colonies in normal cord blood (n=3); sorted CD34+38+Lin− cells from normal cord blood were transduced with either ORF control or ADAR1 overexpression lentivirus. *, p<0.05, p=0.029; as described in detail in Example 1, below.
FIG. 5 illustrates representative pictures of GFP+ colonies; as described in detail in Example 1, below.
FIG. 6 graphically illustrates data showing that ADAR1 knockdown leads to a universal decrease of self-renewal capacity; individual colonies from FACS sorted CD34+38+lin− cord Blood (n=4), CML CP (n=3), and CML BC (n=1) were replated in to 96 well-plates and replate efficiency was observed after 14 days. *, p<0.05; as described in detail in Example 1, below.
FIG. 7 graphically illustrates data showing that ADAR1 expression is significantly positively and negatively correlated with PU1 and GATA1, respectively; FIG. 7A: mRNA from individual colony formed from sorted CD24+38+Lin− CML patient was measured for expression of ADAR1, PU1, and GATA1 using qRT-PCR; FIG. 7B: cord blood sorted CD34+38+Lin− cells were transduced with either ORF control or ADAR1 overexpression lentivirus; the expression levels of ADAR1, PU1, and GATA1 were analyzed; as described in detail in Example 1, below.
Compositions and Methods for Dormant Cancer Stem Cell Detection and Elimination
Sonic Hedgehog Targets as Biomarkers of Prognosis and Response for Human Chronic Myelogenous Leukemia
FIG. 8a graphically illustrates data where GLI1 and GLI2 transcripts were compared by TaqMan RT-PCR in FACS-purified human cord blood and peripheral blood CD34+CD38+Lin−PI− progenitor cells, chronic phase CML and in blast crisis CML patient samples; and comparative qRT-PCR analysis of GLI3 transcript levels was performed on normal, chronic phase and blast crisis CML cells; FIG. 8b graphically illustrates data from a FACS analysis that showed a reduction in leukemic progenitor survival following 7 days of PF-04449913 compared with vehicle (DMSO) treatment in SL/M2 co-cultures; FIG. 8c graphically illustrates data from the down regulation of GLI expression, as analyzed by TaqMan RT-PCR in human LSC engrafted bone marrow derived from PF-04449913 and vehicle treated mice; FIG. 8d graphically illustrates data showing spleen size in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle or PF-04449913; FIG. 8e illustrates images of an immunofluorescence analysis of splenic sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913: Photomicrographs of sections stained with DAPI (upper panel) and antibodies specific for human CD45 (upper middle panel), human GLI (lower middle panel) and the merged image (lower panel); FIG. 8f graphically illustrates data from isoform-level transcriptome measurements of Shh pathway genes in vehicle-treated and PF-04449913-treated CML BC LSC and in normal CD34+CD38+Lin− FACS-purified progenitors; FIG. 8g in tabular format presents data showing a significant concordance was observed in the relative expression of the 50 isoforms in PF-0449913-treated and in normal cells relative to vehicle treated cells; FIG. 8h graphically illustrates data of nanoproteomic (CB1000) traces of total GLI protein after vehicle and PF-04449913 treatment; and, FIG. 8i graphically illustrates the quantification of GLI protein expression in splenic CD34+ cells derived from vehicle (n=3) or PF-04449913 treated LSC engrafted mice; all figures are also further described, below.
FIG. 9a graphically illustrates data showing the frequency of CD45+ cells in hematopoietic tissues of blast crisis CML engrafted mice; FIG. 9b graphically illustrates data from a FACS quantitation of common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP) populations within each hematopoietic tissue; FIG. 9c graphically illustrates data showing a comparison of cell cycle status of human CD45+ blast crisis CML progenitors engrafted mice in the bone marrow and spleen; FIG. 9d illustrates representative FACS plots comparing Ki67 and 7AAD in bone marrow engrafted viable human CD45+ cells after 14 days of vehicle or PF-04449913 treatment; FIG. 9c graphically illustrates data from a gene set enrichment analysis for the significantly down-regulated pathway “Regulators of Cell Cycle”; FIG. 9f graphically illustrates data showing isoform-level transcriptome measurements of cell cycle genes in vehicle-treated and PF-04449913-treated blast crisis CML LSC and in normal CD34+CD38+Lin− FACS-purified progenitors; and, FIG. 9g graphically illustrates data showing the relative expression of the 75 isoforms in PF-0449913-treated and in normal cells relative to vehicle treated cells; all figures are also further described, below.
FIG. 10a graphically illustrates data showing: Left, Differentiation into CFU-Mix (black), BFU-E (red), CFU-G (orange), CFU-M (yellow), CFU-GM (blue) of normal cord blood HSPC was assessed in hematopoietic progenitor assays (n=3) after PF-04449913 (1 μM) or vehicle treatment for 12 days, Right, illustration of representative photomicrographs of cord blood colonies after 12 days of treatment with vehicle (DMSO) or PF-04449913 (1 μM); FIG. 10b graphically illustrates FACS analysis data from experiments where human cord blood (n=3), CD34+38+Lin−PI− cells were plated on SL/M2 stroma and treated with vehicle (DMSO) or PF-04449913 (1 μM) for 7 days followed by FACS analysis; FIG. 10c illustrates representative FACS plots depicting HSPC, myeloid and lymphoid differentiation in human cord blood engrafted mice after 14 days of treatment with vehicle (n=3) or PF-04449913 100 mg/kg (n=4), after staining with murine CD45, CD34, CD13 and CD4; FIG. 10d graphically illustrates data from a FACS analysis used to determine the total human CD45+, HSPC, myeloid and lymphoid cell count in bone marrow after 14 days of treatment with vehicle (n=3, green) or 100 mg/kg of PF-04449913 (n=4, purple); FIG. 10e graphically illustrates data summarizing a FACS quantification of G0 (green), G1 (light blue) and G2/S (navy) human CD45+ cells in cord blood engrafted marrow after 14 days of treatment with vehicle (n=3) or PF-4449913 100 mg/kg (n=4); all figures are also further described, below.
FIG. 11a schematically illustrates in vivo experiments where RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ cells within 48 hours of birth; and after 8 to 10 weeks, blast crisis CML engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or combination (PF-04449913 100 mg/kg and Dasatinib 50 mg/kg); and then hematopoietic tissues were FACS analyzed for leukemia engraftment and qRT-PCR for BCR-ABL1 transcripts; FIG. 11b graphically illustrates a myeloid sarcoma count of blast crisis CML engrafted mice in each treatment group vehicle (n=13, green), PF-04449913 (n=7, purple), dasatinib (n=6, red) and combination (n=3, black) after 14 days of treatment; FIG. 11c graphically illustrates BCR-ABL1 transcripts in the spleens of blast crisis CML engrafted mice after 14 days of treatment with vehicle (green, n=9), PF-04449913 (purple, n=11), dasatinib (n=8, red) or combination (n=5, black); FIG. 11d graphically illustrates Shh gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n=3, green), PF-04449913 (n=4, purple) dasatinib (n=4, maroon), combination (n=3, dark grey) was analyzed via qPCR array (SA Biosciences); FIG. 11e graphically illustrates FACS analysis of percentage of marrow engrafted blast crisis LSC (n=3 patients) after 14-day treatment with vehicle (n=31, green), PF-04449913 (n=25, purple), dasatinib (n=27, maroon) and combination (n=27, grey); FIG. 11f graphically illustrates myeloid sarcoma counts in mice serially transplanted with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n=8, maroon) or combination (n=3, grey) treated human progenitors; all figures are also further described, below.
FIG. 12a schematically illustrates the chemical structure of PF-04449913, a selective smoothened (SMO) antagonist; FIG. 12b graphically illustrates data from a competition-binding assay using a characterized cyclopamine-competitive SMO antagonist. PF-04449913 competes with the radiolabeled SMO antagonist for binding to human SMO (amino acids 181-787) with an IC50 of 4 nM; FIG. 12c graphically illustrates data from a study inhibiting Shh-stimulated luciferase expression using mouse embryonic fibroblasts expressing luciferase under control of an 8× Gli-response element (Gli-Luc MEFs); FIG. 12d graphically illustrates data from a study showing dose-dependent inhibition by PF-04449913 in the Gli-Luc MEF reporter assay; PF-04449913 inhibits Shh stimulated reporter activity with an IC50 of 6.8 nM (n=5); all figures are also further described, below.
FIG. 13a graphically illustrates data from a study showing anti-tumor activity of PF-04449913 against Ptch+/−p53+/− medulloblastoma; FIG. 13b graphically illustrates data from a study showing dose dependent anti-tumor efficacy of PF-04449913 against Ptch+/−p53+/− medulloblastoma allografts; FIG. 13c graphically illustrates data from a study showing Hh pathway inhibition in PF-04449913 treated Ptch+/−p53+/− medulloblastoma allografts; FIG. 13d graphically illustrates data from a study showing which genes are significantly down-regulated by PF-04449913 treatment in Ptch+/−p53−/− mice; FIG. 13e graphically illustrates data from a study showing gene signatures are enriched for within the top 31 PF-04449913-downregulated genes in Ptch+/−p53−/− mice; all figures are also further described, below.
FIG. 14a graphically illustrates data from a study showing the percentage of weight changes in groups of mice over the course of 14 days of treatment with vehicle, PF-04449913, dasatinib, or a combination thereof; FIG. 14b illustrates representative photographs of spleens from no transplant, blast crisis CML engrafted mice treated with vehicle or PF-04449913; FIG. 14c graphically illustrates representative FACS plots of blast crisis CML engrafted mice treated with vehicle, PF-04449913, dasatinib, or combination; FIG. 14d graphically illustrates total blast LSC count in bone marrow, spleen and liver after 14 days of treatment with vehicle (n=15) or PF-04449913 (n=14); all figures are also further described, below.
FIG. 15a graphically illustrates heat-map of normalized expression values on log 2 scale for 10,573 highly expressed genes in FACS-purified primary blast crisis CML CD34+CD38−Lin− and CD34+CD38+Lin−; RAG2−/−g-c−/− marrow engrafted blast crisis CML CD34+CD38−Lin− and CD34+CD38+Lin−; and normal cord blood CD34+CD38−Lin− and CD34+CD38+Lin− samples; FIG. 15b graphically illustrates a gene set enrichment analysis (GSEA) summary table obtained from SOLiD RNAseq data comparing PF-04449913 treated mice (n=4) to control (n=4) (average 24.7-58.0 million mapped reads/sample); FIG. 15c graphically illustrates a GSEA enrichment plot for the significantly down-regulated pathway (regulation of Cell Cycle); FIG. 15d graphically illustrates a cell cycle analysis of bone marrow from blast crisis CML engrafted mice after 14 days of vehicle (n=8) or PF-04449913 (n=8), *p<0.05 for both G0 and G1 population compared with vehicle treatment; all figures are also further described, below.
FIG. 16a schematically illustrates a heatmap from unsupervised agglomerative hierarchical clustering of sonic hedgehog (SHH) pathway genes using RNA Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients, 3 normal cord blood (CB) and 3 normal peripheral blood (NPB) sample; FIG. 16b graphically illustrates a principal components plot derived from RNA Seq data for 41 genes in the SHH pathway, from 8 chronic phase (CP; black triangles) and 9 basic crisis (BC; red circles) subjects, as well as 3 cord blood normal samples (CB; blue diamonds) and 3 normal peripheral blood (NPB; blue circles); FIG. 16c graphically illustrates box plots for GLI2 expression of 7 chronic phase (CP) and 6 blast crisis (BC) non-treated subjects, as well as 3 cord blood normal samples (CB) and 3 normal peripheral blood (NPBc); FIG. 16d graphically illustrates quantitative RT-PCR data where GLI1 and GLI2 transcripts were compared using quantitative RT-PCR in FACS-purified human cord blood and normal peripheral blood CD34+CD38+Lin−PI− progenitor cells (n=9, black), chronic phase CML (n=7, blue) and in blast crisis CML (n=10, red) patients samples; all figures are also further described, below.
FIG. 17a schematically illustrates the chemical structure of PF-04449913, a selective smoothened (SMO) antagonist; FIG. 17b graphically illustrates a FACS analysis, which revealed a significant reduction in blast crisis leukemic progenitor survival (n=4 patients) following 7 days of PF-04449913 (1 mM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures; FIG. 17c graphically illustrates data from a Colony forming unit (CFU) survival experiment where cord blood (n=3) or AML (n=4 patients) CD34+ cells were plated on SL/M2 co-cultures and treated with vehicle (DMSO) or PF-04449913 (1 uM) for 7 days, and colony forming unit (CFU) survival was determined and compared to vehicle treatment; FIG. 17d graphically illustrates spleen weight in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n=16, blue) or PF-04449913 (n=12; 100 mg/kg daily, purple); FIG. 17e graphically illustrates nanoproteomic (CB1000) traces of total GLI2 protein after vehicle (blue) and PF-04449913 (green) treatment; FIG. 17f graphically illustrates a quantification of GLI2 protein expression in sorted progenitors derived from vehicle (n=3) or PF-04449913 (n=3) treated LSC engrafted mice. GLI2 expression was determined after normalizing the area under the curve (AUC) to a β2-microglobulin (b2M) loading control (Student's t-test *p=0.001); FIG. 17g illustrates photomicrographs of confocal fluorescence microscopic images of an analysis of spleen sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913; all figures are also further described, below.
FIG. 18a graphically illustrates a heatmap from unsupervised agglomerative hierarchical clustering of cell cycle pathway genes using RNA Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients sample; FIG. 18b schematically illustrates a network analysis performed on differentially expressed genes between BC and CP revealed CDKN1A as a key hub for cell cycle difference; FIG. 18c graphically illustrates representative FACS plots comparing Ki67 and 7AAD in bone marrow engrafted viable human CD45+ cells after 14 days of vehicle or PF-04449913 treatment; FIG. 18c graphically illustrates data from a cell cycle analysis of bone marrow from blast crisis CML engrafted mice after 14 days of vehicle (n=8) or PF-04449913 (n=8); FIG. 18e schematically and graphically illustrates a GSEA enrichment plot for the significantly down-regulated pathway; FIG. 18f graphically illustrates normalized gene expression values for the 18 genes in the core enrichment subset from the “Regulation of Cell Cycle” pathway; all figures are also further described, below.
FIG. 19a summarizes the characteristics of patients enrolled in clinical trial NCT01546038; FIG. 19b graphically illustrates the clinical response to PF-04449913 in the bone marrow of AML patient samples; FIG. 19c illustrates representative FACS cell cycle plots of Ki67 and 7AAD staining of human CD34+CD38− and CD34+ CD38+ cells derived from primary patient samples after 4 weeks (C1D28) of treatment with PF-04449913 (40 mg) on the Phase I clinical trial; FIG. 19d graphically illustrates cell cycle analysis (peripheral blood-CD45+PI−) from a secondary AML patient that was treated with PF-04449913 (40 mg) for 4 weeks on the Phase I clinical trial; FIG. 19e summarizes the characteristics of patient samples analyzed for their cell cycle study; all figures are also further described, below.
FIG. 20a schematically illustrates in vivo experiments using RAG2−/−γc−/− pups that were transplanted intrahepatically with 50,000 CD34+ cells within 48 hours of birth, as discussed in detail, below; FIG. 20b graphically illustrates a myeloid sarcoma count in blast crisis CML engrafted mice in each treatment group vehicle (n=13, blue), PF-04449913 (n=7, purple), dasatinib (n=6, red) and combination (n=3, black) after 14 days of treatment; FIG. 20c graphically illustrates a FACS analysis of percentage of marrow engrafted blast crisis progenitor LSC (n=3 patients) after 14-day treatment with vehicle (n=31, blue), PF-04449913 (n=25, purple), dasatinib (n=27, maroon) and combination (n=27, grey), Graph shows percentage of CD34+CD38+lin− cells in the bone marrow; *p<0.05 by ANOVA and Tukey post-hoc analysis; FIG. 20d graphically illustrates data showing the amount of BCR-ABL transcripts in the blast crisis CML engrafted marrow mice after 14 days of treatment with vehicle (blue, n=9), PF-04449913 (purple, n=11), dasatinib (n=8, red) or combination (n=5, black); FIG. 20e graphically illustrates a qPCR array assay showing Hedgehog pathway gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n=3, blue), PF-04449913 (n=4, purple) dasatinib (n=4, maroon), combination (n=3, dark grey) was analyzed by hedgehog (SHH) (SAbiosciences); FIG. 20e graphically illustrates data of myeloid sarcoma count from mice serially transplanted with FACS purified human progenitors from LSC engrafted mice treated with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n=8, maroon) or combination (n=7, grey); all figures are also further described, below.
FIG. 21a graphically illustrates data from a competition-binding assay using a characterized cyclopamine-competitive SMO antagonist; FIG. 21b graphically illustrates data showing that the inhibition of Shh stimulated luciferase expression using mouse embryonic fibroblasts expressing luciferase under control of an 8× GLI-response element (GLI-LUC MEFs); FIG. 21c graphically illustrates data showing the dose dependent inhibition by PF-04449913 in the GLI-Luc MEF reporter assay; FIG. 21d graphically illustrates data showing anti-tumor activity of PF-04449913 against Ptch+/−p53+/− medulloblastoma; FIG. 21e graphically illustrates data showing the dose dependent anti-tumor efficacy of PF-04449913 against Ptch+/−p53+/− medulloblastoma allografts; FIG. 21f graphically illustrates data showing genes significantly down-regulated by PF-04449913 treatment in Ptch+/−p53−/− mice; FIG. 21g graphically illustrates data showing Hh pathway inhibition in PF-04449913 treated Ptch+/−p53+/− medulloblastoma allografts; FIG. 21g summarizes the gene signatures enriched for within the top 31 PF-04449913-downregulated genes in Ptch+/−p53−/− mice); all figures are also further described, below.
FIG. 22a is a GSEA analysis summary table obtained from RNA sequencing data comparing PF-0449913 treated engrafted mice (n=4) to control (n=4) (average 24.7-58.0 million mapped reads/sample); FIG. 22b summarizes the characteristics of patients enrolled and sequenced using the gene expression profile by Affymetrix GENECHIP 1.0 STTM after PF-04449913 treatment for 28 days (C1D28); FIG. 21c summarizes a GSEA analysis summary table obtained from patients (n=8) sequenced after PF-04449913 treatment (C1D28); all figures are also further described, below.
FIG. 23a graphically illustrates data showing the differentiation into CFU-Mix (purple), BFU-E (red), CFU-G (orange), CFU-M (yellow), CFU-GM (blue) of normal cord blood progenitors, as assessed in hematopoietic progenitor assays (n=3) after PF-04449913 (1 mM) or vehicle treatment for 14 days; FIG. 23b graphically illustrates data from a FACS analysis used to determine the total human CD45+, hematopoietic stem and progenitor cell (HSPC), myeloid and lymphoid cell count in bone marrow after 14 days of treatment with vehicle (n=3, green) or 100 mg/kg of PF-04449913 (n=4, purple); FIG. 23c graphically illustrates data from a FACS quantification of G0 (green), G1 (light blue) and G2/S (navy) human CD45+ cells in cord blood engrafted marrow after 14 days of treatment with vehicle (n=3) or PF-04449913 100 mg/kg (n=4); FIG. 23c illustrates representative FACS plots depicting HSPC, myeloid and lymphoid differentiation (panel B) in human cord blood engrafted mice after 14 days of treatment with vehicle or 100 mg/kg of PF-04449913; all figures are also further described, below.
Spliced Isoform Biomarkers to Assess Responses to Cancer Stem Cell Targeted Therapies
FIG. 24 illustrates a mechanism of action analysis using nanoproteonomics (CB1000) technology to show the nanoproteomics of SAR302503, panels show phospho-JAK2 protein (upper left); total JAK2 protein (upper right); phospho-STAT5A protein (lower right) and β2-microblobulin (lower right) status after vehicle (blue) or selective JAK2 inhibitor (SAR302503; green) treatment; all figures are also further described, below.
FIG. 25A graphically illustrates RNA-seq-based expression levels of isoforms involved in the Jak/Stat pathway, in vehicle-treated (blue) and SAR302503-treated (red) blast crisis CML sorted progenitors; FIG. 25B graphically illustrates specific isoform expression after treatment with SAR302503 (JAK2 inhibitor) and dasatinib (BCR-ABL inhibitor), relative to vehicle treatment; all figures are also further described, below.



Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
RNA Editing as a Novel Cancer Stem Cell Target
The invention provides compositions and methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, e.g., leukemia or Chronic Myeloid Leukemia (CML). While the invention is not limited by any particular mechanism of action, compositions and methods of the invention can slow or inhibit RNA editing by, e.g., inhibiting or slowing the expression of or the activity of an ADAR1 gene (adenosine deaminase acting on RNA 1), an ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 polypeptide.
Unlike many known treatments for CML, which focus directly on the Bcr-abl protein pathway, this invention targets the RNA editing events that lead to Chronic Myeloid Leukemia (CML) progression. ADAR1 is an RNA editing enzyme, required for hematopoiesis; and levels of ADAR1 were assessed in isolated human normal and cancer stem cells (CSC) at various times during the progression of CML. Data described herein demonstrates a crucial role for ADAR1 in both cell differentiation and self-renewal of hematopoietic stem cells. Hence, in alternative embodiments, the invention provides compositions and methods that target, or inhibit ADAR1, and treat or ameliorate diseases and conditions responsive to the inhibition of cell differentiation and/or self-renewal of hematopoietic stem cells, such as cancer, e.g., CML. In alternative embodiments, the invention provides a model for the development of therapeutics for treating CML patients, as well as for diagnosing and monitoring CML patients.
Experiments described herein using cells isolated from normal cord blood, CML chronic phase (noted as “CP” in figures), and CML blast crisis (noted as “BC” in figures) indicate that the expression of ADAR1 p150 is increasing as CML progresses from Chronic to Blast phase. Results include: qRT-PCR data that show that blast crisis leukemia stem cells harbor higher levels of ADAR1 p150 isoform, (vs. chronic phase or normal); increased ADAR1 expression (in vitro transduction of lentiviral ADAR1 p150) changes normal and chronic phase progenitors to a preferred differentiation to a GMP (Granulocyte-macrophage progenitor) population found in the leukemia stem cells in CML; and ADAR1 knockdown (shRNA) leads to a universal (blast and chronic phase) decrease of self-renewal capacity.
ADAR1 is significantly upregulated in cancer stem cell population as CML progresses from chronic phase to blast crisis, the final phase of the disease, and because ADAR1 deletion in CML patient sample reduces cancer stem cell renewal capacity, the compositions and methods of the invention are effective for diagnosing, treating and ameliorate diseases and conditions responsive to the inhibition of cell differentiation and/or self-renewal of hematopoietic stem cells, such as cancer, e.g., CML.
Polypeptides and Peptides
In alternative embodiments, the invention provides compositions and methods for treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells, e.g., leukemia or Chronic Myeloid Leukemia (CML), comprising use of polypeptides, e.g., antibodies, and/or peptides, e.g., aptamers, that inhibit or slow the expression of or the activity of: an ADAR1 gene (adenosine deaminase acting on RNA 1), and ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 enzyme.
Polypeptides and peptides used to practice the invention (e.g., an ADAR1 enzyme-inhibiting peptide or polypeptide) can comprise a recombinant protein, a synthetic protein, a peptidomimetic, a non-natural peptide, or a combination thereof. Peptides and proteins used to practice the invention can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides used to practice the invention can be made and isolated using any method known in the art. Polypeptide and peptides used to practice the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) including any automated polypeptide synthesis process known in the art.
Antibodies
In alternative embodiments, compositions and methods of the invention comprise use of antibodies to inhibit or slow the expression of or the activity of: an ADAR1 gene (adenosine deaminase acting on RNA 1), and ADAR1 gene product, an ADAR1 transcript, and/or an ADAR1 enzyme.
In alternative embodiments, an antibody for practicing the invention can comprise a peptide or polypeptide derived from, modeled after or substantially encoded by an ADAR1 gene (SEQ ID NO:1) or transcript (SEQ ID NO:2), or a peptide or polypeptide derived from, modeled after a protein as set forth in SEQ ID NO:3, or subsequences thereof, or immunogenic fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
In alternative embodiments, an antibody for practicing the invention includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen (e.g., a Bel-2 family protein, or immunogenic fragments thereof) including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”
Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
In alternative embodiments, antibodies used to practice this invention comprise “affinity matured” antibodies, e.g., antibodies comprising with one or more alterations in one or more hypervariable regions which result in an improvement in the affinity of the antibody for antigen; e.g., an ADAR1 protein, or immunogenic fragments thereof. In alternative embodiments, antibodies used to practice this invention are matured antibodies having nanomolar or even picomolar affinities for the target antigen, e.g., a targeted transcriptional activating factor. Affinity matured antibodies can be produced by procedures known in the art.
For example, one embodiment, antibodies used to practice this invention are designed to bind to, or affinity matured to bind to, a polypeptide encoded by SEQ ID NO:1, or subsequences thereof:








(SEQ ID NO: 1)










    1
gaccagacca ttgattcccg actgaaggta gagaaggcta cgtggtgggg gagggtgggg



 


   61
ggagggtcgc ggccgcactg gcagoctccg ggtgtccggc cgtgtcccga ggaagtgcaa


 


  121
gacccggtaa gagcctccgt ccttctcggc tacacctgcc tgggctggaa cgcgcggccc


 


  181
atgcggcctc tccagtctct ggcgccgatg ttagaggaag cgtgggggcg ccccggcagg


 


  241
gcactgaggg tggccgcagg gctgggtggg gacgcagctg gtaggggcac agtggccggt


 


  301
ctcggcagcc ttccaggagg cggacgcccg ggccggtgta cttttgtgcg tgtgtgcgcg


 


  361
cccgtgtgtg cgcgagtgtg ggcggcagag gctgcgcacg gatgctccgg cgctcgtgcc


 


  421
agccggagcc cagcagctgg gtaccaaagg cccaacagct gggtaccaaa ggcccttgtt


 


  481
tccctctcgc cggctccccc gcctcggaga gtgactggag agtgagctgc ctgggactcg


 


  541
ccgcggtagg cgcttttgct gcgccttcta coacaaactg cgttaggacc ggccctttat


 


  601
cccagagata acatcgccgc cctggcgtgc cttccacagg gaaggcgtag gaggccactg


 


  661
tggaaagctc actgcggggt cacggccgcc gcgctcagcc tgtctggcct ggtgcaggag


 


  721
gcctagttgc gtcacctcct cttccttctc tgtttagctt gatttggggg ccccactaga


 


  781
gggtaatccg gccccaggtg tttcgtgttg gtatcagagt ttgggaaact tgccttccaa


 


  841
aaagggatac ctgtcattgg attttgaatg ttgtgtggag gagcccaagt atgcaattag


 


  901
cggagggagc agagtctagg ctgccacggg aggagacttg caaacagagg cctacacagt


 


  961
gtcttgttgc tagagaggga ggcaggattg ctagagcggg tcattggggg cagagaaggc


 


 1021
aggggcgtca aactgtcagc agttgtgatg ccaacttctt tctccaccag agaagccttg


 


 1081
ttcccatcct tttaaagatc tcttgaaata tcttggtcgt tatttacaaa cagttagtgc


 


 1141
ttgctatatg ggaaaaaggg aaatagagtg gaaaggggac atgaactttg gagtcaggtc


 


 1201
tgccactttg cagctgtttg acattgaatg agttatttaa cctccccact cctacttggg


 


 1261
cttatctaca aaatgaagat aattatatct accttgcccg aggtgtcaga attcgaagtg


 


 1321
tctagctatt attagtaagg gcccagtacc caggagcttg gacttgctcc cttgctgaag


 


 1381
gtttcctatt ggtacagctg cttgagaagc aggggaactt tttgctattt tagatgtttg


 


 1441
ctattctgcc agatatgata tgcagacttg ggggtggtag tggaggggaa atctcaaaat


 


 1501
ccataatctc tctgctgtac acttcattaa attctagaac tgcaaagagg tgaagatctc


 


 1561
actttaggcc gggtgcagtg gctcatgcct gtaatcccag cactttggga ggccgaggca


 


 1621
ggtggatcat gtgaggtcag gagttcgaga ccagcctggc caacatggtg aaaccccgtc


 


 1681
tctactaaaa atacaaaaat tagaccagca tgatggtgca catctgtgat cccagctact


 


 1741
tgggaggctg aggcaggacc atctctcaaa cccaggtggt ggaggttgca gtgagccaat


 


 1801
atccaccatt gcaccccagc ctggacgaca gcgaaactgt atctcaaaaa aaaaaaaact


 


 1861
cactttatta attatagctt ctcattttat tttattattt ttcaatttta tcccgagtag


 


 1921
ctgggatcac agatgtgcac caccatgcct ggctaatttt tttttttttt tttttttgag


 


 1981
acagagtctc actccatcgc gcaggctgga gtgcaatgat gcgatctcag ctcactgcaa


 


 2041
actctacctc ccgggttcaa gtgagtctcc ctcctcagcc tcccgagtag ctgggattac


 


 2101
aggagcatgc caccatgcct ggctaatttt tgtattttta gtaagacagg gtttcaccat


 


 2161
gttggccagg ctggtctcga actcctgacc tcaggtgatc cacctgcctc ggcctcccaa


 


 2221
actgctggga ttacaggtat gagccaccgt gcctggccta tgcccggcta atttttgtat


 


 2281
ttttagtaga gatggggttt caccatgttg gccaggctgg tctcgaactc ctgacctcaa


 


 2341
gtgatctgcg cacttaggcc tcccagagtg atgggattac aggcatgagc caccgcgcca


 


 2401
ggcctttcgc ttctctttag cacttgctat gtacctgaca cggtgttgag cactttacat


 


 2461
atactaactt atttcatccc tctaacaact gagtgcacta aatattatcc ccattttata


 


 2521
gatgcagaag tgaatcttgg ctaaggtcct gacattaatt aacggcaaag ctggaacttg


 


 2581
aagccagctg tgtggtttga gtcttgctct ttctcagtgt ctagcaagtc ttgctcttgc


 


 2641
ccttgctcat caggctatgt ggtttgagtc ttgctctttc tcagtgacta gtatcccagc


 


 2701
aatgtaaacc tggtgcagtg ggggaaagga ggcccagaca gagtttagtg ggccaggatt


 


 2761
ggctgcaaga ataacatctc aaattgtcta atgatacatt atcttttatt ttacattttt


 


 2821
tactcagcat tgtccagata tgaaccaagg tttggccttt tcacccagag aaaaaggaca


 


 2881
ggctcatggg tacactgtgc tgggatgacg ggcacaaagt aggtattcgg aaaatgcttg


 


 2941
tggaaaaaag aaatcctgag gcattgttat ttctgccaga aggaggccca gtgcttatta


 


 3001
cacacaggct ccttaagcca gctattttta tactataaca cactgtaata tgagcatttt


 


 3061
tctgattcat ggatttaaag atttgaagcc ctgtttcaga ccaagattgg tagtattatc


 


 3121
tgtgaccaac tgattagagt tctcaaatat gtgaacaaca gaatctgagc ttttgggctt


 


 3181
ctgtagtaat ctcttgagat agagcaattt gttttgctat aacatctgtt agacttggac


 


 3241
cttaatgggt acaactgctt gagttttctg gagaacactg gatgattgtt tgacatcagg


 


 3301
gataaagagt tcaaaatatt aagtttgtct caaaattaaa tatttgggaa agacctgtga


 


 3361
ttgatgtgca ttcattgtag gaatagtaca ctagttttaa acagatgata ttcctggtat


 


 3421
ttttgatgag ctattctgtg tcttaaagat gtttaaagat gacttgtagt tgtatagtga


 


 3481
actattagga acatacaaaa tttatataac ccatccatat agactgtgac ttacaccccc


 


 3541
ctcagttcct ccacccaagg aggtctagtt gctgcttcta taccctctgg ttaccttgaa


 


 3601
tctacctatt tcaccatctt tatcagactt tatcagactt aataggtgag accctctgac


 


 3661
atcagcctcc ctttccttct gtaccacccc ttgccacatg cacacagaat gaagctcttc


 


 3721
tccaaaagga ctgtatatta atctacttgc aagtggcaca aacccagtgt aaattaactt


 


 3781
aagcaaaaaa aggttttttc ctccctttga ttatttaact agaaagtcca aaggcaagct


 


 3841
tcaggcatgg ctggatcctg gggctaaaat gatgtcatga gggctttctt ggcccttcct


 


 3901
ttttcggctc cgttttcttt attgacttca ttctctcttg ctgctttctt tttctttttt


 


 3961
tttttttgag atggagtctt gctcgtcttg ctctgtcgcc cccaggctgg agtgcagtgg


 


 4021
cacgatcttg gctggcggca acctccacct cccaggttca agtgattctc ctgcctcagc


 


 4081
ctcccgggta gctaggatta caggagcccg tcactacccc cggctaattt ttgtattttt


 


 4141
agtagaaatg gggtttcact atgttggcca ggctggtctt gaactcctga cctcaggtga


 


 4201
tccacccgcc tcagcctccc aaagtgctgg gattacaggc atgagctact gtgcccggcc


 


 4261
ttcttgctgc tttctctatg catactgaaa aagctccctg tcagcaaccc caagcctatg


 


 4321
acctgatggc tgtcttttag cgttgtaaat taaccattgc tgacaaggaa atagggagac


 


 4381
cacaattgga gaggtctaca tcataggctc actcttggga tgaggaagta aactgcccgt


 


 4441
ccataacctt atgggatggg gaagttcaga ggtcctaggc aggcagaaac agcacatgac


 


 4501
tactgcaggg gtgtgtgtat gcacacatgt gtgcttacat gtgtgctctt atctcacttc


 


 4561
tttggggtcc ttctagagcc ttccaactgg aatgaatcca gtgggttgtg tttgtccatc


 


 4621
aaactttgac tcccactgtg gcatccatta tgtgctctcc gtcctttctg tacctttgca


 


 4681
tctgctgttt gggttgccct tccccattgc ttcatctggc tgatccttct agattcaggt


 


 4741
catgcttctc ccagaagcct tccttgacac cttccttaat gagctaggca tcccttctct


 


 4801
ctgctcccat aaacacctga gcacaacccc cttgtagatg ttatttctgt ttgataatta


 


 4861
tctgtttatt ggtcatgtct ccactagacg gagagcccct tgaggacaga gtgtcctttg


 


 4921
ctttgtattc cttatctcct acccttagct agtattattt gtcttcatag cactcataac


 


 4981
tgacattgta tatttatttg tttatttatt gcctgtctct cctcattgac tgtctttcat


 


 5041
tagcttcatg agaacaatgt ggaaaggaat tttctttgat caaagcaaaa caaaactacc


 


 5101
ttgaggccta ggacagtgcc tcatactggc atatagtata tgttcagtaa atgtttatta


 


 5161
aatgaatgaa tggaaatctt cccacaggaa gaaaacactg gcattatgtt gatgaacatt


 


 5221
ccagtctgca tgatagatac acttctcact gtaaagatct gaaacttgag taccatggca


 


 5281
tcccaggaag gtagcacacc tcttcccata tgtgtttgaa cctgcagagg tcaggcccaa


 


 5341
ggagcctcca gctggaaatt ctaccctgtg tgatgtttca ctcctctcaa acttttctga


 


 5401
actttgatag gttgtatatt aaggccttct ccatattttg gggccctttc cttggagtat


 


 5461
ttatttttaa aacatttatt taagcactta caaggtgaca ggcactgttc ttagtccttt


 


 5521
ataaatagca actcactctt ggactggagt ttggggacca agggttgtca actttaaaac


 


 5581
ttttttcttt ttaagtacca aaacactcaa gaacagaaat aagactactc tgcatctagt


 


 5641
ccatcttgcc ctccagaagt agagctatct aacagcctga tacaggtctt gtccctgccc


 


 5701
tttcctcagg tttttacagc catagctagt aaatataaca tactgcatct catcgccttt


 


 5761
gtttggtctt cacccaaacg cagccacgct ctgccgcagg atatgtggca gagccaggtg


 


 5821
gggaatcagt ggctcagagc ccactctgct tttcaggaca taggctgctc aagcatctgt


 


 5881
cttcagccct ctcccaggga gggcctaaac ccacatcctc aggcccctgc agagcacatc


 


 5941
catcttcctg gttacatgtt atgccttatc tgtgggaacc ctagccacgc cacacccaca


 


 6001
cctcaacaca tgaagaaagg gaaaaaatac ccctttcttt cttttgacaa caacaaaaaa


 


 6061
gtacttctcc tacaaataga gacttgaaat gacagggtta ttatttaacc ccgctcttac


 


 6121
tccaaacatc caagcggcag tttcagccta gctgcagtac tctgttgtag tgcttagtcc


 


 6181
tgatttgctt cctaagaagt gaataccagt ttccactgcc atcagcaatg cctgagtact


 


 6241
tactgtgcct ctccctcacc ctaccctcct ctccagaact gagtatttta ctaaaaaagg


 


 6301
aaaatcttag ctaaatttga aatagcatct cattttaatt tgcatttttt attataattg


 


 6361
tttgagccct ttttacccat actgagccat ttttatttct actttttaaa tttttggtta


 


 6421
atgttttgtc tattcactta ttgtgtttaa gtgattttta aaatatacaa atattctgaa


 


 6481
aaacatgagt ttatcctcat ggtaaaaagc aaacaatttt atttgcagtt aattcttttt


 


 6541
agtttattgt ttgccatcta attttggctt tatcattaaa aatacaaatc tatttttcat


 


 6601
tcttttttag acaaatttta ttctttttga tttcttttgc ttgtgagcat aaaaaggttt


 


 6661
ttttccttat tctatattct agtttttttt atggtttgca tttttacata tgacttttaa


 


 6721
aaagctagca ttcagtttta tttcagattt taataagata tttgatgaaa atagtgtaat


 


 6781
attttaaata aaatcagttt taattaaagg tctatgtttt gttttaagga tgtttatatt


 


 6841
actgtactta tattttttct ttttaaataa tttatcagaa tagcgaacag cttgcttgac


 


 6901
aaaactaaga ttcacatata tagataatca taattttagt gttcttagcg tttccataaa


 


 6961
ttgctgcatg aaaagatatt tatgtgtggc tgtctggttt aaagaggcac tcgtgcttgg


 


 7021
caacagcttt tcagcatgta gccaagatga caaattcagc ctcttccagt tcctcttcct


 


 7081
cactatttcc ctaagatttt tttcatggga gcataactat gtgtatctct gtgcacagat


 


 7141
tttatgtgta cctttcaatg tattttaaca aaaatatatc catttaacta tccaaatcaa


 


 7201
gaaataaaac attttcatta cctcagaaag ctcccctgtg gtagtcacca ctacccccaa


 


 7261
aggccaacac tattgatgtc tgatatctat cactatagat tagattagtt ttgcctgttc


 


 7321
taaaatttca tgtcagtatg tgcttttgtt tgtggcttct ttcagtgagg atcaaccatg


 


 7381
ttgcatgtgt cagtatccat tgtgtgaata tgcatcaagt tatttattca ttagttgatt


 


 7441
gacatttggg ttattccagt ttggggctat taagaagaat aaagctctat gaacagtttt


 


 7501
ttttcctgtt ttttattgta gtaaaataca caacataaaa tttaccatct taaaccattg


 


 7561
tttgtttttt tttttgagac ggagccttgc tctgttaccc aggctggagt gcagtggcac


 


 7621
gatcttggct ccctgcaacc tccacctccc aggttcaagc aattctctgc cttagcctcc


 


 7681
caagtagctg ggattacagg cacccaccac catgcccagc taatgtttgt atttttaata


 


 7741
gagacggagt ttcatcacct tggccaggct ggtcttgaac tcctgacctt gtggtccacc


 


 7801
cgccttggcc tcccaaagtg ctgggattac aggcatgagc caccgcgcct ggcccgtaaa


 


 7861
ccatttttaa gtgtatagtt cagtagtgtt aggtatagtc acattgttgt gcaacaaatc


 


 7921
tccagaactt tttcatcttg cagatctaaa actatattca ttaacaactc ctcttttccc


 


 7981
ccatcctcca gcccctggtt ctatggacat tcttgtacaa gtctttttgt ggccattttt


 


 8041
cttgggtaag tacccaggag tataaatgct gggtctagcc ttagaagaaa ctgccttaca


 


 8101
gttttctcga atagttagtc gtaccatttt atgttcccac agtatacaaa aatgctagtt


 


 8161
tgcttcatat cctcaccaac attaggtatt gtgagtcttt ttttatttta gccattctgt


 


 8221
agctgtgaag cggcatccca ttgtggtttt attacatgtt ctagtcagtt atttacatgt


 


 8281
ctgttcccat tactagactg tgaacttcat aagggaaggt tcatgcagta agtagttttg


 


 8341
ttttttttgt tgttgttctt attaaccttc ctcttcttat tttgagggtg atacatgctt


 


 8401
tattattaaa aagaacttaa aaattataga aagctattaa aaagccaata aaatcacagg


 


 8461
ttagcccacc tctcagacat catcaggagt attttggtct ttcaggcagt gctgtgttgg


 


 8521
ttcgtacaga cagaacagtt cccatgatgg ttgattcagc aacccagcat tgttccagcc


 


 8581
cactgccttg ctatcctctg gacatcactt attcccagcg aggagccctt ttctcagaca


 


 8641
gccccagaca gattgttttt gtggtttgct ggccacaaat gtatcacatg tatcactgac


 


 8701
aaaagaagta gaatcctcaa tactggatta aattagatta attaacatcc ttcctctggg


 


 8761
gctggggagg gaacctggcc ttcttaggaa agtggacaga atcagggcac tctcagaaag


 


 8821
ggaggcatag ttctagcgta ggccactaac attatctgcc tctttttttt tgagatggag


 


 8881
tctcactctg ttgcccaggc tggagtgcag tggcacaatc tcagctcact gcaacctctg


 


 8941
cttcccaggt tcaagcgatc ctcctgcctc tgccccacta gtagctggga ttacaagtac


 


 9001
ctgccaccac gcccggctaa tgtttgtatt tttagtagag acggggtttc gccatgttgg


 


 9061
ccaggctggt ctcaaactcg tgacctcagg tgatccacct gcctcggact tccaaagtgc


 


 9121
tgagattaca ggcataagcc accacggccg gcctgcttct tggtgttttt atttaatttt


 


 9181
tttacactta gtctgtcaat acatttgcaa tttgttttgg cttattaata taaggctaga


 


 9241
ctctagactt tttttttttc caaatagcca ttcagttggc ccagctccat ctgttgaata


 


 9301
acgctttttc tcactgctgt ttttggttcc tcatcatgca ttggatttct gcacatacta


 


 9361
ggctctgttt ggagggaggt ggttcttttc tgttagtctg aagctctacc cttgctttat


 


 9421
accagtgctg ccttggttta attagcgtag ctgtcttctt tttaaaaaac tgttttgttt


 


 9481
gttcttgtac ttgaacttta tttattccca tccaatgacc tattaattgc atctgaactt


 


 9541
tagagtaatc attttgtcag gtatccactc cttactgccc caacctcata gtatttgcag


 


 9601
taaatcgtta tttaatttga tattaattag tattcataaa gtattctgcc atcacatttg


 


 9661
agactggtat gtccttctat ttaagacata tattcctcag cagagttttg taattttcca


 


 9721
catgtaagct cttcacattt cttagggtta gtcctagata cttctttttt ttaatttaat


 


 9781
gttttatttg ctattaagaa tatggtattc ttaatagcaa ataaaacatt aaagtatata


 


 9841
aagaatatat acttactata tgaagaaggt atacttatat attactatag taatgtatat


 


 9901
attactatat aaagaatata tactttagat gcatatatac atatgcatat aaagaagcta


 


 9961
ttgtgtttta tatgtttctc ttatattcaa ccatattcct tgactggtat tggttctaag


 


10021
agtttttcag ttgatcctct tgaatcaact agggttatga tgatatcatc tgcaaataat


 


10081
ctatttttct ttttaatagt ttatctctta tttttgtttc ataatgtaga atatgtaaga


 


10141
atttccatag gtagaaaaga tattaaatat atatatttaa catgtgttaa ataccatatt


 


10201
tgttttaaga ccactgagca taaagacttg ctattgattt aataacagat ttggggggca


 


10261
ctatattaaa tgtatactta cactgtaatt catattccaa tatcagaaat gttaggatgt


 


10321
aaaaagcagt gcatcttaga attaaggaaa ataacaatgg taatggtagc cctccttttt


 


10381
ctcgtttgag tggtagtgtc taactgttcc attctacata tgatattggc tgttgattta


 


10441
tgatagacag tctttattat taaggggtcc tcacacatct tcctgtttgg aagaggtttt


 


10501
gtgttggttt aatcagctta ggagggaaag gtgatcatca gggtcacttt cgtgcagaat


 


10561
cccacctctg cccacaattc ctgccttctt tgaactttct ttgttaaagt aagaatctag


 


10621
actctggcat caaagattga tatgctaagc agaagtttac tgcccagcag agaaccagtt


 


10681
gagtgcaaaa gttaggctct gagatctaag ccttgagggt ggcaatgagg gatgaggtag


 


10741
gtttttaaaa ctcatggtct ccatcccata ttaggaaatc aaagtctcca aatgctactt


 


10801
attggccagt gttatctgga ctaggcattg tctaagcaac aagcaggaca ggacgcttgc


 


10861
aggggttttt gataagttat ctctcactat gtctatgagt caggataata gtgccccagt


 


10921
cattgttttg gtagaagtcc agtccactgg ctaggcagcc atgttgggat tcatcacttt


 


10981
gtagtttcta actttttact gtattccata ttggaaagtt cagccctcct ctgagatacc


 


11041
cattgtccac tagaactcca gacagaagtg ctggagaggg cagtgggcca ggtcaggaag


 


11101
acataagcca gagttgaagt acaacagtgg aaagaacaaa gtctttggag cagtgcctct


 


11161
taaccttcgt tctgggtaca cagatctatc tgggagtctg aaagctatgg gctgtctctt


 


11221
gagggaaatg cgcatgttgc gtttttcagt aaatattttt gagcacgctg aaagcaaaac


 


11281
acttgtatag ccctagcttc ttggaacttg tagtaagaga gacagatatt taccaaaaaa


 


11341
ccacatgaag aaatgtaaaa ttacaagtgt aaatatgctc caaaagagag gagggggtct


 


11401
tgaaatcatg cgatgggagg gggtaggggt tagagctcaa gctcttcctc cagaagtgtt


 


11461
gactaccctg agatctgaag ggcaaatcat aggatagtgg aaaggaggag aaataatcca


 


11521
gagagagggg caagtatgtg gcaggaggga gcatggcaat ctagagggag gaaaagaagg


 


11581
ctcagggagg ctgggggaca gagagatggg gcatggtcca acagaaggga gaaggatggc


 


11641
agggctaggc cacgtaaggc tttggagcca tcgtgtaaag atgtttgcct ttatcttaag


 


11701
agcattagga agccatcaaa gtattttttg ttaataatgt aaatatttga tatgcataaa


 


11761
agattcctgc agcatatatg tatcactgaa atacaacaat gaaacgaatg cacgtgaacc


 


11821
taacaccttc ccctgaatga gaaccttatc tgtattgctg aagcctccct tgtcgcctct


 


11881
tggatcccat ctgcctgtgt tccccctccc cccttaattg ctaggttaga attttcttga


 


11941
attccatgca tgacatgatc agattttggt tttcagagat cacacaagct gtgtcgagaa


 


12001
gactggatag aaaagaggcc aaagtacatg cggggaaacc agtttggagg ttgtaatcac


 


12061
aaaacacaat cttactaaaa gtttgtctgc attttatttt attgccttgg aaatttattt


 


12121
ctttcacaga tgatttattt tcatcatttt aaaaatataa attaatggta tcttcctaga


 


12181
aaaaaataaa atcagataaa actaaaagct ctaattctgt tggacaataa tgagaaggct


 


12241
gtactgtttg gcttaactaa aaaatgtggt gaggcatggt ggctcacgcc tgtaattcca


 


12301
gcactcaggg aggccaaggc aggcagattg cttgagccca ggagttcgtg actggcctgg


 


12361
gcaacatagt gcggccctat ttctacgaaa aaaaattttt ttaattagcc aagcatggtg


 


12421
gtatgtgcct gtagtcccag ctgcttaaga agctgaggca ggaggattgc ttgagcccag


 


12481
gagtttgaga ttgcagtgag acatgactgt accactgcac tctggcctgg gtgacagaga


 


12541
ccttgtcttt taaaaaataa ataaaggaca tcatggctgt gcataagtac atctatgaca


 


12601
gctagtctgt tttggccttt tccctttgat gtacccaaat gcagaaactc ctatctcgcc


 


12661
tagcaaagcc tctggcagcc ctggacttgg agctccctgg tttgtattgt ccagcaaaga


 


12721
tgctgtagta gtttcccgtg gctgtctgct gtaaaaaaat aaccacaaac taggtggctt


 


12781
aaaacaatgg aaatttatta tctgacagtg ctggaggcca gaaatctgaa atcagtatca


 


12841
ctgggccatg ttggcagggc tgtgctccct ccagaggctg taggggagaa tccattcctt


 


12901
gcctctccca acctctggtg ggtgctggca tcgcttgtgt tgcggctgcg tcgctctagt


 


12961
cttcaaggct agcatcttcg aatcattctc tactctgtct tcacatggcc ttttcctctg


 


13021
tgtgtgtagg tggaaatttt tttgaacttg ccaacactaa aagaaacact tttaaagacc


 


13081
agtgttcact tgaaaatggc tctctgtcaa attccaagaa aaccagttca ctgaaagtca


 


13141
gttctacaaa agcccctcca tcctacccct ttgcctcctc agtttcctcc tcaatcttct


 


13201
gctctacagc agcagggaga cagcagagca tatttcctct aaattctatt aagaagacag


 


13261
aataaaaact ggtcaattaa gactagggag atttaagaaa aaagtaaatg caacatattg


 


13321
gttgtttcgt aaattggtta tccaaggaat tgaccatttg gcaaactgac tttcggcaaa


 


13381
ttggctgttg gtgaaatcag cctatttccc tgagaaacac tgcagaaggc agggcagtgg


 


13441
ctgccttgag cctgcccagg acaggactgt gactgtcccc tcctgctttc tacaagccat


 


13501
ggagataggg gcattgctct tgcatgaggc tggggctgag agcagccccc tacaggctgg


 


13561
atctggatcc tagggaagaa gaagatggga gatctccccc tttgggtcct gactcaatag


 


13621
aacccaaatg taggccagta gcggaacctc tgtgctagcc agagtcaggc cagaagtcag


 


13681
tcaggtgctg catccaagaa caatctagca tcggagaagc ggcttaaggg tgtcagaata


 


13741
taatgtataa aaccacaagt tgtatagggt caccccgtgg ggtagttatc cgactgaatg


 


13801
tacatttatt agttatacat acttgcaaaa gattgcacac aggcctgtta gtcattatta


 


13861
ttagtattat tttacatatg taatattatt gcaaatgtta tatctttatt atataatact


 


13921
taagcctggc acatatagct agttgataaa taccactttt tttctttgtt actagataac


 


13981
ttactggagt ggataaatgc acttaatagc ttttggagac ctctttttct tctgggggta


 


14041
cctgaggcat ttcttgcttt cttttttttt ttttttgaga tggagtttcg ctcttgttgc


 


14101
tcagggtgga gtgcagtggt gcgatctcgg ctcactgcaa cctccgcctc ccaggttcaa


 


14161
gtgattctcc tgcctcagcc tccctagtag ctaggattac aggcatgtgc caccacaccc


 


14221
ggctaatttt gtatttttag taaagacgga gtttctctat gttagtcagg cgggtctcga


 


14281
actcccgacc tcaggtgatc cacccacctc ggtgtcccaa agtgctggga ttacaggcat


 


14341
gagccaccgc gcctggccac atttcttgct ttcttgtaac ttcaaaagcc agttttagct


 


14401
gggagcaatg gttcacgcct gtaatcccag catttttgga ggccgaggca ggcggatcac


 


14461
ctgagatcag gagtttgcga ccagcctggc caacatggtg agaccccacc tctactaaaa


 


14521
atataaaaat tagccaggca tgatagcgcg tgcctgtagt cccagctact tgggaggctg


 


14581
aggcaggaga atcacttgta cctgggaggt ggaggttgca gtgagccgag atcatgccac


 


14641
tgcactggag cctggataac agagtgagat tctggctcaa aaaaaaaaaa aaaaaaaaag


 


14701
ccatttgtga tcattaacat caatagaata tatgtcagca taaatactgc cacagagcac


 


14761
tactcagcca gttgggcaac tcactcttct ctaccaaaag ctttacaggt tatcaaagca


 


14821
agtgggttta ctgtgggagg ctactgtgaa ttttggaaat taatgttgag ggactagcgc


 


14881
agtggctcac acctgtaatc ccaacatttt gggaggccga ggcaggcgga tcacgaagtc


 


14941
aggagattga gaccatcctg gctaacacgg tgaaacgccg tctctactaa aaatacaaaa


 


15001
aattacccag gcatggtggc atgtgcctgt agtcccagct actcgggagg ctgagacagg


 


15061
agaatcactt gaacctggga ggtggaggtt gcagtgagct gagatcgcac cactgcactc


 


15121
caacctgggc aacagagcga gactctatct caaaaaaaga aagaaagaaa gaaaattaat


 


15181
gttgttagga actgttgtag tggaataaaa actcagtgat aaagaattga ggagaaacaa


 


15241
cgataacaaa aaaggagggc aataaatttg tgcctagcaa ctctgaattg cttactatac


 


15301
atttcctaag agttattcta aaatgatgcc ctggtactta aaaataactg agcaggctgg


 


15361
gcgtggtggc tcacacctgt aatcccagca ctttgggagg ccaagaccgg cagatgacga


 


15421
ggtcaggaga tcgagaccat cctggctaac acagtgaaac cccgtctcta ataaaaatac


 


15481
aaaaaaatag ccgggtgtgg tggcaggcac ctgtagtccc agccactcgg gaggctgagg


 


15541
caggagaatg gcgtgaaccc aggaggcgga gcttgcagtg agccaagatt gcaccactgc


 


15601
actccagcct gggtgacaga gccagactcc atctcaaaaa aaaaaaaaaa aaaaaaaaaa


 


15661
aactgagtaa tttattactt tctttgataa tatgttgcaa attgtttaga atacattcac


 


15721
aggcagtaca tttctgtccc agtatcactg tatgagcaaa tactgaagga gtttgcagac


 


15781
atctgcttta cagtagacag gagatagatg ttccaattgg atatagccca tgtgtgtagt


 


15841
aaagtctggt tataggaaat tacagattaa aaaagactgt tgttgtaaac atgtatccct


 


15901
gcacgcagta aggttaaaca gaagatccac aacaaaccaa attaacaatt actaagtcag


 


15961
actaacagtt acttatagtt aaaaagcaag aatagcaggg attggaaaac aaaaccaact


 


16021
caggagccct gataatagca tgtcctttcc tataggtggg gaagtctcac cccctgaatg


 


16081
cacctataga gctagagctt ggcactgcta tgtggaagcc agacttctga gaatactgag


 


16141
tttctgagta cttcattttt agacagtaac tcttaatcat ttaggtggtt tttaataagt


 


16201
agacctaggt ggtatagata catctggggt catttcacag gcccctgggg aggtgagcca


 


16261
ttctttatag gaagagctcc tcccttgaga ggactgacca cccatgtgtt ggcagtgccg


 


16321
ttacgcacgg ggaagttacg cacagggaaa atgctcgaag ggtaggcacg gatgaatgat


 


16381
gaattaacaa tgggtgattt ttattgtgtt tgcttgtctg tattttctga aataaaccta


 


16441
ttatttttgg ttaatttttt taaagacttt catccaactc gtcaagttcc tacaatagaa


 


16501
cagttaaaaa aaaaaagaca tcatcacctc acaaccatgg gggagttaaa acaattactg


 


16561
agggggagga gatgaaacgt ttatgaataa gactgtctgc agttacttga gctgctaaga


 


16621
ttaaatgaca ataggagttg ttgttctttc aaggcataaa ctgaccttct gttgaggaaa


 


16681
gttcactgct ttgccactgt gtcctatgtt agattaaaag gggtgggggg agcagcggtg


 


16741
cagcttccct cggacagtag ttgtgattaa agcttttctg gatcatttct gaagaagggt


 


16801
aaaggctttc agattgtctt gcctgaagac aagaaagcag aggtaagata taactcctgt


 


16861
tacttaaacg gaggaaaaaa aacatgggga agtggactgg aattgcaaca gcaagagttt


 


16921
gttgtggttc ttgtttgttt ttacctttta aatttgtgaa atacagcata tatatacaga


 


16981
aacatgaata aaacgtaaat gtataatgaa attattggaa agcaaacata tgtagccact


 


17041
acccaagtca agagctagaa catcaccggt acccaaaaga tttcttccca gtcaaaactc


 


17101
ccacctttct ccctagaagt gattaaccac aattttgtct tttttttttt ttttttcttt


 


17161
tttttttttt ttgagacggt gtcttgctct gtctccaggc tggagtgcaa tggcacaatc


 


17221
ttggctcact gcaacctccg actccctgct tcaagcgttt ctaccacgtc agcctcccca


 


17281
gtagctggga ctacaggcac acgccaccat gcccagctaa ttttttgtac ttttagtaga


 


17341
gatggggttt cactatgttg gccaggatgg tctcgatccc ctgacctcgt gatccgccca


 


17401
cctcggcctc ccaaagtgct gggattacag gcatgagcca ctgcgcccgg cctcattttg


 


17461
tcttttatgc agagcatact atagtttggt tttgcctggc ttgggacttt ctgtaagtgg


 


17521
gatcatatag tatacatatg gcttttccca ctcagtatca tatttagata attagctacc


 


17581
ttgttgcatg cgtagatctt tcatttttgt tgctgtattg tattccatca tgggcatatg


 


17641
ccaccattta tccattttac ttttgatgga catttgggtt gtttccagtt tgggggctat


 


17701
taattacaaa taatgcaacc atgaacttct tggggtttac cattaccttt ctagataatg


 


17761
gtaaactggt ttcctgtagt gactaagagc tgctctgcat ccttgctgcc acctggtcta


 


17821
atcagatggc taatgctgtc tgtctggtga atggacagta gctcactgta gctatactgt


 


17881
gcatttctct gatgtgatgg ggttgagaac cttttcatat gtttgatatt ttttatctcc


 


17941
tgtgaagttg tctttcgatt gtttttgccc attgttatac ctactgaggg gtctgttatt


 


18001
ctttcaacta accagccctc tgaaggcgaa tataaggata caaaacatgc ttcctgcccc


 


18061
cgaggagatg acttgacaat tctagaggca gatatatgaa caagaagtac atgataattg


 


18121
tgattactga ggcatccatc ttccctagca gactagctaa aggatggcgc tcttatttgt


 


18181
gtcatatcct gggtgcttaa cacagtatct gccacataat taggtgctta gtaaagtttt


 


18241
gttaaatgat cgaatggggc gtttttagcg cagtgtgcaa gtgccctatt aggggtaggc


 


18301
gcccagtaac tcgagaagca tggagtagga aaccacaaac agcacctgct ccccctcctc


 


18361
tccccctacc tgctgtgggg aaggcctccc ttgtaaattt gaaaggttga ttcacgggaa


 


18421
gccgtggagg aggtgcatgc taggccaacg aatagaatgt gcaaaggccc agaaggaaga


 


18481
cagagcccag cctgcaaggg aatgttaatt tggagtgact aacaccatga aagggcatca


 


18541
gctggagata ctgctataaa gggactgcct tgtaatttca taagcatggg ggtaaaatta


 


18601
agattaaaca cagggaaaga acattctcac aggtgaggat ggtgttgaac cctagaataa


 


18661
tcgtttccca gataccttga acaaaaatcc agcagttaga gaagcctgac catgaagcaa


 


18721
atttgacttt tgtccctcta gataacaaaa gttatctttt tgaaagtaat ggtgtaattt


 


18781
gaatgagtgt agagaagcgc tgaagactga gctttactaa agccttcaga cctggatttg


 


18841
gcagcagcgt ggccttagtc aagcctcggt ttctacacct gcaaagtggg aataatgcct


 


18901
accttggagg gctgttgtga agattaaggg agataataca tgtaaagtac ttaaccattt


 


18961
gcctggtggg gtagttttta tgacctagat cctaaattgt tcactgctgc tgttgctact


 


19021
cttggtactt tttactggct ggcatctgct tgcttaagtt tataacatag taggagcatt


 


19081
aacaaggtcc cacggtgggg accttggtcg tttgacgaga tctgcgctcc cgcccatccc


 


19141
ctcccccccc cctccacatt ggagacgcgg ccaccaccgc gctggcgcgg agagagggag


 


19201
gaccgggcgt catgctgttt ctggcctgag gttttgtgtg cctttgtttt ccttttgctc


 


19261
tattcgtgta ttcctgccta cggcctgtgc ggggaattag gagctcagta ctgaaacggc


 


19321
ggttttccta aacagtaccg gacgggcgcg ggggctgacg cctgtaatcc caacactttg


 


19381
ggaggccgag gtgggcggat ctcttgaagc cgggagttcg agaccaccct ggctaacgtg


 


19441
gtgaaaccct gttcttacta aaaatacaaa aaaaaaaaaa aaaaaaaagc caggagtgat


 


19501
ggcgctcgcc tgtaatccca gctactccgt aggctgaggc aggagaatcg cttgaacccg


 


19561
gggggcagag gttgcagtga gccgagattg cgccattgca ctccagcctg ggcaaaaaga


 


19621
gcgagactcc gcctcaaaaa aaaaaaaaaa agtaccttcc gtagttctca tgcagcggag


 


19681
gggttcgact tgtaaccggc ctgaaaccaa gcgtggcgca agatttgctc aagcccctcc


 


19741
tcttggccaa actttccgga ggggaaggct ttccgaggaa acgaaagcga aattgaaccg


 


19801
gagccatctt gggcccggcg cgcagacccg cggagtttcc cgtgccgacg ccccggggcc


 


19861
acttccagtg cggagtagcg gaggcgtggg ggcctcgagg ggctggcgcg gcccagcggt


 


19921
cgggccaggg tcgtgccgcc ggcgggtcgg gccgggcaat gcctcgcggg cgcaatgaat


 


19981
ccgcggcagg taagccgggc cggccttgga ccttcgccgc cgtctgggtt ctgggacaac


 


20041
ctcacaggct ttgtgttgca gtgcgtagcg tgtgcgtctt gtgagtgtta gagtgtgtgt


 


20101
gtgtgtgtcg tcttgccaag cagcattgct ggtttaggaa tttgtgcgtc ttgtgagtgt


 


20161
gtgtgtgtgg gtgtgtgtcg tcttgccaag cagcattgct ggtttaggaa tttgtgcgtc


 


20221
ttgtgagagt gtgtgtgtgt gtgtgtgcgt gtgtgtgtag tcttgccaag cagcattgct


 


20281
ggtttaggaa tttgtgaatt tgtatcctgc tcattaattc tgcagaatgg agcagtgcgt


 


20341
gaagagggct tgggggaaaa tgcgcccccg tctgagtagg aaggcctgag cccatgtcaa


 


20401
ggcagacaca tcgtctccct ttctgctagg gccccttgtg gaacccccta cccccgcttt


 


20461
agccccactt gaacaacgtt cggactttga gcagcgcaca ctatcctcag ctcaccttat


 


20521
ccacctcctg aaggccttct gggagttaaa aatggcactt aagctgtagg agaaagcttg


 


20581
ttaaccactt tatagctaaa aactgggaaa acacaaatgg ccttcagcag gttaacagat


 


20641
aaactgaaat acatccacat aatgggatac tgcttagtag tgaagaggaa atactgttac


 


20701
aagtaacaac acgggtgact cgcaagtgcg ttatgctaag cacgagaagc cagactcaaa


 


20761
aggctgcata ctgtatgagt ccatttatat gacattctgg aaaaaaaaaa ccacagttat


 


20821
agggatggaa agtggatccg tgggtgccag ggactgggta tcgtggaaga aattgattgt


 


20881
tgagtggcat gaaaaagctt tttagggaaa tagaaatgtt ctatatcttg attgtggtgg


 


20941
cgattgtcga aattcataga tttatacact taaaaggata aattttactg tatctaaatt


 


21001
atatacctca attttgttaa gatatatata tatttttttt tttttaagca ctcctttgaa


 


21061
aggattaagg acgcctaact tgaaggaaaa gcatttctgc acaggtgtca gtgtattgca


 


21121
ctgtggaacc tgtgtggtaa aggcaaaggg ggtagtgctt atctcttgat cctaaatatg


 


21181
tgagaccaga ttaaagtgaa atctgggagg caatgaatgt taaatgagtt gttatgtaat


 


21241
ttgcatagag gtgatgctga gagatttaga aaggatcact gtgggttgct tgctcacttt


 


21301
cttgctctcc tattccgtag ctttccaaat ggctgtactc aacggtggct tggtgtttag


 


21361
gggatttaag gggggcaaaa agaaagatta ataatctcct cctctccctc taaccctact


 


21421
gccctaagat atccttagca aacttacatc tcctttcttt tctctgtgtt cattccattg


 


21481
tgcgcacaca tacacattca tggattttct ctttttgttt agggaaaaaa attataatgt


 


21541
acatactatt ctacaacttt ttgttgtttt attgaacatt atatgattcc taaattatcc


 


21601
ccaggtgaat acaaatagat atgacacatt ttaaaaaaat aaaataactg gccgggcgtg


 


21661
gtagctcatg cctgtaatcc cagcactttg ggaggccgag gagggcggat caggagatcg


 


21721
acaccatcct ggccagcatg gtgaaacccc atctctacta aaaatacaaa aattagctgg


 


21781
gtgtggtggc gtgcgcctgt aatcccagct actccggagg ctgaggcagg agaatcactt


 


21841
gaacccggga ggcggagatt gcagtgagct gagatcacac tgcactccag cctgattgca


 


21901
gtgagccgag atcatgccac tgcactccag cttggcaaca gagcgagact ccgtctcaca


 


21961
agaaaaaaaa taaccgtgtg agtactattc catagaatga atgtttcata atttaattct


 


22021
tctatagaca gacattaaaa tattttccag atttgggcca agagtagcag tttaaaaaac


 


22081
atttagcttt taactgactc tagccacttt gaaacacaat ttttttttcc caaggtcact


 


22141
caaagagcta ataggagaac ccctaagtcc cataattcag ctctgggagc cagcactcac


 


22201
tctgtacaca catttgcctc tgtccctagc aatatggtgg gcgtgagggt gcagcaagag


 


22261
gaacaagaaa gaaatgattg cttgcatagt ggcgtcttgt tcatgcagtc attaattcaa


 


22321
caaatgtttg ttgagaatca gctttgtgcc aagtgctaga gaggttgaga tgattgaagc


 


22381
atagtccttg acccccaaga gctcaccatg gaatcaactg aagcccctca tcagtactgt


 


22441
gttgggaata ttgagagtgg agagttgagt ataacttata ggacacctaa tgttaattac


 


22501
ctttcagaca ctgcaatgtg tgtgtgccat aaaaaaaaaa aaaatccagt agctctgata


 


22561
cgagggaaag taaatggttt aacaggtgct gagtaggaga agctcaagga gaggaaaccc


 


22621
caagggctga agaaggtggg agtcaggagt ctcctgaagc aagtggcatt taaggagctc


 


22681
tataaggaaa gggtcagagt tgtgataggt ggctgtggag ggagatgtgc cacctggatt


 


22741
ggcatgtaga gggatagaaa gattataggc ctttgcaatg gcccagtaag aggtaatgag


 


22801
gggctggaac tggaagaaaa cacatttaag acacagtaca gaggtggcag acaaggtggg


 


22861
acttggcaac tacctgatga gatccaggag atgaggccag gaggcgggca gcaaagatga


 


22921
cgcaggtttc tagccttaat aggctaggag gagagtgatg ccattagcaa taagaactac


 


22981
aggagaagga gctgagtttg agggaactat taatttggtt cagaatatgt gctgtttgag


 


23041
ttatggcagg atatttaagt ggacagactg tcgacatagt tggaaattca gatcttaagc


 


23101
tcccacacaa ggtagtggct ggacatagta gatttgagtg ctcttgcttc agagggctag


 


23161
tttaggttgg ggcagtgatt aaagcaacct aggaaataaa ttataaagga agaagaggtc


 


23221
cttaaaacct tggagactga ttatataaag ggtggatccg ttaatacagt agctattaaa


 


23281
aaattataag gggtgggaaa aagggacaaa gaagaaaaaa gaggtgaaag acctttgctg


 


23341
tgtcaccaaa ccctgggagg agaatttttt aaaagaagag tactcaatcc acagtgaact


 


23401
aaggcatgtt tggttaacac aattgaccac cacacagcga agacccaaat gaggttcaag


 


23461
agaaggaatt tttatggacc ctgttagcac aagtcaaggt ccttctccag taccactggg


 


23521
aagctttgga gaagaaaagg gggacagtgg gccttgggtg gagaagggaa ctgaccatga


 


23581
gatccaggtg gggtgaggag gtgtgtgaag tcagagtggg gaagaatcag ggtggcttac


 


23641
tggcagcttc accggggtca tgcgaggagc aggttccacc agagtaagga gtgaagttgt


 


23701
agaaacacag ggaagtcacc atcagaaaag agcaggagtc aagaaactat ggcccacagg


 


23761
tacaaactac ctgtttttat aaataaagct tcatggtaaa tgaattgtaa ataaagtttt


 


23821
gcatattgtg tgaggctgtt tttgtgctac agtggcagag ctgtctggcc ctttatagaa


 


23881
aaagtttatc agccactgga aaagagttgc aggatttgag gtcttggtgt gatggcctag


 


23941
gttagagtca tagtgagatt gaaggagtag ctagacaagc caattgtgtg gcagaaaggt


 


24001
agggatggag atcactgggt taaggatcct tgtagccagg acaccatgag agtgattgac


 


24061
aagaatgctg aaatccccta agtgtgtcag gatgtggaag agtagaaggc tagagttatg


 


24121
gaagaaaggg tcccacctct acctgtgcag cctccaggag agtctgagga gggggcggtg


 


24181
agtgtgagta aatgttgtca gaatccctcc tcccagtcta caagccccag ggaaaggaag


 


24241
caaggtgttt actgacaccc ccaggcttat aagtacttcc tggctccatc accctccagt


 


24301
gaacagccct ggggagaaga cagtactggt ttgcaggtgg gtgggtgggg aaaggggtca


 


24361
caggtgcttg gtgttctggt aaatgtgcat atgagaacat ggggttgctt gtgccctgtc


 


24421
cttcagggtt cagaagaacg tgcagtggaa gcagctatgg ggaagtagct agggaaggta


 


24481
ggactggtct gaggtggtga ggagcagatg ctgccagctc cacacatcca ggagagcctg


 


24541
ggtggtttgg gcaaaagtct ctggcatccc ttctgagcct gggtaccaca ctgaagagtg


 


24601
aggacagtgt gccattttta tcaggaaacc ctccagctcc ctgaagacca aattctgatc


 


24661
ctcctgggat ggcagtgaag agccacagag atgactctga ggtcccgtgg ccttttccca


 


24721
cctggagatt gttttcgtta ctgcgctgtt acagccttgg aggactgggg ttcagtttca


 


24781
tccaatcaca tttcttcttt tgtcatagtc atctaaacga tagatcttag agacaggtgg


 


24841
gcaaggggtg cactggtgag cctgacttaa ggagaggtca tctcgtccct tccctagtcc


 


24901
catctccctt ggttattgtt atttcatgtg attgttctgg ttatttcagg ttattctgtt


 


24961
tttgttttca aaacaataac atatatttgt tgttctgatt ttaaaggggt aattgttttg


 


25021
gtaactagaa aattaccttc tcaactccct taaattctgt caaagggaaa agtaagttag


 


24081
gttgctggag aggctatgct gaggcctcag aacctctgta ttcctggaag ttctgcgtgc


 


25141
tttgcctcct gctcccctct ctgtgttcct gttggcaggc ccctaggcag gatttaggag


 


25201
gtaggcaagt caccctagcc aagtcataag cccatggctc aattgccttc tcagcccttc


 


25261
aagggctgtt ccacaggcag caaagggagg ggcctccaca ggttcaccac tgcagcccta


 


25321
attcattttc tttttccact gtcttattct gcaggggtat tccctcagcg gatactacac


 


25381
ccatccattt caaggctatg agcacagaca gctcaggtac cagcagcctg ggccaggatc


 


25441
ttcccccagt agtttcctgc ttaagcaaat agaatttctc aaggggcagc tcccagaagc


 


25501
accggtgatt ggaaagcaga caccgtcact gccaccttcc ctcccaggac tccggccaag


 


25561
gtttccagta ctacttgcct ccagtaccag aggcaggcaa gtggacatca ggggtgtccc


 


25621
caggggcgtg catctcggaa gtcaggggct ccagagaggg ttccagcatc cttcaccacg


 


25681
tggcaggagt ctgccacaga gaggtgttga ttgcctttcc tcacatttcc aggaactgag


 


25741
tatctaccaa gatcaggaac aaaggatctt aaagttcctg gaagagcttg gggaagggaa


 


25801
ggccaccaca gcacatgatc tgtctgggaa acttgggact ccgaagaaag aaatcaatcg


 


25861
agttttatac tccctggcaa agaagggcaa gctacagaaa gaggcaggaa cacccccttt


 


25921
gtggaaaatc gcggtctcca ctcaggcttg gaaccagcac agcggagtgg taagaccaga


 


25981
cggtcatagc caaggagccc caaactcaga cccgagtttg gaaccggaag acagaaactc


 


26041
cacatctgtc tcagaagatc ttcttgagcc ttttattgca gtctcagctc aggcttggaa


 


26101
ccagcacagc ggagtggtaa gaccagacag tcatagccaa ggatccccaa actcagaccc


 


26161
aggtttggaa cctgaagaca gcaactccac atctgccttg gaagatcctc ttgagttttt


 


26221
agacatggcc gagatcaagg agaaaatctg cgactatctc ttcaatgtgt ctgactcctc


 


26281
tgccctgaat ttggctaaaa atattggcct taccaaggcc cgagatataa atgctgtgct


 


26341
aattgacatg gaaaggcagg gggatgtcta tagacaaggg acaacccctc ccatatggca


 


26401
tttgacagac aagaagcgag agaggatgca aatcaagaga aatacgaaca gtgttcctga


 


26461
aaccgctcca gctgcaatcc ctgagaccaa aagaaacgca gagttcctca cctgtaatat


 


26521
acccacatca aatgcctcaa ataacatggt aaccacagaa aaagtggaga atgggcagga


 


26581
acctgtcata aagttagaaa acaggcaaga ggccagacca gaaccagcaa gactgaaacc


 


26641
acctgttcat tacaatggcc cctcaaaagc agggtatgtt gactttgaaa atggccagtg


 


26701
ggccacagat gacatcccag atgacttgaa tagtatccgc gcagcaccag gtgagtttcg


 


26761
agccatcatg gagatgccct ccttctacag tcatggcttg ccacggtgtt caccctacaa


 


26821
gaaactgaca gagtgccagc tgaagaaccc catcagcggg ctgttagaat atgcccagtt


 


26881
cgctagtcaa acctgtgagt tcaacatgat agagcagagt ggaccacccc atgaacctcg


 


26941
gtaagagacc acccaggaac tgtacctagg gttggggtca ggtgcttttg ctcctgacgc


 


27001
agtcttggct gatttgtgag cagtgctgtt tggtggcgcc tatcttttcc tccttccctt


 


27061
ctgcctttta gctaaattcc ccttgattgg ccctttctcc agatattgag cagggaatat


 


27121
agaccttgga ccagccagaa tcttggctga acaaggggga ggttgactct gttggctgta


 


27181
atgaagcttc tttagaaatg attggttttg gccgtacgcg gtggctcatg cctgtaatcc


 


27241
cagcactttt tgaggccgag gcaggcatat cacgaggtca ggagtttgag accagcctgg


 


27301
ccaacatggt gaaaccctgt ctctactaaa aatacaaaaa ttagctgggc gtggtggcgt


 


27361
gcacctgtag tcccagctac tcaggaagct gagacaggag aatcacttga acccaggagg


 


27421
cagaggttgc agtgaactga gattgcgcca ctgcactcca gcctgggcca cagagcaaga


 


27481
ctccatctca aaaaaaaaaa agaaagaaat gattggtctt gggggccggg gcggtggctt


 


27541
acgactgtaa tcccagcact ttgggaggcc aaggcaggca gatcatgagg tgaggaattc


 


27601
gagaccagcc tggccaacat ggtgaaaccc catctctact aaaaatacaa aaattagctg


 


27661
ggggtggtgg tgcttgcctg taatcccagc tactcgggag gctgaggcag gagaatcact


 


27721
tgaacccagg aggtggaggt tgtagtgagc cgagattggc gccactgcac tccagcctgg


 


27781
gcgacagagt gagactccat cttggaaaaa gaaagaaaaa agaaaaacat gattgatctc


 


27841
catgcatcaa tatcatgcct gcctcctaag gcagaggtaa tgaagactta attcccttct


 


27901
gtaggccttc ccctcctccc taagccgttt tctgagagag gtgcaggagc aggtgggttg


 


27961
gggcaggctg catacacagt gggggtgggt tgtgctgcta agcagcagca ggtccacaat


 


28021
cccccctctg catagctcct ggggggaaag gatggaggag cgtgtgcacg gctgcctgcc


 


28081
tgttgaaggt ggtggttcta attttataaa cctcctctgc acagatgggt aggctagcac


 


28141
ttgctgccac tcctgagctg tgaagtcagc ctttacctca ctcagatagc tggtcaggcc


 


28201
ctgcactgta ggtcctaata ggccagtgga cagattgagg aaaacaggag cttctgaagg


 


28261
gcataacaga gagcaaaacc actgaagctg agtggctgca gctgcagcca gggaaagagc


 


28321
cagtaggatg ggggagaatt ccactgacct ttatgtttac ctagcctggt ttctaggggt


 


28381
gtagattcct ggctagggcc cttattcctt gtcttgactg tcttcatgac accaatttgg


 


28441
catttcagga gagcggttaa gaaaaggagt tgtgtctgtc caaaagctgg caaggccaga


 


28501
gctggattgt ttggggtaga gactggatgg ccgtcattct cttttgcctc catccctcct


 


28561
ccccagagtt ggaggaaagc agtggatttt gtggttagtc attctttgga ctcacactaa


 


28621
aagaaacatt ggtgccatgt tcaaatatat cagaagacct aggaaataag aaatttgacc


 


28681
tacttttcta aatgaaatcc cagactgagc aaagagctca ccacatttga aagcttgaac


 


28741
aaagggggcc taggctaagt ccagaggcct agaacaaatg cttttttatt ttctacataa


 


28801
caaggggaaa ttccttgtta tgtagaaaat agctggagac aaatggtgct atagagtgac


 


28861
tcataacaaa ctacggtgat ataggtctag ggacaaaagc aggccactga taagtggcag


 


28921
atgcctgatc cccctaggta gtggggagtg tgagactggg ttataagaag ccttcactat


 


28981
cttttaggac cctcccttga ggaggccagt ctaccacaat tgctttagaa tgaaggtctt


 


29041
ttggttgctc acaagactat aatggtaatt tttggctcat catttttgtg tgtgtgggtt


 


29101
ttatcttaat tcattgttaa agagatagtg ggtttcccct gagctagttt cctatcatct


 


29161
gtgcctatgt ttgcttcact gagctatggg gaaagagtca ctggctgctt tgtttacaaa


 


29221
aagaaaggac aggctgagcc ttaaggagta ggaaggagtt ccttggccta cccttcatct


 


29281
ccacaagtga aaagcccctt agcgtagcag aaaattccag gttgaaggtc tctttggaga


 


29341
aggcagaagg agtgacctag actcctgttc acacatctaa tcactttccg tcaagattta


 


29401
aattccaggt tgtcatcaat ggccgagagt ttcccccagc tgaagctgga agcaagaaag


 


29461
tggccaagca ggatgcagct atgaaagcca tgacaattct gctagaggaa gccaaagcca


 


29521
aggacagtgg aaaatcagaa gaatcatccc actattccac agagaaagaa tcagagaagg


 


29581
taggtgtcct cctgccatct gggaggatca gttccctgtc agtgtctgga ttgtaactga


 


29641
ctcccttgag gcaatcccag cctaaacaac tgacaccatc tcggagtcag cttcccactc


 


29701
ttccctctac cctatctcgc cagcccacct ttcctcctct catacccagc tcctctgtct


 


29761
gctcctgtcc caacataatt ggatttacag aatttcggac ttgaaagaga cctgaaagtt


 


29821
tattgaggcc aatctcctct cttggaagat gcggaatgag agctccagca gcccctttaa


 


29881
ctttgggaag ccaacccctt gacaggtggt gggaataaag tcccccatcc ccatcccttc


 


29941
ttctgtgatg gactaaccag tgttttctta gttttgtttt cttcttgtct ctcattttct


 


30001
ctagactgca gagtcccaga cccccacccc ttcagccaca tccttctttt ctgggaagag


 


30061
ccccgtcacc acactgcttg agtgtatgca caaattgggg aactcctgcg aattccgtct


 


30121
cctgtccaaa gaaggccctg cccatgaacc caagtatgtc ctacgtgtcc tctgtccagc


 


30181
tgaggttttc tcagaaagaa aaagagacac attttcttcc ttgcctcctc agggatggca


 


30241
gattgaccaa tttctcctgt ttcaaaatgg ggaaaggagg gctctgaggt cctggttgct


 


30301
gcttgtgagg cagacaatta gggattagga attcaaaggg aattcctggc ccaccctcta


 


30361
tctcctctat aagcactagg gaggttcacg gcttggaggt cactcactgt tggtgggaca


 


30421
gaaaggtaca ggacctgaga agctctctgc ctgtgggtct atagactcac atgttcaaga


 


30481
gaagtgttcc tggaagaggt gaaactaggt tgatgcttaa atgttgaaag cggtcagcca


 


30541
ggctaagaga cactggctgt tcaaggcaga agtgactgca tgagatgtga ctgcacagag


 


30601
gtgatgtgtt caaggaaatt accagtagtg gaggatggca gcctggcaga ggctaggtca


 


30661
ggctcctcag tcaaaaccat tttaatcctt ctacgtgctt catctcctgt caggttccaa


 


30721
tactgtgttg cagtgggagc ccaaactttc cccagtgtga gtgctcccag caagaaagtg


 


30781
gcaaagcaga tggccgcaga ggaagccatg aaggccctgc atggggaggc gaccaactcc


 


30841
atggcttctg ataaccaggt agggcgtttt cctactcaaa agatacaggt catttttagc


 


30901
aactgagtgg tttaagattg ccagtgactc cctcaacatt tcctgaagtg tttatggctc


 


30961
ctctgtttga tggattctcc tttagcctga aggtatgatc tcagagtcac ttgataactt


 


31021
ggaatccatg atgcccaaca aggtcaggaa gattggcgag ctcgtgagat acctgaacac


 


31081
caaccctgtg ggtggccttt tggagtacgc ccgctcccat ggctttgctg ctgaattcaa


 


31141
gttggtcgac cagtccggac ctcctcacga gcccaagtga gtgtcctagt cctggctaat


 


31201
gcatgtgtca ccagttgggg atggtctgta acccagggaa aacaagggtg tgctttagct


 


31261
gtgtaggaca gaaggggcga gttgagggaa acaagtccag ccctgtctcc acggcctctt


 


31321
agaagacaat agacctgcca agagtgaatg cgttcactct tccagtaagc atgatccttt


 


31381
ttaatttttt gactagtttt aatttttaaa gaaatgtata ggtacattaa aaaatcattc


 


31441
aggccagaca tggtggctca cgcctgtaat cccagcactt tgggaagttg aggcaggtag


 


31501
attacctgag gtcgggagtt caagaccagc cggaccatat gaggaaaccc tgtctctact


 


31561
aaaaatacaa aaattagctg ggcgtggcag cgggtgcctg taatcccagc tactcgggag


 


31621
gcagacagga gaattgattg aagctgggag gcggaggttg cagtgagctg agatcgcacc


 


31681
actgcactcc agcctgagca acagagcaag actccatctt gaaaaaaatc attcaagctg


 


31741
attgaaaaag tagtcatttc aatgaaaagt ctcatttccg tctcagactt ctagtttccc


 


31801
tccctagacc catatattac tatagccctg cattagcaac tgggatgcgg tctgagaagc


 


31861
atggttttgt tgttgtgaga acatcagtgt gtatttacat aaacctagat ggcatgggct


 


31921
cctacacacg tacaggctgt atggtatggc cggttgctcc taggctacaa acctgtacag


 


31981
catgtaactg tagtgaatac tgtgggcagc tgcaacacac tggtaagtat tggtgtctat


 


32041
ctaaacagca aaaagataca gtaaaaagac agcataaagg attaaaaaaa tactacacct


 


32101
gtatagggcg cctgccatga atggagcttg caggactaga agttgctgtg ggtgagtcag


 


32161
ccagtgagtg gagaatgaat atgagggcct aggacatgac tgtacactaa tgtaggcttt


 


32221
gtaaacactg ttcacttagg ctaactacat ttatttaaaa tatttttctt taataaatta


 


32281
accttagctt actgtaactt ttttacttta tttttacttt attttttact ttattaactt


 


32341
ttttattttt tgttcctttt gtaataatac ttagcttaaa acacaaacat gttgtactgc


 


32401
tatccaaaaa tatttccttt gtttatatcc ttaattctat agtctttttt ctgtttgtaa


 


32461
atttttttat ttttttaact ttttaaactt ttttgttgaa agctaagatg aatatacatt


 


32521
agctgttagc ctaggcctac acagggtcag gatcctcagt atcactgtct tccacctcca


 


32581
cgtcttgtcc cactgtaagg tgttcagggg caataacatg catgggggcc gtcatctcct


 


32641
atggtaacag tgctttctgg aatacctgcc tgaggctttt tttagttact tatttttcta


 


32701
gaagtagaag gagtatacgc tgaaataatg ataaaaatat agtaaacaca taaggagcgt


 


32761
gtagcctaga tccctcgcat tcacagttca cagtcgagtt cacactccta caagaatctc


 


32821
atgctgctgc tgatcccaca ggaggcggag ctcggaccag aatgctttct tgctcgccac


 


32881
tcacctcttc ctgtgtgccc aggttcctaa caggttacag agccctgcac ttggtgataa


 


32941
gaatttttca gctccatcat aatcttacgg ggcccctgtc atatgtgtga tttatcattg


 


33001
aacaaaacac tgttatgcag tacatgacta taccagtttc ttgagtcctt ctagaaattg


 


33061
ctgtgcataa ctagcacatt ttcccttttg tctttttaaa caagtatatc ttctgtttta


 


33121
tacgttgact cctttactta ccagacttct tgcatagctg catagtattt cattgaatag


 


33181
aagctgacaa attcagtttc tcagaagtaa acatttaata gggacttagg aacagaaatg


 


33241
atgtcttggg tggctgcaag atggtggatc cctgcactta ccctccagaa agtatgcttt


 


33301
ctatagaggc ttttttggtt aaacatgcag ctggtcacac attatacttt cttgtgaaac


 


33361
ttatgagcac tagctggtgg ggaggcttgg tagacatctt tgtgtggaat tatttatgct


 


33421
acccaacact ttgggatgcg ggagtcagat attggtggtc atagtggttt tgcatcaaga


 


33481
tggcatcact cttgccatgc aaccggcatg ttttcttaat ctctaaggtc tatcacaaag


 


33541
tggaaaagca aggtgtaaag tgaggggaga ggtgaagatt gtctgtatat gtctagaata


 


33601
ttttcttgaa ggatcgaaaa aactggcaac tgtgtttgct tctggggaag gagggacact


 


33661
tttattgtac actcttaact gtttgaattt tcacatgttt ggcatccaac ttgtccattt


 


33721
taaagtcatt aaggaaaaaa gcttataaaa atgctatctt gttactttaa atttgtctct


 


33781
ccttaataga agtaatgtca gccgggcgca gtggcttacg cctataatcc cagcactttg


 


33841
ggaggccgag gtgggtggat catttgagct cagtaatttg agaccagcct ggccaacatg


 


33901
gtaaaacccc atctctacta aaaatacaaa aaatagctga gcatggtggt atgcccctgt


 


33961
atgccagcta ctccggaggc tgagcaggag aattacttga acccaggagg cggaagttgc


 


34021
agtgacctga gattgtgcca ctgcactcca gcctgggtga tggagtgaga ctccgtctca


 


34081
aaaaaaaaaa caaaaaaatc agaagtaatg gcatttcatt tattcatatt tatgtgttat


 


34141
ttgcatttcc ttttctgtga accacctgtt cttgtccttt tccagttttc tttcagattg


 


34201
ttactctcat ggatatgtaa gaacttttaa cataattttt aaaagtagcc ctttgcccaa


 


34261
catgacccaa tttagaacac acatctgccc actgtttaag ccatctggaa aaggagaggc


 


34321
ccagtctccc tccagctgct cttctactaa ttcatatcct tcatctcaat aagcacctcc


 


34381
ttatctttaa gcacagtcct tcagatgata ggttcagtgc atttcctctt tctcttctgt


 


34441
gaaatcttct gtgaaatcct ttctaggact tcaagtcagc cattcattga gaatgtaaga


 


34501
acttactatg aactagatgg aagatacaaa aaattgctgc ccctgagtgt gtagcagaca


 


34561
tatagccata gcagtattga aatgtgccat tggaggtgaa tgcagagtgc tgtggggaca


 


34621
cagaggacac tgctgccact gtctgccagg gagctgagga aggttgcata gagaggggaa


 


34581
atgctaattg agtaactacg tttattaact atctcgtaca ataatatact ttagttgctt


 


34741
cttaatgtga attatgctgg gtcttttttt cctttctgtc tactgtggct ggtgacgtta


 


34801
actaatttta tgacttcttt tctgtttttt tttttttttt tttttggaga tggagtctca


 


34861
ctctgtcacc caggctggag tgcagtggcg cagtctcagc tcactgcaac ctccgcctcc


 


34921
tgggttcaaa caattctctt gcctcagctc ctcgagtagc tgggattaca ggcatctgcc


 


34981
accacatctg actatttttg tatttttagt agagacagag tttcaccatg ttggccaggc


 


35041
tggtcttcaa ctcctgacct cagatgatcc acccgcctca gcctcccaaa gtgctgggat


 


35101
tacaggcatg aaccaccgcg acagccatga cctttaatat ctaaatgctg gagaccactc


 


35161
agagctcccg attcctccat ctagttgctt acttgtcacc tctgtttgga ttaacatccc


 


35221
taatatgaca tgtccaaagc agaactcttg ggttttgccc ccttcgcttc atttccccca


 


35281
agtctttccc agcttagtaa gtgataccac tgtctaccca gtggctcaag tcagaaacct


 


35341
gaggatcatc ttttcctcta tctcctttac ctccttcgtc aactcatcct taagtcctat


 


35401
tggttatact tagaatttct atcccaaatc ccttcctctt ctttccacct ccagcgtcag


 


35461
cactctaatc caagccactg tctcatctag actgtcacaa tagcctccta actgatcttt


 


35521
atactttatt tactcaacac ttatttatgg agtatcttct atttttttta gaacaggaac


 


35581
caaacattaa ccaatagtca cctaaagaaa tgtaagatct catatttgat aagtattcca


 


35641
aagggctgga gttgcagcga gaatcaatta tagcacagtt tgacttagga aggagagggg


 


35701
gaagggcttc cccaagaagt actgcctgag ctgagacttt aaggatgatg aagagagaaa


 


35761
gactgagggt tctaggcgaa ggacactgtt aaatcctgtg gtgggagaag cgcagagctg


 


35821
gcaaagaggc tgagaggagg cacttatggc aatagcccca tttggctagt ggctaccata


 


35881
ttggatagca cagctctaaa ggattctagt tgcataatta acaaacttta tcatatatgt


 


35941
aaatatatca gtataaaact ttgcacgtaa gagttcatat tcttttcaaa cacatggggc


 


36001
agtcatgaaa ttctcctgga tccctgaggt aaagatacta cacaatctgt ccctcctgac


 


36061
cgccttcacc atcttctttt catgccactg tccctctcat tctgaactta agcccactgg


 


36121
cctgcacggg cctgccagca tggacaaaga aaagagtgaa ggcatttagt tgcttcggat


 


36181
catcaaggaa cagtgagaaa ctccatgtgg ttgaacataa acgttgattc cttttactgc


 


36241
ctgagctgct aagtcttgaa actgaagcct ctgttatatg cagttagagt atccaaagct


 


36301
gggttctcag ccagtagatt aggatctggt tgaaagctag gattcttgat gccttgtctg


 


36361
gtacatttcc aagctgtttt gctccaccat gctcttccct ctgtgtttgg aaattaccag


 


36421
ccggagcggt agaagccacc tccctgctcc acttgggcac tttattgtca gcgcagctca


 


36481
tgcgcccaag atccattcct taccccagag ttagcttaag cctgccagga gtcaaaaaaa


 


36541
caactcccag caaaacctgg gtccctggcc taaggccgac cttgacctaa tgacctctcc


 


36601
aggtcaagtc cccttgagct aataggaagt aaatgaggga atggagttgg gcgctttaaa


 


36661
aatcagacag tgtatgaagt aacagtgact tgtggggaga aaggtagtcc cttctgattt


 


36721
ctgcttaaga agagagtcca agttggtgct agcatttcgt taagccttcc tcatacttca


 


36781
taatcttcct ttatagtaag gacgggctca gagcctttgc aggtaacagt atctagtcag


 


35841
aattttataa catgatttta tcatattctg ttttacagcc accttcaata tgtggcaagt


 


36901
gatactggtt ttccatttac agtagtgaaa caaagtttcc tatttcagtg tgtttacatt


 


36961
tttatctctt ttttcatacc tagtattacg gatatttaca tttttagagt ctgtttgttt


 


37021
attttagaga tggggatctt gctgtctcgc gcaggctgga gtgcagtgat atggtcatag


 


37081
ctcactgcag ccttgacctc cagggctcaa gtgatcctct caccccagcc tcccaagtag


 


37141
ctgagactat aggcacagac caccgtgccc agctaaattt tttttttttt ttttaagaga


 


37201
caggggtctc gctatgttgc ctgggggatt actatgttgc ccaggctggt cttgaactcc


 


37261
tggcctcaag tgatctccca ccttagctcc tcaagttgct gggattacag gtgtgagcca


 


37321
caatggctgg ctctaagaag tcaatttaga gaaaaatatt aattaatagt ccaagtaata


 


37381
cctggatgtg gcacaaatca tgtggtgatt catgaatgac caaaatctag gaaatgctgc


 


37441
cttagaaaca gcttctgttt gacgggtcca tatgtttgca agactggcca catcttcagc


 


37501
aaaactaacc cttccttgga acaatgtcag ggtctggcac ttgtcttcat tctctgtttc


 


37561
tcatcccaaa ggttcgttta ccaagcaaaa gttgggggtc gctggttccc agccgtctgc


 


37621
gcacacagca agaagcaagg caagcaggaa gcagcagatg cggctctccg tgtcttgatt


 


37681
ggggagaacg agaaggcaga acgcatgggt ttcacagagg taaccccagt gacaggggcc


 


37741
agtctcagaa gaactatgct cctcctctca aggtccccag aagcacagcc aaagacagtt


 


37801
aagacgtcta cttttggtgc cttttttggg gcgggggggt cctcctaact cctaagtgga


 


37861
ggtggctctt gctgtcatgc gagttattcc taggctttac tcttagcctc gagagagcag


 


37921
taactgggac actagatgta agaaggaaaa gatgactcac acgacaagta gagcttgatc


 


37981
tccctgccca cggtgaatat ggtggacaca gcctcagctt tgtggtgctg acacagcctc


 


38041
ttttccccac agctccctct cactggcagc accttccatg accagatagc catgctgagc


 


38101
caccggtgct tcaacactct gactaacagc ttccagccct ccttgctcgg ccgcaagatt


 


38161
ctggccgcca tcattatgaa aaaagactct gaggacatgg gtgtcgtcgt cagcttggga


 


38221
acaggtgagt gaggctctga gacatgccgc ctcccatggc gcctgaaagc gggtgcctct


 


38281
catcctcccc tggagtccat gcatgtaagt ccaaggcagg gagaagagac ttcattttag


 


38341
ctacagtcaa ttcagagtga ggaatgagtt cttagttcct agaggagaga atatgggagt


 


38401
ctaggatctg agaaactgag gctgtttctg ccttgaagct ttcagaacaa atagccttca


 


38461
tcctgttttc catcggtttc cttccattat tctatttctg ttttaaacac cttcctaggg


 


38521
aatcgctgtg tgaaaggaga ttctctcagc ctaaaaggag aaactgtcaa tgactgccat


 


38581
gcagaaataa tctcccggag aggcttcatc aggtgagcga ggtcagagct gtggcccggc


 


38641
tgcccggctg tggagagctc cagttccctg ccccacatgg ctctgacacg gcctctgaat


 


38701
ccccctcaga cagacgggtc atgatgtggc agtggcagcc tttgcttttc acccgtccat


 


38761
ttgaacctgt ctgatggaat ccatcccctc tgtgagctga gctgcctccc actgctcggc


 


38821
ctgtttttaa atgctgtcct tttttctgct aactctgctg cttcatgttc ttttctaaaa


 


38881
acacaaaatg accttttagt cctcagggcc ttgaggatga ggcagctttc catttccgtt


 


38941
tgaggaccta cacaaccttg atgcccctgc cagctttctc ctctagctca ccttttcttt


 


39001
aatttatgaa gggagagact tagaaaggag caacagcttc ctgtagtcct tgaatcagtt


 


39061
tgctctgctc tagaatccct gtagccgcca tagcgaggag ccctcagcag aaatgaagga


 


39121
gaccaaaaag gctaactatg ctttatgaaa tgctgaggtc tcccctggag aatttccacc


 


39181
tgataaactg tgaaacgtct gcaacattga gacttttcct tactttctca tttggaggtc


 


39241
agattataga aacaactgct tttcccagaa ttgaacctgc cttcctaacc agactttctt


 


39301
tttgtaggtt tctctacagt gagttaatga aatacaactc ccagactgcg aaggatagta


 


39361
tatttgaacc tgctaaggga ggagaaaagc tccaaataaa aaagactgtg tcattccatc


 


39421
tgtatatcag gtctgtacag ttcctgttgc tgccagggtg ggccctgcca ggctgttaga


 


39481
attgggtatc caaatgctct cctggcctgt aaatcgaacc tgatacaata agccacactc


 


39541
cactgtgggt ttgaggtcca tattcaggtg tagatgactc acatgtactg ctgtccacct


 


39601
ccagtctccc atggtaggcc ttagaaaaca tcccttgctt ctgtcacatc tgactgtttt


 


39661
ggagccccac gaaattgcag atttcccaca ggtgagtttt aacagccacc cctgtttttc


 


39721
agcactgctc cgtgtggaga tggcgccctc tttgacaagt cctgcagcga ccgtgctatg


 


39781
gaaagcacag aatcccgcca ctaccctgtc ttcgagaatc ccaaacaagg aaagctccgc


 


39841
accaaggtgg agaacggtga gtgatacatg cccccgcctc ctttcctcaa aaggctctgc


 


39901
aaggtccagg gaccccaagt ctctacaagg ctgctaggat tttaccatta gtcactgggc


 


39961
acagaggtgc tgtttacagg aaagggaaga cctgggtcag ggagctgtgt ggtaagatca


 


40021
gggttctatt ttgaatgtgt tagtttggga gatctgggag atccccaagt caaataggag


 


40081
gtgggggttc ccatgtggag ctcagaggag ggagcttggc tggaaataga aatatgggag


 


40101
tcatccccct atagggtctt catggccatg agaatgcata ggattacctc aagaaagcgg


 


40201
ggaggaaatg aagagtgcgg cacaaccaag ccctgaggag ttgacagatg aggatgccaa


 


40261
atgctggggt cccctcctgt ctagctggca gttgactctg ccttgtccac tggctccttc


 


40321
tctcctatcc tctcctgtct ccttactgtc tcttcgcatc cactccattg cgttcaggcc


 


40381
acgtcagcag tcatcatggt ggtcctgaaa ccttgctaaa taccctaaag tatagacaca


 


40441
gttaccatgg agccggtgct ccactcctag gtatatgctg cagagagatg gagatctgtg


 


40501
tccacacgga aactaatatg tgaatgttca tggcagcatt actcacaaga gccaaaaaag


 


40561
tggaaacaac ccagacgtcc atcagctgat ggattcataa ataaaacatc aaatatatcc


 


40621
ataaattgaa tattattttg gccataaaaa gaagtgaagt gctgatacat gcttacaata


 


40681
tggatgcact tgaaaacttg atgccaaatg aaaaaagcca gtcacaaaag atcacatatt


 


40741
gtatgattcc atttatatga aatgtccaga atagacaaat ccatagagac agaaagtaga


 


40801
tgagtggttg ccagggccag gagtgggaga gttggagaga tgaggagtga ctgcgccaat


 


40861
gggtagaagg tttctttttg gagcgatgaa atgttctaaa attgactgtg gtgacagttg


 


40921
cagaactctg tgaatatact aaaaatgact gaattgtatg ctttaaatgg gtgaactgta


 


40981
tggcatatga actatatctc agtaaagcat ttttttttgt ttttttttaa acccgatagt


 


41041
ggtttcccac tgcactgcat atacaagcaa aacctgctgt gatcacccgg cctcctctca


 


41101
tgccactttc cccatccctc gcatatgctc tgtccacact ggctctctgt caggcgcctg


 


41161
aacagccaat ccgctggctg ccttggggcc tttgcttttg tcctgtgtgc ctgaaacact


 


41221
tactccagct gtcctcctcc atgtctgggc tcccctgctg ctgctgtccc aggaggtgtc


 


41281
ccctgtggtc ctccgtcata gctgcctcag agccttcaga gcactgtcag catctggaat


 


41341
tcttccgtat gtactggcta ctggtttagt gtctattctc ttccctttca atctcctcac


 


41401
caccatagat gttttaagag cacaaagatc ttacttggtt ttgctcaccg ttctttccca


 


41461
gcaccaagca tagtgcctgg cgcatagcag gggctgtgaa atatgtgaag aatgaatgaa


 


41521
tgtagcctgt ggcccaagct taaggaggat agaaaccacg ccagggagtg gtttggtcca


 


41581
ttggcgcctg tgggtctgac ccaagtctct cacacaggag aaggcacaat ccctgtggaa


 


41641
tccagtgaca ttgtgcctac gtgggatggc attcggctcg gggagagact ccgtaccatg


 


41701
tcctgtagtg acaaaatcct acgctggaac gctctgggcc tgcaaggggc actgttgacc


 


41761
cacttcctgc agcccattta tctcaaatct gtcacattgg gtaagggcc tgccttgggga


 


41821
tctggaactg gtctgtcctt cttgtgccca gatcccaaac tgcatgcttt attgccaggt


 


41881
gttttgtctc ccttatcaaa gtgagcatga ttcactcctc agtaattgat tgagtgtcca


 


41941
gtctgctgtg gtaggaagat cctggtagcc ccagtcagaa ggtgcttcct aacaaggcag


 


42001
ctgtttctct ttcttgacaa ctatatcttg tacctccaaa atccccacat gcttctgcct


 


42061
cttaacagca tttggtgcaa acacaggtat atatgtttct cttttttgta ctcaggttac


 


42121
cttttcagcc aagggcatct gacccgtgct atttgctgtc gtgtgacaag agatgggagt


 


42181
gcatttgagg atggactacg acatcccttt attgtcaacc accccaaggt gctataaccc


 


42241
ccttctattt tccctgacat tttcctcctt ttcaagcagt catgtaaaca gaggaaaaat


 


42301
gtacactgtg ggcaagggga acattgccca cagtggtagc ccacaaggga acattgccca


 


42361
cagtggccca ccaccaacat tggttggtct cccaagaact taaactttct tccttttgga


 


42421
tgccaagggc ttttcttctc ttagtctgga attaatctga atcgaggtgg agttagtatg


 


42481
tctagagggt gctcagtctt agccaaacag aaccctaaat acaggggaaa gatcatgacc


 


42541
ccacacttcc tctctcctat gagtcttgag tccctgcttc agaatcttat tcctgaaagg


 


42601
tttccatctt tctcccgttg cttctgggat tcctaggttg gcagagtcag catatatgat


 


42661
tccaaaaggc aatccgggaa gactaaggag acaagcgtca actggtgtct ggctgatggc


 


42721
tatgacctgg agatcctgga cggtaccaga ggcactgtgg atgggtaagg agacaggaga


 


42781
gcgcagtgag gaccaagcct ctgccctgac ttgcaagggt gcatcatacc tctgcagtct


 


42841
cagggcttga gagccgcctc ccctcccacg gtgtctccac tgtgagctcc ttatcttaca


 


42901
ggtcccaggt gaataatgag tgcttttgtt tctctaggcc acggaatgaa ttgtcccggg


 


42961
tctccaaaaa gaacattttt cttctattta agaagctctg ctccttccgt taccgcaggg


 


43021
atctactgag actctcctat ggtgaggcca agaaagctgc ccgtgactac gagacggcca


 


43081
agaactactt caaaaaaggc ctgaaggata tgggctatgg gaactggatt agcaaacccc


 


43141
aggaggaaaa gaacttttat ctctgcccag tatagtatgc tccagtgaca gatggattag


 


43201
ggtgtgtcat actagggtgt gagagaggta ggtcgtagca ttcctcatca catggtcagg


 


43261
ggattttttt ttctcctttt tttttctttt taagccataa ttggtgatac tgaaaacttt


 


43321
gggttcccat ttatcctgct ttctttggga ttgctaggca aggtctggcc aggcccccct


 


43381
tttttccccc aagtgaagag gcagaaacct aagaagttat cttttctttc tacccaaagc


 


43441
atacatagtc actgagcacc tgcggtccat ttcctcttaa aagttttgtt ttgatttgtt


 


43501
tccatttcct ttccctttgt gtttgctaca ctgacctctt gcggtcttga ttaggtttca


 


43561
gtcaactctg gatcatgtca gggactgata atttcatttg tggattacgc agacccctct


 


43621
acttcccctc tttcccttct gagattcttt ccttgtgatc tgaatgtctc cttttccccc


 


43681
tcagagggca aagaggtgaa cataaaggat ttggtgaaac atttgtaagg gtaggagttg


 


43741
aaaactgcag ttcccagtgc cacggaagtg tgattggagc ctgcagataa tgcccagcca


 


43801
tcctcccatc ctgcacttta gccagctgca gggcgggcaa ggcaaggaaa gctgcttccc


 


43861
tggaagtgta tcactttctc cggcagctgg gaagtctaga accagccaga ctgggttaag


 


43921
ggagctgctc aagcaatagc agaggtttca cccggcagga tgacacagac cacttcccag


 


43981
ggagcacggg catgccttgg aatattgcca agcttccagc tgcctcttct cctaaagcat


 


44041
tcctaggaat attttccccg ccaatgctgg gcgtacaccc tagccaacgg gacaaatcct


 


44101
agagggtata aaatcatctc tgctcagata atcatgactt agcaagaata agggcaaaaa


 


44161
atcctgttgg cttaacgtca ctgttccacc cggtgtaata tctctcatga cagtgacacc


 


44221
aagggaagtt gactaagtca catgtaaatt aggagtgttt taaagaatgc catagatgtt


 


44281
gattcttaac tgctacagat aacctgtaat tgagcagatt taaaattcag gcatactttt


 


44341
ccatttatcc aagtgctttc atttttccag atggcttcag aagtaggctc gtgggcaggg


 


44401
cgcagacctg atctttatag ggttgacata gaaagcagta gttgtgggtg aaagggcagg


 


44461
ttgtcttcaa actctgtgag gtagaatcct ttgtctatac ctccatgaac attgactcgt


 


44521
gtgttcagag cctttggcct ctctgtggag tctggctctc tggctcctgt gcattctttg


 


44581
aatagtcact cgtaaaaact gtcagtgctt gaaactgttt cctttactca tgttgaaggg


 


44641
actttgttgg cttttagagt gttggtcatg actccaagag cagagcaggg aagagcccaa


 


44701
gcatagactt ggtgccgtgg tgatggctgc agtccagttt tgtgatgctg cttttacgtg


 


44761
tccctcgata acagtcagct agacacactc aggaggacta ctgaggctct gcgaccttca


 


44821
ggagctgagc ctgcctctct cctttagatg acagaccttc atctgggaac gtgctgagcc


 


44881
agcaccctca gatgatttcc ctccaaactg ctgactaggt catcctctgt ctggtagaga


 


44941
cattcacatc tttgctttta ttctatgctc tctgtacttt tgaccaaaaa ttgaccaaag


 


45001
taagaaaatg caagttctaa aaatagacta aggatgcctt tgcagaacac caaagcatcc


 


45061
caaggaactg gtagggaagt ggcgcctgtc tcctggagtg gaagaggcct gctccctggc


 


45121
tctgggtctg ctgggggcac agtaaatcag tcttggcacc cacatccagg gcagagaggt


 


45181
ctgtggttct cagcatcaga aggcagcgca gcccctctcc tcttcaggct acagggttgt


 


45241
cacctgctga gtcctcaggt tgtttggcct ctctggtcca tcttgggcat taggttctcc


 


45301
agcagagctc tggccagctg cctcttcttt aactgggaac acaggctctc acaagatcag


 


45361
aacccccact cacccccaag atcttatcta gcaagcctgt agtattcagt ttctgttgta


 


45421
ggaagagagc gaggcatccc tgaattccac gcatctgctg gaaacgagcc gtgtcagatc


 


45481
gcacatccct gcgcccccat gcccctctga gtcacacagg acagaggagg cagagcttct


 


45541
gcccactgtt atcttcactt tctttgtcca gtcttttgtt tttaataagc agtgaccctc


 


45601
cctactcttc tttttaatga tttttgtagt tgatttgtct gaactgtggc tactgtgcat


 


45661
tccttgaata atcacttgta aaaattgtca gtgcttgaag ctgtttcctt tactcacatt


 


45721
gaagggactt cgttggtttt ttggagtctt ggttgtgact ccaagagcag agtgaggaag


 


45781
acccccaagc atagactcgg gtactgtgat gatggctgca gtccagtttt atgattctgc


 


45841
ttttatgtgt cccttgataa cagtgactta acaatataca ttcctcataa ataaaaaaaa


 


45901
aacaagaatc tgaattctta gaaa






In another embodiment, antibodies used to practice this invention are designed to bind to, or affinity matured to bind to, a polypeptide encoded by SEQ ID NO:2, or subsequences thereof:








(SEQ ID NO: 2)










   1
gaccagacca ttgattcccg actgaaggta gagaaggcta cgtggtgggg gagggtgggg



 


  61
ggagggtcgc ggccgcactg gcagcctccg ggtgtccggc cgtgtcccga ggaagtgcaa


 


 121
gacccggggt attccctcag cggatactac acccatccat ttcaaggcta tgagcacaga


 


 181
cagctcaggt accagcagcc tgggccagga tcttccccca gtagtttcct gcttaagcaa


 


 241
atagaatttc tcaaggggca gctcccagaa gcaccggtga ttggaaagca gacaccgtca


 


 301
ctgccacctt ccctcccagg actccggcca aggtttccag tactacttgc ctccagtacc


 


 361
agaggcaggc aagtggacat caggggtgtc cccaggggcg tgcatctcgg aagtcagggg


 


 421
ctccagagag ggttccagca tccttcacca cgtggcagga gtctgccaca gagaggtgtt


 


 481
gattgccttt cctcacattt ccaggaactg agtatctacc aagatcagga acaaaggatc


 


 541
ttaaagttcc tggaagagct tggggaaggg aaggccacca cagcacatga tctgtctggg


 


 601
aaacttggga ctccgaagaa agaaatcaat cgagttttat actccctggc aaagaagggc


 


 661
aagctacaga aagaggcagg aacaccccct ttgtggaaaa tcgcggtctc cactcaggct


 


 721
tggaaccagc acagcggagt ggtaagacca gacggtcata gccaaggagc cccaaactca


 


 781
gacccgagtt tggaaccgga agacagaaac tccacatctg tctcagaaga tcttcttgag


 


 841
ccttttattg cagtctcagc tcaggcttgg aaccagcaca gcggagtggt aagaccagac


 


 901
agtcatagcc aaggatcccc aaactcagac ccaggtttgg aacctgaaga cagcaactcc


 


 961
acatctgcct tggaagatcc tcttgagttt ttagacatgg ccgagatcaa ggagaaaatc


 


1021
tgcgactatc tcttcaatgt gtctgactcc tctgccctga atttggctaa aaatattggc


 


1081
cttaccaagg cccgagatat aaatgctgtg ctaattgaca tggaaaggca gggggatgtc


 


1141
tatagacaag ggacaacccc tcccatatgg catttgacag acaagaagcg agagaggatg


 


1201
caaatcaaga gaaatacgaa cagtgttcct gaaaccgctc cagctgcaat ccctgagacc


 


1261
aaaagaaacg cagagttcct cacctgtaat atacccacat caaatgcctc aaataacatg


 


1321
gtaaccacag aaaaagtgga gaatgggcag gaacctgtca taaagttaga aaacaggcaa


 


1381
gaggccagac cagaaccagc aagactgaaa ccacctgttc attacaatgg cccctcaaaa


 


1441
gcagggtatg ttgactttga aaatggccag tgggccacag atgacatccc agatgacttg


 


1501
aatagtatcc gcgcagcacc aggtgagttt cgagccatca tggagatgcc ctccttctac


 


1561
agtcatggct tgccacggtg ttcaccctac aagaaactga cagagtgcca gctgaagaac


 


1621
cccatcagcg ggctgttaga atatgcccag ttcgctagtc aaacctgtga gttcaacatg


 


1681
atagagcaga gtggaccacc ccatgaacct cgatttaaat tccaggttgt catcaatggc


 


1741
cgagagtttc ccccagctga agctggaagc aagaaagtgg ccaagcagga tgcagctatg


 


1801
aaagccatga caattctgct agaggaagcc aaagccaagg acagtggaaa atcagaagaa


 


1861
tcatcccact attccacaga gaaagaatca gagaagactg cagagtccca gacccccacc


 


1921
ccttcagcca catccttctt ttctgggaag agccccgtca ccacactgct tgagtgtatg


 


1981
cacaaattgg ggaactcctg cgaattccgt ctcctgtcca aagaaggccc tgcccatgaa


 


2041
cccaagttcc aatactgtgt tgcagtggga gcccaaactt tccccagtgt gagtgctccc


 


2101
agcaagaaag tggcaaagca gatggccgca gaggaagcca tgaaggccct gcatggggag


 


2161
gcgaccaact ccatggcttc tgataaccag cctgaaggta tgatctcaga gtcacttgat


 


2221
aacttggaat ccatgatgcc caacaaggtc aggaagattg gcgagctcgt gagatacctg


 


2281
aacaccaacc ctgtgggtgg ccttttggag tacgcccgct cccatggctt tgctgctgaa


 


2341
ttcaagttgg tcgaccagtc cggacctcct cacgagccca agttcgttta ccaagcaaaa


 


2401
gttgggggtc gctggttccc agccgtctgc gcacacagca agaagcaagg caagcaggaa


 


2461
gcagcagatg cggctctccg tgtcttgatt ggggagaacg agaaggcaga acgcatgggt


 


2521
ttcacagagg taaccccagt gacaggggcc agtctcagaa gaactatgct cctcctctca


 


2581
aggtccccag aagcacagcc aaagacactc cctctcactg gcagcacctt ccatgaccag


 


2641
atagccatgc tgagccaccg gtgcttcaac actctgacta acagcttcca gccctccttg


 


2701
ctcggccgca agattctggc cgccatcatt atgaaaaaag actctgagga catgggtgtc


 


2761
gtcgtcagct tgggaacagg gaatcgctgt gtgaaaggag attctctcag cctaaaagga


 


2821
gaaactgtca atgactgcca tgcagaaata atctcccgga gaggcttcat caggtttctc


 


2881
tacagtgagt taatgaaata caactcccag actgcgaagg atagtatatt tgaacctgct


 


2941
aagggaggag aaaagctcca aataaaaaag actgtgtcat tccatctgta tatcagcact


 


3001
gctccgtgtg gagatggcgc cctctttgac aagtcctgca gcgaccgtgc tatggaaagc


 


3061
acagaatccc gccactaccc tgtcttcgag aatcccaaac aaggaaagct ccgcaccaag


 


3121
gtggagaacg gagaaggcac aatccctgtg gaatccagtg acattgtgcc tacgtgggat


 


3181
ggcattcggc tcggggagag actccgtacc atgtcctgta gtgacaaaat cctacgctgg


 


3241
aacgtgctgg gcctgcaagg ggcactgttg acccacttcc tgcagcccat ttatctcaaa


 


3301
tctgtcacat tgggttacct tttcagccaa gggcatctga cccgtgctat ttgctgtcgt


 


3361
gtgacaagag atgggagtgc atttgaggat ggactacgac atccctttat tgtcaaccac


 


3421
cccaaggttg gcagagtcag catatatgat tccaaaaggc aatccgggaa gactaaggag


 


3481
acaagcgtca actggtgtct ggctgatggc tatgacctgg agatcctgga cggtaccaga


 


3541
ggcactgtgg atgggccacg gaatgaattg tcccgggtct ccaaaaagaa catttttctt


 


3601
ctatttaaga agctctgctc cttccgttac cgcagggatc tactgagact ctcctatggt


 


3661
gaggccaaga aagctgcccg tgactacgag acggccaaga actacttcaa aaaaggcctg


 


3721
aaggatatgg gctatgggaa ctggattagc aaaccccagg aggaaaagaa cttttatctc


 


3781
tgcccagtat agtatgctcc agtgacagat ggattagggt gtgtcatact agggtgtgag


 


3841
agaggtaggt cgtagcattc ctcatcacat ggtcagggga tttttttttc tccttttttt


 


3901
ttctttttaa gccataattg gtgatactga aaactttggg ttcccattta tcctgctttc


 


3961
tttgggattg ctaggcaagg tctggccagg cccccctttt ttcccccaag tgaagaggca


 


4021
gaaacctaag aagttatctt ttctttctac ccaaagcata catagtcact gagcacctgc


 


4081
ggtccatttc ctcttaaaag ttttgttttg atttgtttcc atttcctttc cctttgtgtt


 


4141
tgctacactg acctcttgcg gtcttgatta ggtttcagtc aactctggat catgtcaggg


 


4201
actgataatt tcatttgtgg attacgcaga cccctctact tcccctcttt cccttctgag


 


4261
attctttcct tgtgatctga atgtctcctt ttccccctca gagggcaaag aggtgaacat


 


4321
aaaggatttg gtgaaacatt tgtaagggta ggagttgaaa actgcagttc ccagtgccac


 


4381
ggaagtgtga ttggagcctg cagataatgc ccagccatcc tcccatcctg cactttagcc


 


4441
agctgcaggg cgggcaaggc aaggaaagct gcttccctgg aagtgtatca ctttctccgg


 


4501
cagctgggaa gtctagaacc agccagactg ggttaaggga gctgctcaag caatagcaga


 


4561
ggtttcaccc ggcaggatga cacagaccac ttcccaggga gcacgggcat gccttggaat


 


4621
attgccaagc ttccagctgc ctcttctcct aaagcattcc taggaatatt ttccccgcca


 


4681
atgctgggcg tacaccctag ccaacgggac aaatcctaga gggtataaaa tcatctctgc


 


4741
tcagataatc atgacttagc aagaataagg gcaaaaaatc ctgttggctt aacgtcactg


 


4801
ttccacccgg tgtaatatct ctcatgacag tgacaccaag ggaagttgac taagtcacat


 


4861
gtaaattagg agtgttttaa agaatgccat agatgttgat tcttaactgc tacagataac


 


4921
ctgtaattga gcagatttaa aattcaggca tacttttcca tttatccaag tgctttcatt


 


4981
tttccagatg gcttcagaag taggctcgtg ggcagggcgc agacctgatc tttatagggt


 


5041
tgacatagaa agcagtagtt gtgggtgaaa gggcaggttg tcttcaaact ctgtgaggta


 


5101
gaatcctttg tctatacctc catgaacatt gactcgtgtg ttcagagcct ttggcctctc


 


5161
tgtggagtct ggctctctgg ctcctgtgca ttctttgaat agtcactcgt aaaaactgtc


 


5221
agtgcttgaa actgtttcct ttactcatgt tgaagggact ttgttggctt ttagagtgtt


 


5281
ggtcatgact ccaagagcag agcagggaag agcccaagca tagacttggt gccgtggtga


 


5341
tggctgcagt ccagttttgt gatgctgctt ttacgtgtcc ctcgataaca gtcagctaga


 


5401
cacactcagg aggactactg aggctctgcg accttcagga gctgagcctg cctctctcct


 


5461
ttagatgaca gaccttcatc tgggaacgtg ctgagccagc accctcagat gatttccctc


 


5521
caaactgctg actaggtcat cctctgtctg gtagagacat tcacatcttt gcttttattc


 


5581
tatgctctct gtacttttga ccaaaaattg accaaagtaa gaaaatgcaa gttctaaaaa


 


5641
tagactaagg atgcctttgc agaacaccaa agcatcccaa ggaactggta gggaagtggc


 


5701
gcctgtctcc tggagtggaa gaggcctgct ccctggctct gggtctgctg ggggcacagt


 


5761
aaatcagtct tggcacccac atccagggca gagaggtctg tggttctcag catcagaagg


 


5821
cagcgcagcc cctctcctct tcaggctaca gggttgtcac ctgctgagtc ctcaggttgt


 


5861
ttggcctctc tggtccatct tgggcattag gttctccagc agagctctgg ccagctgcct


 


5941
cttctttaac tgggaacaca ggctctcaca agatcagaac ccccactcac ccccaagatc


 


6001
ttatctagca agcctgtagt attcagtttc tgttgtagga agagagcgag gcatccctga


 


6061
attccacgca tctgctggaa acgagccgtg tcagatcgca catccctgcg cccccatgcc


 


6121
cctctgagtc acacaggaca gaggaggcag agcttctgcc cactgttatc ttcactttct


 


6181
ttgtccagtc ttttgttttt aataagcagt gaccctccct actcttcttt ttaatgattt


 


6241
ttgtagttga tttgtctgaa ctgtggctac tgtgcattcc ttgaataatc acttgtaaaa


 


6301
attgtcagtg cttgaagctg tttcctttac tcacattgaa gggacttcgt tggttttttg


 


6361
gagtcttggt tgtgactcca agagcagagt gaggaagacc cccaagcata gactcgggta


 


6421
ctgtgatgat ggctgcagtc cagttttatg attctgcttt tatgtgtccc ttgataacag


 


6481
tgacttaaca atatacattc ctcataaata aaaaaaaaac aagaatctga attcttagaa


 


6541
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In another embodiment, antibodies used to practice this invention are designed to bind to, or affinity matured to bind to, a polypeptide SEQ ID NO:3, or subsequences thereof:








(SEQ ID NO: 3)










   1
mnprqgysls gyythpfggy ahrqlryqqp gpgsspssfl lkqieflkgg lpeaprigkq



 


  61
tpslppslpg lrprfpvlla sstrgrqvdi rgvprgvhlg sgglqrgfqh psprgrslpq


 


 121
rgvdclsshf qelsiyqdge qrilkfleel gegkattahd lsgklgtpkk einrvlysla


 


 181
kkgklqkaeg tpplwkiavs tqawnqhsgv yrpdghsqga pssdpslepe drnstsvsed


 


 241
llepfiavsa qawnqhsgvv rpdshsqqsp nsdpgleped snstsatedp lefldmaeik


 


 301
ekicdylfnv sdssalnlak nigltkardi navlidmerq gdvyrqgttp piwhltdkkr


 


 361
ermqikrntn svpetapaai petkrnaefl tcniptsnas ssmvttekve ngqepvikle


 


 421
srqearpepa rlkppvhysg pskagyvdfe ngqwatddip ddlnsiraap gefraimemp


 


 481
sfyshglprc spykkltecg lknpisglle yaqfasqtce frmieqsgpp heprfkfqvv


 


 541
ingrefppae agskkvakqd aamkamtill eeakakdsgk seesshyste kesektaesq


 


 601
tptpsatsff sgkspvttll ecmhklgnsc efrllskegp ahepkfqyev avgaqtfpsv


 


 661
sapskkvakq maaeeamkal hgeatnsmas dngpegmise sldnlesmmp nkvrkigelv


 


 721
rylntnpvgg lleyarshgf aaefklvdqs qpphepkfvy qakvggrwfp avcahskkqg


 


 781
kqeaadaalr vligenekae rmgftevtpv tgaslrrtml llsrspeaqp ktlpltgstf


 


 841
hdqiamlshr efntltnsfq psllgrkila aiimkkdsed mgyvyslgtg nrcvkgdsls


 


 901
lkgetvndch aeiisrrgfi rflyselmky nsqtakdsif epakggeklq ikktvsfhly


 


 961
istapcgdga lfdkscsdra mestesrhyp vfenpkqgkl rtkvengegt ipvessdivp


 


1021
twdgirlger lrtmscsdki lrwnvlglqg allthflqpi ylksvtlgyl fsqghltrai


 


1081
ccrvtrdgsa fedglrhpfi vnhpkvgrvs iydskrgsqk tketsvnwcl adgydleild


 


1141
gtrgtvdgpr nelsrvskkn ifllfkkles fryrrdllrl sygaekkaar dyetaknyfk


 


1201
kglkdmgygn wiskpqeekn fylcpv






Generating and Manipulating Nucleic Acids

In alternative embodiments, compositions and methods of the invention use nucleic acids for treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation and/or self-renewal of hematopoietic stem cells or cancer stem cells. In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of the ADAR1 gene (e.g., SEQ ID NO:1) or an ADAR1 gene transcript (e.g., SEQ ID NO:2). In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript comprises: an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA), or a ribozyme.
In alternative embodiments, nucleic acids of the invention are made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
The nucleic acids used to practice this invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
Alternatively, nucleic acids used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
Techniques for the manipulation of nucleic acids used to practice this invention, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I, Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids.
Nucleic acids or nucleic acid sequences used to practice this invention can be an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. Compounds use to practice this invention include “nucleic acids” or “nucleic acid sequences” including oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practice this invention include nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. Compounds use to practice this invention include nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to practice this invention include “oligonucleotides” including a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that may be chemically synthesized. Compounds use to practice this invention include synthetic oligonucleotides having no 5′ phosphate, and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
In alternative aspects, compounds used to practice this invention include genes or any segment of DNA or RNA involved in producing a polypeptide chain; it can include regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening sequences (introns) between individual coding segments (exons). “Operably linked” can refer to a functional relationship between two or more nucleic acid (e.g., DNA or RNA) segments. In alternative aspects, it can refer to the functional relationship of transcriptional regulatory sequence to a transcribed sequence. For example, a promoter can be operably linked to a coding sequence, such as a nucleic acid used to practice this invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. In alternative aspects, promoter transcriptional regulatory sequences can be operably linked to a transcribed sequence where they can be physically contiguous to the transcribed sequence, i.e., they can be cis-acting. In alternative aspects, transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
In alternative aspects, the invention comprises use of “expression cassette” comprising a nucleotide sequence used to practice this invention, which can be capable of affecting expression of the nucleic acid, e.g., a structural gene or a transcript (e.g., encoding a DRP or antibody) in a host compatible with such sequences. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, in one aspect, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers.
In alternative aspects, expression cassettes used to practice this invention also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a “vector” used to practice this invention can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice this invention can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors used to practice this invention can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). In alternative aspects, vectors used to practice this invention can include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice this invention can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.
In alternative aspects, “promoters” used to practice this invention include all sequences capable of driving transcription of a coding sequence in a cell, e.g., a mammalian cell such as a brain cell. Thus, promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter used to practice this invention can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
“Constitutive” promoters used to practice this invention can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation. “Inducible” or “regulatable” promoters used to practice this invention can direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions.
Antisense Inhibitory Nucleic Acid Molecules
In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of the ADAR1 gene (e.g., SEQ ID NO:1) or an ADAR1 gene transcript (e.g., SEQ ID NO:2). In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript comprises: an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA), or a ribozyme.
Naturally occurring or synthetic nucleic acids can be used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids.
RNA Interference (RNAi)
In one aspect, the invention provides RNAi inhibitory nucleic acid molecules capable of decreasing or inhibiting expression of one or a set of ADAR1 transcripts or proteins, e.g., the transcript (mRNA, message) SEQ ID NO:2 or isoform or isoforms thereof. In one aspect, the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi molecule can comprise a double-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA (microRNA) and/or short hairpin RNA (shRNA) molecules.
In alternative aspects, the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi, e.g., siRNA for inhibiting transcription and/or miRNA to inhibit translation, is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence.
In one aspect, intracellular introduction of the RNAi (e.g., miRNA or siRNA) is by internalization of a target cell specific ligand bonded to an RNA binding protein comprising an RNAi (e.g., microRNA) is adsorbed. The ligand can be specific to a unique target cell surface antigen. The ligand can be spontaneously internalized after binding to the cell surface antigen. If the unique cell surface antigen is not naturally internalized after binding to its ligand, internalization can be promoted by the incorporation of an arginine-rich peptide, or other membrane permeable peptide, into the structure of the ligand or RNA binding protein or attachment of such a peptide to the ligand or RNA binding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003; 20060025361; 20060019286; 20060019258. In one aspect, the invention provides lipid-based formulations for delivering, e.g., introducing nucleic acids of the invention as nucleic acid-lipid particles comprising an RNAi molecule to a cell, see e.g., U.S. Patent App. Pub. No. 20060008910.
Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA, for selectively degrade RNA are well known in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
Methods for making expression constructs, e.g., vectors or plasmids, from which an inhibitory polynucleotide (e.g., a duplex siRNA of the invention) is transcribed are well known and routine. A regulatory region (e.g., promoter, enhancer, silencer, splice donor, acceptor, etc.) can be used to transcribe an RNA strand or RNA strands of an inhibitory polynucleotide from an expression construct. When making a duplex siRNA inhibitory molecule, the sense and antisense strands of the targeted portion of the targeted IRES can be transcribed as two separate RNA strands that will anneal together, or as a single RNA strand that will form a hairpin loop and anneal with itself. For example, a construct targeting a portion of a gene, e.g., an NADPH oxidase enzyme coding sequence or transcriptional activation sequence, is inserted between two promoters (e.g., mammalian, viral, human, tissue specific, constitutive or other type of promoter) such that transcription occurs bidirectionally and will result in complementary RNA strands that may subsequently anneal to form an inhibitory siRNA of the invention.
Alternatively, a targeted portion of a gene, coding sequence, promoter or transcript can be designed as a first and second antisense binding region together on a single expression vector; for example, comprising a first coding region of a targeted gene in sense orientation relative to its controlling promoter, and wherein the second coding region of the gene is in antisense orientation relative to its controlling promoter. If transcription of the sense and antisense coding regions of the targeted portion of the targeted gene occurs from two separate promoters, the result may be two separate RNA strands that may subsequently anneal to form a gene-inhibitory siRNA used to practice this invention.
In another aspect, transcription of the sense and antisense targeted portion of the targeted gene is controlled by a single promoter, and the resulting transcript will be a single hairpin RNA strand that is self-complementary, i.e., forms a duplex by folding back on itself to create a gene-inhibitory siRNA molecule. In this configuration, a spacer, e.g., of nucleotides, between the sense and antisense coding regions of the targeted portion of the targeted gene can improve the ability of the single strand RNA to form a hairpin loop, wherein the hairpin loop comprises the spacer. In one embodiment, the spacer comprises a length of nucleotides of between about 5 to 50 nucleotides. In one aspect, the sense and antisense coding regions of the siRNA can each be on a separate expression vector and under the control of its own promoter.
Inhibitory Ribozymes
The invention provides ribozymes capable of binding and inhibiting, e.g., decreasing or inhibiting, expression of one or a set of ADAR1 transcripts or proteins, e.g., SEQ ID NO:1 or SEQ ID NO:2, or isoform or isoforms thereof.
These ribozymes can inhibit a gene's activity by, e.g., targeting a genomic DNA or an mRNA (a message, a transcript). Strategies for designing ribozymes and selecting a gene-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it can be released from that RNA to bind and cleave new targets repeatedly.
Kits and Instructions
The invention provides kits comprising compositions and/or instructions for practicing methods of the invention. As such, kits, cells, vectors and the like can also be provided. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
Compositions and Methods for Dormant Cancer Stem Cell Detection and Elimination
In alternative embodiments, the invention provides compositions and methods to detect dormant cancer stem cells, e.g., Chronic Myelogenous Leukemia (CML) stem cells. In alternative embodiments, the invention provides compositions and methods for use to, e.g., therapeutically, initiate, stimulate or force a dormant cancer stem cell, e.g., a Chronic Myelogenous Leukemia (CML) stem cell, into cycle so that it can be targeted by a therapeutic agent or procedure, e.g., chemotherapy, radiation therapy or targeted tyrosine kinase inhibitors, or any agent or procedure that targets dividing cells.
In alternative embodiments, the invention provides compositions and methods comprising or comprising use of sonic hedgehog inhibitors, e.g., inhibitors of Smoothened (SMO), an integral membrane protein mediator of Hedgehog signaling (see e.g., Shi et al. (2011) Development, Epub 2011 Aug. 18; Su, et al. (2011) Sci. Signal. July 5; 4(180):ra43), which activate, stimulate or initiate in a stem cell, e.g., a cancer stem cell, a transition from G0 to G1 of the cell cycle, thereby sensitizing the stem cells to agents that target dividing cells.
Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−γc−/− mice with a novel selective Shh inhibitor, designated PF-04449913 (see Supplementary FIG. 1a for structure), reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation.
Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory gene expression and splice isoform analysis demonstrated reversion toward a normal splice isoform signature for many cell cycle regulatory genes.
Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected.
Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators.
Our findings demonstrate that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors.
The invention also provides novel stem cell, e.g., LSC, splice isoform detection platforms, e.g., as kits of the invention, to assess the efficacy of Shh inhibitor-mediated sensitization. In alternative embodiments, stem cell splice isoform detection platforms and compositions (e.g., kits) of the invention are used to identify “conversion” of a stem cell to a “normal cell”, or to determine or define a molecularly targeted therapy for dormant cancer stem cell elimination strategies that ultimately avert relapse. In alternative embodiments, stem cell splice isoform detection platforms and compositions (e.g., kits) of the invention are used to identify compounds or treatments that can successfully inhibit Shh expression.
In alternative embodiments, compositions and methods of the invention can be used synergistically to sensitize dormant stem cells, e.g., LSC, to therapeutic agents that target dividing cells including e.g., tyrosine kinase inhibitors, chemotherapy. In alternative embodiments, compositions and methods of the invention can be used sensitize, e.g., radiosensitize, a cancer stem cell, e.g., a LSC and other cancer stem cell populations, through cell cycle induction or stimulation.
When CML stem cells were treated with a Smoothened (SMO) protein inhibitor (SMO being a key component of the Hedgehog signaling pathway), dormant stem cells entered the cell cycle, as evidenced by the RNA isoform pattern of a leukemic cancer stem cell reverting to the pattern of a normal cell. Hence, in alternative embodiments, this RNA isoform pattern can be used as a biomarker of response to chemotherapy and a target for drug development, e.g., successful or effective inhibition of Shh give a particular, detectable RNA isoform pattern.
Our findings demonstrate that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel cancer stem cell, e.g., a LSC, splice isoform detection platforms of this invention can assess the efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapies. Implementation of novel LSC splice isoform detection platforms of this invention can identify and determine the effectiveness of dormant cancer stem cell elimination strategies that ultimately avert relapse.
In alternative embodiments, the invention provides splice isoform detection kits or arrays for stem cells, e.g., LSC stem cells. In alternative embodiments, the invention provides nanoproteomic detection kits or arrays for stem cells (to determine an altered proteome caused by an altered RNA splicing pattern, or alternatively spliced transcripts) to determine a response to a stem cell therapy, e.g., a LSC targeted therapy.
Kits and Instructions
The invention provides kits comprising compositions and/or instructions for practicing methods of the invention. As such, kits, cells, vectors and the like can also be provided. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
Antisense Inhibitory Nucleic Acid Molecules
In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitor to expression of the Shh or a Shh gene transcript. In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the Shh gene or Shh gene transcript comprises: an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA), or a ribozyme.
Naturally occurring or synthetic nucleic acids can be used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids.
RNA Interference (RNAi)
In one aspect, the invention provides RNAi inhibitory nucleic acid molecules capable of decreasing or inhibiting expression of one or a set of Shh transcripts or proteins, e.g., the transcript (mRNA, message) or isoform or isoforms thereof. In one aspect, the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA (microRNA) and/or short hairpin RNA (shRNA) molecules.
In alternative aspects, the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi, e.g., siRNA for inhibiting transcription and/or miRNA to inhibit translation, is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence.
In one aspect, intracellular introduction of the RNAi (e.g., miRNA or siRNA) is by internalization of a target cell specific ligand bonded to an RNA binding protein comprising an RNAi (e.g., microRNA) is adsorbed. The ligand can be specific to a unique target cell surface antigen. The ligand can be spontaneously internalized after binding to the cell surface antigen. If the unique cell surface antigen is not naturally internalized after binding to its ligand, internalization can be promoted by the incorporation of an arginine-rich peptide, or other membrane permeable peptide, into the structure of the ligand or RNA binding protein or attachment of such a peptide to the ligand or RNA binding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003; 20060025361; 20060019286; 20060019258. In one aspect, the invention provides lipid-based formulations for delivering, e.g., introducing nucleic acids of the invention as nucleic acid-lipid particles comprising an RNAi molecule to a cell, see e.g., U.S. Patent App. Pub. No. 20060008910.
Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA, for selectively degrade RNA are well known in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
Methods for making expression constructs, e.g., vectors or plasmids, from which an inhibitory polynucleotide (e.g., a duplex siRNA of the invention) is transcribed are well known and routine. A regulatory region (e.g., promoter, enhancer, silencer, splice donor, acceptor, etc.) can be used to transcribe an RNA strand or RNA strands of an inhibitory polynucleotide from an expression construct. When making a duplex siRNA inhibitory molecule, the sense and antisense strands of the targeted portion of the targeted IRES can be transcribed as two separate RNA strands that will anneal together, or as a single RNA strand that will form a hairpin loop and anneal with itself. For example, a construct targeting a portion of a gene, e.g., a Shh coding sequence or transcriptional activation sequence, is inserted between two promoters (e.g., mammalian, viral, human, tissue specific, constitutive or other type of promoter) such that transcription occurs bidirectionally and will result in complementary RNA strands that may subsequently anneal to form an inhibitory siRNA of the invention.
Alternatively, a targeted portion of a gene, coding sequence, promoter or transcript can be designed as a first and second antisense binding region together on a single expression vector; for example, comprising a first coding region of a targeted gene in sense orientation relative to its controlling promoter, and wherein the second coding region of the gene is in antisense orientation relative to its controlling promoter. If transcription of the sense and antisense coding regions of the targeted portion of the targeted gene occurs from two separate promoters, the result may be two separate RNA strands that may subsequently anneal to form a gene-inhibitory siRNA used to practice this invention.
In another aspect, transcription of the sense and antisense targeted portion of the targeted gene is controlled by a single promoter, and the resulting transcript will be a single hairpin RNA strand that is self-complementary, i.e., forms a duplex by folding back on itself to create a gene-inhibitory siRNA molecule. In this configuration, a spacer, e.g., of nucleotides, between the sense and antisense coding regions of the targeted portion of the targeted gene can improve the ability of the single strand RNA to form a hairpin loop, wherein the hairpin loop comprises the spacer. In one embodiment, the spacer comprises a length of nucleotides of between about 5 to 50 nucleotides. In one aspect, the sense and antisense coding regions of the siRNA can each be on a separate expression vector and under the control of its own promoter.
Inhibitory Ribozymes
In alternative embodiment, compositions and methods of the invention comprise use of ribozymes capable of binding and inhibiting, e.g., decreasing or inhibiting, expression of one or a set of Shh transcripts or proteins, or isoform or isoforms thereof.
These ribozymes can inhibit a gene's activity by, e.g., targeting a genomic DNA or an mRNA (a message, a transcript). Strategies for designing ribozymes and selecting a gene-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it can be released from that RNA to bind and cleave new targets repeatedly.
Bioisosteres of Compounds Used to Practice the Invention
In alternative embodiments, the invention also provides bioisosteres of compounds used to practice the invention, e.g., compounds having a structure as set forth in FIG. 17a, or PF-04449913. In alternative embodiments, the invention provides compositions that inhibit or slow the expression of a Shh gene, a Shh gene product, a Shh transcript, and/or a Shh polypeptide comprising a PF-04449913, or an equivalent thereof, or a bioisostere thereof.
In alternative embodiments, bioisosteres of the invention are compounds of the invention comprising one or more substituent and/or group replacements with a substituents and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to a compound of the invention, or stereoisomer, racemer or isomer thereof. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structures.
For example, in one embodiment, bioisosteres of compounds of the invention are made by replacing one or more hydrogen atom(s) with one or more fluorine and/or deuterium atom(s), e.g., at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place. Because the fluorine atom or deuterium is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.
Sonic Hedgehog Targets as Biomarkers of Prognosis and Response for Human Chronic Myelogenous Leukemia
In alternative embodiments, this invention compositions and methods to analyze or measure biomarkers in human leukemia cells to predict the amount of blastic transformation, the severity of the disease, the progress (e.g., regression) of the disease in light of a particular treatment or drug administration. In alternative embodiments, this invention compositions and methods to analyze or measure biomarkers which are predictors of response to selective Sonic Hedgehog (Shh) inhibition.
This invention is the first to analyze Shh gene expression in human leukemic versus normal progenitors and to identify an increase in GLI2 and commensurate decrease in GLI3 as predictor of blastic transformation. In alternative embodiments, compositions and methods of the invention measure GLI1 and GLI2 transcript and/or GLI1 and GLI2 protein levels to predict a response to selective Shh inhibition, where GLI1 and GLI2 presence is a positive predictor of a response to selective Shh inhibition. In alternative embodiments, GLI1 and/or GLI2 are used as predictive biomarkers, individually or together, of a response to an inhibitor of the Sonic Hedgehog (shh) pathway, including inhibitors of Smoothened (SMO), an integral membrane protein mediator of Hedgehog signaling (see e.g., Shi et al. (2011) Development, Epub 2011 Aug. 18; Su, et al. (2011) Sci. Signal, July 5; 4(180):ra43). Both qRT-PCR and nanoproteomics confirmed these functional biomarkers.
In alternative embodiments, compositions and methods of the invention measure (determine) levels of GLI2 (increasing) and/or GLI3 (decreasing) as prognostic biomarkers (individually or together) for chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and tyrosine kinase inhibitor resistance. Both qRT-PCR and nanoproteomics confirmed these functional biomarkers.
In alternative embodiments, the invention provides compositions and methods comprising or comprising use of sonic hedgehog inhibitors, e.g., inhibitors of Smoothened (SMO), which activate, stimulate or initiate in a stem cell, e.g., a cancer stem cell, a transition from G0 to G1 of the cell cycle, thereby sensitizing the stem cells to agents that target dividing cells.
Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−γc−/− mice with a novel selective Shh inhibitor, designated PF-04449913 (see Supplementary FIG. 1a for structure), reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation.
Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory gene expression and splice isoform analysis demonstrated reversion toward a normal splice isoform signature for many cell cycle regulatory genes.
Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected.
Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators.
Our findings demonstrate that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors.
The invention also provides novel stem cell, e.g., LSC, splice isoform detection platforms, e.g., as kits of the invention, to assess the efficacy of Shh inhibitor-mediated sensitization. In alternative embodiments, stem cell splice isoform detection platforms and compositions (e.g., kits) of the invention are used to identify “conversion” of a stem cell to a “normal cell”, or to determine or define a molecularly targeted therapy for dormant cancer stem cell elimination strategies that ultimately avert relapse. In alternative embodiments, stem cell splice isoform detection platforms and compositions (e.g., kits) of the invention are used to identify compounds or treatments that can successfully inhibit Shh expression.
In alternative embodiments, compositions and methods of the invention can be used synergistically to sensitize dormant stem cells, e.g., LSC, to therapeutic agents that target dividing cells including e.g., tyrosine kinase inhibitors, chemotherapy. In alternative embodiments, compositions and methods of the invention can be used sensitize, e.g., radiosensitize, a cancer stem cell, e.g., a LSC and other cancer stem cell populations, through cell cycle induction or stimulation.
When CML stem cells were treated with a Smoothened (SMO) protein inhibitor (SMO being a key component of the Hedgehog signaling pathway), dormant stem cells entered the cell cycle, as evidenced by the RNA isoform pattern of a leukemic cancer stem cell reverting to the pattern of a normal cell. Hence, in alternative embodiments, this RNA isoform pattern can be used as a biomarker of response to chemotherapy and a target for drug development, e.g., successful or effective inhibition of Shh give a particular, detectable RNA isoform pattern.
Our findings demonstrate that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel cancer stem cell, e.g., a LSC, splice isoform detection platforms of this invention can assess the efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapies. Implementation of novel LSC splice isoform detection platforms of this invention can identify and determine the effectiveness of dormant cancer stem cell elimination strategies that ultimately avert relapse.
In alternative embodiments, the invention provides splice isoform detection kits or arrays for stem cells, e.g., LSC stem cells. In alternative embodiments, the invention provides nanoproteomic detection kits or arrays for stem cells (to determine an altered proteome caused by an altered RNA splicing pattern, or alternatively spliced transcripts) to determine a response to a stem cell therapy, e.g., a LSC targeted therapy.
In alternative embodiments, biomarkers of the invention can be detected using arrays, microarrays, proteomic arrays and the like, e.g., as described by: Oehler, et al., (2009) Blood, “The derivation of diagnostic markers of chronic myeloid leukemia progression from microarray data”, October 8; 114(15):3292-8. Epub 2009 Aug. 4; Fan, et al., Nature Medicine, May 2009, “Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens,” Volume 15, Number 5: 566-571; O'Neill, et al., Proc. Natl. Acad. Sci. USA, October 2006, “Isoelectric focusing technology quantifies protein signaling in 25 cells”, Volume 103, Number 44: 16153-161158.
Kits and Instructions
The invention provides kits comprising compositions and/or instructions for practicing methods of the invention. As such, kits, cells, vectors and the like can also be provided. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
Spliced Isoform Biomarkers to Assess Responses to Cancer Stem Cell Targeted Therapies
In alternative embodiments, the invention provides compositions and methods for determining the pattern of alternatively spliced transcripts and their protein products to, e.g., distinguish leukemic progenitors from their normal counterparts, e.g., by determining the pattern of alternatively spliced Stat5a specific splice isoforms. In alternative embodiments, the pattern of alternatively spliced Stat5a specific splice isoforms is used as a novel leukemic stem cell (LSC) identification marker.
In alternative embodiments, the invention provides compositions and methods to assess LSC specific responses to targeted agents such as JAK2 inhibitors, e.g., Stat5a specific splice isoforms can be used as biomarkers of response to JAK2 inhibition.
In alternative embodiments, one or both phosphoStat5a and phospho-JAK2 are used as biomarker or biomarkers of a response to a selective JAK2 inhibition, e.g., when selective JAK2 inhibition is used as a clinical treatment or when selective JAK2 inhibitors are tested in in vitro, in animals, or in clinical trials. In alternative embodiments, both signatures of response (biomarker of response in clinical trial) are quantitatively measured; and if the alternative spliced forms do not decrease, the self-renewing LSC are resistant to treatment; and, if STAT5a isoforms are decreased or inhibited, LSC self-renewal capacity is decreased.
We have identified novel isoforms of phosphoStat5a and phospho-JAK2 via RNA-seq that will provide functional LSC markers and will help to determine if different therapies will target this specific population. Our invention is the first to analyze specific isoform expression of STAT5 as a paradigm for human leukemia stem cell identification and to identify splice isoforms that predict cancer stem cell response. In addition, reductions in phosphoStat5a and/or phosphoJAK2 proteins are positive predictors of response to selective JAK2 inhibition. Both qRT-PCR confirmed decreases in transcripts and nanoproteomics confirmed decreases in these alternatively spliced proteins as functional biomarkers of response.
In alternative embodiments, the invention provides compositions and methods that comprise use of splice isoform specific qPCR, and equivalents, and/or nanoproteomics, to detect isoforms that sustain LSC self-renewal, including Stat5a isoforms and other pathway LSC specific splice isoforms in a prognostic kit for cancer stem cells (CSC).
In alternative embodiments, the invention provides compositions and methods that comprise use of splice isoforms such as Stat5a isoforms as predictors of LSC response to selective JAK2 inhibition.
In alternative embodiments, the invention provides compositions and methods that comprise use of splice isoform patterns by qPCR and nanoproteomics to predict response to CSC targeted therapy.
In alternative embodiments, the invention provides compositions and methods to detect cancer stem cell specific JAK/STAT signaling pathway splice isoforms by RNA sequencing, qRT-PCR and nanoproteomics, validated in CML, but applicable to cancer stem cells in the primary and metastatic niches, particularly in the setting of inflammatory cytokines and interleukins elaborated in cancer.
In alternative embodiments, biomarkers, or alternatively spliced forms, used to practice the invention can be detected using e.g., arrays, microarrays, proteomic arrays and the like, e.g., as described by: Oehler, et al., (2009) Blood, “The derivation of diagnostic markers of chronic myeloid leukemia progression from microarray data”, October 8; 114(15):3292-8. Epub 2009 Aug. 4; Fan, et al., Nature Medicine, May 2009, “Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens,” Volume 15, Number 5: 566-571; O'Neill, et al., Proc. Natl. Acad. Sci. USA, October 2006, “Isoelectric focusing technology quantifies protein signaling in 25 cells”, Volume 103, Number 55: 16153-16158.
FIG. 24: Nanoproteomic SAR302503 mechanism of action analysis. Sorted progenitors from mice spleen that where treated where analyzed using nanoproteonomics (CB1000) technology. Panels shows phospho-JAK2 protein (upper left); total JAK2 protein (upper right); phospho-STAT5A protein (lower right) and β2-microblobulin (lower right) status after vehicle (blue) or selective JAK2 inhibitor (SAR302503; green) treatment.
FIG. 25: Identification and quantification of transcript isoforms in LSC treated with vehicle or a selective JAK2 inhibitor. A) RNA-seq-based expression levels of isoforms involved in the Jak/Stat pathway, in vehicle-treated (blue) and SAR302503-treated (red) blast crisis CML sorted progenitors. B) Specific isoform expression after treatment with SAR302503 (JAK2 inhibitor) and dasatinib (BCR-ABL inhibitor), relative to vehicle treatment demonstrating synergistic inhibition of self-renewal gene isoforms such as phosphoSTAT5a and increases in interferon response genes.
Kits and Instructions
The invention provides kits comprising compositions and/or instructions for practicing methods of the invention. As such, kits, cells, vectors and the like can also be provided. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.
EXAMPLES
Example 1
Inhibiting ADAR Enzyme to Decrease in Self-Renewal Capacity or Stem Cells
RNA Editing as a Novel Cancer Stem Cell Target
This example provides data demonstrating that the methods and compositions of the invention are effective for treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation and/or self-renewal of dysfunctional cells, cancer cells, leukemia cells, hematopoietic stem cells or cancer stem cells.
Our research focused on dissecting the role of RNA editing in both normal HSC development and the progression of human chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC). The qRT-PCR data shown here demonstrates that blast crisis LSCs harbor higher levels of IFN responsive ADAR1 p150 isoform than chronic phase progenitors and normal cord blood progenitors (p=0.014). An in vitro study of lentiviral ADAR1 p150 transduced progenitors from normal cord blood and chronic phase showed a significant change for preferred differentiation to GMP (Granulocyte-macrophage progenitor) population, which has been shown to be the leukemia stem cells in CML. A similar inclination was observed in lentiviral shRNA ADAR1 transducer progenitors from blast crisis phase and chronic phase. ADAR1 may also play a role in self-renewal, as a significant of decrease in self-renewal capacity was observed in shRNA transducer chronic phase progenitors. The data shown herein illustrates a crucial role for ADAR1 in both cell differentiation and self-renewal of hematopoietic stem cells.
Example 2
Inhibiting Shh and Breaking the Dormancy of Cancer Stem Cells/Biomarkers for Assessing Cancer Stem Cell Populations
Compositions and Methods for Dormant Cancer Stem Cell Detection and Elimination
This example provides data demonstrating that the methods and compositions of the invention are effective for: inducing in a stem cell susceptibility to BCR-ABL inhibition; activating, stimulating or initiating in a cancer stem cell a transition from G0 to G1 of the cell cycle; or initiating cell cycling in a cancer stem cell; or breaking dormancy in a cancer stem cell; or radiosensitizing of a cancer stem cell; or sensitizing a cancer stem cell to a treatment or protocol that targets dividing cells; or sensitizing a cancer stem cell to a chemotherapy, a radiation therapy or a targeted tyrosine kinase inhibitor.
Sonic Hedgehog Targets as Biomarkers of Prognosis and Response for Human Chronic Myelogenous Leukemia
This example provides data demonstrating that the methods and compositions of the invention are effective for measuring or determining, or predicting, chronic myelogenous leukemia (CML) progression, Leukemic Stem Cell (LSC) generation and/or tyrosine kinase inhibitor resistance, and for measuring or determining, or predicting, a response to an inhibitor or inhibitors of a Sonic Hedgehog (Shh) pathway, or a targeted Shh inhibition, or a selective Shh inhibition, comprising measuring or determining, individually or together, levels or amounts of GLI1 and/or GLI2 transcript and/or protein.
Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−γc−/− mice with a novel selective Shh inhibitor, PF-04449913 (see FIG. 17a, Supplementary FIG. 1a), reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls.
In addition, RNA sequencing after the Shh inhibition revealed significantly decreased cell cycle regulatory gene expression and splice isoform analysis demonstrated reversion toward a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected.
Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators.
Our findings demonstrate that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse.
In this study, we investigated whether Shh signaling links human blast crisis LSC quiescence and self-renewal and whether these traits can be uncoupled with a therapeutic Shh antagonist, as a strategy to enhance sensitivity to BCR-ABL1 tyrosine kinase inhibitors (TKI).
First, to determine if Shh pathway activation fuels blastic transformation and LSC generation, chronic phase (n=7), blast crisis CML (n=21) and normal progenitor (n=15) samples (Supplementary Table 1, below) were analyzed by full transcriptome RNA sequencing (Supplementary Table 2, below), qRT-PCR and nanoparticles. Supplementary Table 1: shows CML patient and normal sample characteristics. Supplementary Table 2: shows Sample Characteristics for Full Transcriptome RNA Sequencing.







SUPPLEMENTARY TABLE 1







CML SAMPLES













Sample



WBC Count 
% Blasts, 



ID
Date
Sex/Age
Sample Type
(K/mm3)
PB
Treatment





C001
26 Oct. 2007
N/A
Chronic Phase CML
N/A
N/A
N/A


C002
09 Jan. 2007
M
Chronic Phase CML
689
4%
Imatinib


C003
N/A
N/A
Chronic Phase CML
N/A
N/A
N/A


C004
05 Feb. 2007
F
Chronic Phase CML
N/A
N/A
N/A


C01
13 Nov. 2006
M/60
Chronic Phase CML
189
1.4%  
none


C02
23 May 2008
F/63
Chronic Phase CML
326
5%
none


C03
10 Dec. 1999
M/57
Chronic Phase CML
49
4.1%  
none


C04
14 Oct. 2006
M/44
Chronic Phase CML
306
5.8%  
none


C05
21 Sep. 2009
M/26
Chronic Phase CML
231
<1%  
none


C06
25 Sep. 2009
F/68
Chronic Phase CML
88
<5%  
Imatinib


C07
25 Mar. 2007
F/33
Chronic Phase CML
37.1
<1%  
N/A


C08
29 Jan. 1999
M/56
Chronic Phase CML
381
<5%  
Imatinib


C11
14 Jan. 2009
F/44
Chronic Phase CML
9.5
<1%  
Hydroxyurea


C12
26 Aug. 2009
N/A
Chronic Phase CML
390
N/A
N/A


C13
21 Sep. 2007
F/50
Chronic Phase CML
N/A
N/A
Imatinib


B001
15 May 2008
M/50
Blast Crisis CML
N/A
N/A
N/A


B002
N/A
N/A
Blast Crisis CML
N/A
N/A
N/A


B04
29 Jul. 2008
M/20
Blast Crisis CML
622
68% 
Imatinib


B05
08 Dec. 2003
M/51
Blast Crisis CML
82.4
32% 
Imatinib


B06
26 Oct. 1993
M/30
Blast Crisis CML
170
94.1%  
Hydroxyurea


B07
29 Oct. 1993
M/48
Blast Crisis CML
209
86.1%  
Hydroxyurea


B08
27 Jul. 2000
M/53
Blast Crisis CML
98
82.6%  
Hydroxyurea


B09
17 Oct. 1991
M/65
Blast Crisis CML
72
41.7%  
none


B10
21 Sep. 1993
M/40
Blast Crisis CML
133
82% 
none


B11
16 Mar. 2006
M/31
Blast Crisis CML
40.1
79% 
Hydroxyurea


B12
26 Jul. 2009
F/47
Blast Crisis CML
262
45% 
Hydroxyurea


B13
16 May 2008
M/49
Blast Crisis CML
8.4
15% 
Imatinib


B14
16 Apr. 2004
M/40
Blast Crisis CML
47.7
47% 
none


B15
8 Mar. 2005
F/31
Blast Crisis CML
11.4
55% 
none


B16
22 Jul. 2002
F/52
Blast Crisis CML
60.3
14% 
none


B20
N/A
?/37
Blast Crisis CML
N/A
N/A
N/A


B26
N/A
?/78
Blast Crisis CML
N/A
N/A
N/A
















SUPPLEMENTARY TABLE 2







Cytogenetics on primary patient samples used for RNAseq










Patient 





ID
Treatment
Cytogenetics
Immunophenotyping





CP-01
None
t(9; 22)(q34; q11)
N/A


CP-02
None
46, XX, t(9; 22)(q34; q11.2)[20], nuc ish(ABL1x3), 
N/A




(BCRx3), (ABL1 con BCRx2)[194/200]



CP-04
None
46, XY, t(9; 22)(q34; q11.2)[20]
N/A


CP-05
None
46, XY, t(9; 22)(q34; q11.2)[20]
N/A


CP-06
None
t(9; 22)(q34; q11.2)
N/A


CP-12
None
N/A
N/A


CP-13
Imatinib
46, XX, t(9; 22)(q34; q11.2), add(17)(p11.2~13) & T315I
CD34+ 6% of CD45+


CP-19
None
t(9; 22)(q34; q11.2)
N/A


BC-02
None
t(9; 22)(q34; q11.2)
CD11b 90%; CD13 99%; CD33 99%; CD34 88%; CD56 95%; 





HLA-DR 95%; CD10 21%; CD19 15%


BC-05
None
t(9; 22)(q34; q11.2)
CD11b 95%; CD13 95%; CD33 95%; CD4 95%; CD117 48%; 





HLA-DR 90%


BC-06
Hydroxyurea
46, XY, t(9; 22)(q34; q11.2)
CD13 70%; CD14 .28; CD33 74%; CD19 48%; HLA-DR 49%


BC-07
Hydroxyurea
t(9; 22)(q34; q11.2) abn 7
CD13 76%; CD33 17%; CD19 90%; HLA-DR 88%; CD34 





82%; CD10 84%


BC-08
Hydroxyurea
46, XY, t(9; 22)(q34; q11), add(18)(q?21).nuc ish
CD11 49%; CD13 100%; CD33 95%; CD56 91%; HLA-DR 




9q34(ABLx3), 22q11(BCRx2)
92%; CD34 85%


BC-09
None
45, XY, −7, t(6; 17; 18)(p21.3; q23; p11.3), t(9; 22)(q34; q11)
CD13 45%; CD14 68%; CD33 67%; HLA-DR 58%


BC-17
Imatinib
t(9; 22)(q34; q11.2)
CD34 46%; HLA-DR 83%; CD10 99%; CD19 99%; TdT 99%


BC-19
Imatinib 
t(9; 22)-T315I
CD34+, CD13+, HLA-DR+, CD117+, MPO+, CD33+, weak 



followed 

CD79a, aberrant CD7+



by dasatinib




BC-25
Imatinib & 
t(9; 22) and inv3(q21q26)
CD117−, HLA-DR−, CD33dim, CD38dim



Hydroxyurea






















SUPPLEMENTARY TABLE 3





Sample 
Mouse 
#
Treat-
RNA 
Amount 
# Reads


#
#
Cells
ment
(ng/ul)
(ng)
(Millions)





















1
197
50k
Vehicle
16.06
225.9
96.9


2
197
23k
Vehicle
15.6
232.5
73


3
147
50k
Vehicle
16.91
253.65
21.4


4
147
50k
Vehicle
21.83
327.45
79.7


5
167
50k
Vehicle
27.12
406.8
109.8


6
167
50k
Vehicle
11.98
179.7
78.2


7
176
50k
SMO
16.46
246.9
88.3


8
176
30k
SMO
12.69
190.35
150.2


9
189
50k
SMO
13.54
203.1
157.9


10
191
50k
SMO
14.09
211.35
162.5


11
182
50k
Vehicle
14.05
210.75
187.1


12
187
50k
SMO
13.39
200.85
196.7









Compared with normal progenitors, both chronic phase and blast crisis CML progenitors were typified by diminished expression of GLI3, a transcriptional repressor. Progression of chronic phase to blast crisis was marked by elevated GLI2, a critical Shh pathway activator (FIG. 1a). While seminal studies have linked Shh activation to cancer stem cell generation4-6, others have shown that Shh signaling modulates the malignant niche7.
However, the role of Shh signaling in niche dependent human LSC maintenance had not been established. To recapitulate extrinsic growth regulatory cues provided by the LSC niche, blast crisis LSC were co-cultured on human SCF, IL-3 and G-CSF (SL/M2) stromal layers. Then, a novel small molecules smoothened (SMO) antagonist, PF-04449913, shown to compete for binding to human SMO (amino acids 181-787) with an IC50 of 4 nM (Supplementary FIG. 1a); to inhibit Shh stimulated luciferase expression in mouse embryonic fibroblasts with an IC50 of 6.8 nM (n=5) (Supplementary FIG. 1b, c); and to significantly reduce medulloblastoma growth in a Ptch1+/−p53+/− allograft model (Supplementary FIG. 2a, b) at doses that decreased Shh target gene expression (Supplementary FIG. 2c-e) was utilized to selectively inhibit Shh signaling in human LSC co-culture experiments. In these studies, LSC survival was significantly reduced by targeted SMO inhibition with PF-04449913 (p=0.047) (FIG. 1b).
To delineate whether these LSC inhibitory effects could be recapitulated in vivo and if Shh driven LSC survival was niche dependent, human LSC engrafted immune deficient RAG2−/−γc−/− mice were treated for 14 days with PF-0444913 (100 mg/kg) by oral gavage. Human LSC engrafted mice were able to sustain treatment with no evidence of weight loss (Supplementary FIG. 3a). Progenitors purified by FACS from PF-04449913 treated LSC engrafted mice displayed reductions in GLI1 (p=0.056) and GLI2 transcript levels (p=0.08) (FIG. 1c) in the splenic niche and a corresponding significant decrease (p=0.006) in spleen weight (FIG. 1d) and size (Supplementary FIG. 3b). Molecular mechanisms of response were investigated with full transcriptome RNA sequencing analysis which revealed marked differences in gene expression between primary and bone marrow engrafted CML indicative of niche specific effects on gene expression (Supplementary FIG. 4a). Following PF-044499913 treatment of mice engrafted with human leukemic progenitors, there was a reversion to a normal progenitor splice isoform pattern for 37 of 50 sonic hedgehog pathway genes in PF-04449913 (Fisher exact test, p=0.0048) (FIG. 1f, g). Of particular interest, Shh pathway gene isoforms that adopted a pattern similar to normal progenitors after Shh inhibitor treatment included pathway regulators such as SUFU, ARRB1, BTRC, CSKN1G1, FBXW11, and PRKACB. Moreover, PF-04449913 treatment of human blast crisis LSC engrafted RAG2−/−c−/− mice reduced human GLI protein expression compared with vehicle treated controls as demonstrated by both immunofluorescence (FIG. 1e) and nanoproteomics (p=0.001) analyses (FIG. 1h, i). However, FACS analysis revealed that Shh inhibitor responses were more robust in extramedullary than medullary niches (Supplementary FIG. 3c, d) suggesting that resistant LSC were more prominent in the marrow perhaps as a consequence of quiescence induction.
Because previous mouse model studies linked Shh signaling to modulation of stem cell cycle control8, we investigated whether Shh signaling induced dormancy by employing cell cycle FACS and full transcriptome RNA sequencing analysis of blast crisis LSC engrafted in RAG2−/−γc−/− mice. While human CD45+ leukemic cells homed to liver, spleen, myeloid sarcomas (tumor) and marrow (FIG. 2a), FACS analysis revealed that blast crisis GMP (LSC) were more prevalent in the marrow than other niches (FIG. 2b). In addition, cell cycle FACS analysis demonstrated that a significantly (p=0.045) greater proportion of marrow resident human leukemic cells were dormant compared with those in the splenic niche (FIG. 2c). Full transcriptome RNA sequencing analysis of engrafted leukemic progenitors revealed repression of cell cycle regulators (family wise p-value 0.02) in response to PF-04449913 compared with vehicle treatment (FIG. 2c and Supplementary FIG. 4b, c) and a shift toward a normal progenitor cell cycle gene splice isoform expression pattern for 75 of 110 isoforms (Fisher exact test, p=0.0006) (FIG. 2f, g). Moreover, cell cycle FACS analysis demonstrated that PF-04449913 treatment of LSC engrafted mice reduced the fraction of leukemic progenitors in G0 commensurate with an increase in the G1 fraction (FIG. 2d and Supplementary FIG. 4d) suggesting that the dormant LSC population had been induced to enter the cell cycle through selective Shh inhibition potentially rendering them sensitive to agents that target dividing cells.
For Shh inhibition to be effective in LSC eradication, normal hematopoietic stem cells must be spared. While some mouse model studies suggest that Shh signaling is dispensable for adult hematopoiesis9, others demonstrate that Gli1 regulates hematopoietic stem cell fate decisions10.
However, the role of Shh signaling in normal human hematopoietic stem and progenitor cell (HSPC) maintenance had not been examined extensively in vitro or in primary sample xenograft (primagraft) models that permit robust engraftment. Thus, we examined the effects of PF-04449913 treatment on normal HSPC phenotype and function. Following, PF-04449913 treatment the differentiation capacity of normal human HSPC in hematopoietic progenitor assays (FIG. 3a) was unimpaired and the percentage of HSPC remained constant after 7 days of SL/M2 stromal co-culture period with PF-04449913 (FIG. 3b). When CD34+ cord blood engrafted NSG mice were treated with PF-04449913 (100 mg/kg for 14 days), the frequency of HSPC as well as myeloid and lymphoid cell fate commitment remained comparable to vehicle treated controls (FIG. 3c, d). Moreover, human hematopoietic cell cycle status was unaltered (FIG. 3e). These data suggest that, unlike LSC, normal human HSPC survival, cell fate decisions and cell cycle regulation are Shh independent thus, explaining their resistance to Shh inhibition and providing a therapeutic window between normal HSC and LSC.
The reduced dormancy of blast crisis progenitors following Shh inhibition compared with their normal progenitor counterparts provided the impetus for determining if LSC were rendered sensitive to dasatinib, a potent TKI (FIG. 4a). Combination therapy with dasatinib and PF-04449913 reduced myeloid sarcoma (FIG. 4b) formation (p<0.0001) and BCR-ABL expression (p=0.0331) (FIG. 4c) in LSC engrafted marrow. Furthermore, Hedgehog PCR array analysis revealed that dasatinib synergized with PF-04449913 by significantly increasing expression of negative regulators of the Shh pathway (p<0.05), including NUMB, PRKACB, FKBP8, CSNK1A1 and CSNK1D (FIG. 4d). Combination therapy led to a marked reduction in marrow LSC (p=0.0016) (FIG. 4e) and LSC serial myeloid sarcoma transplantation potential (p=0.02) (FIG. 4f) providing the impetus for developing combination BCR-ABL and Shh inhibitor clinical trials for imatinib resistant and advanced phase CML patients.
While Ptc-1+/− mouse model experiments have linked Shh modulation of cell cycle regulators to hematopoietic stem cell regeneration8, the role of Shh signaling in human normal progenitor and LSC dormancy had not been established. In robust primagraft assays, we show, for the first time, that marrow resident GLI expressing human blast crisis LSC become dormant thereby enabling them to evade therapy. Following treatment with a clinical Shh antagonist, PF-04449913, dormant LSC were activated to enter the cell cycle thereby rendering them susceptible to agents that target proliferating cells, such as dasatinib, and reducing LSC self-renewal potential. In contrast, normal hematopoietic progenitor cell cycle status and cell fate were unaffected. Together these data support clinical implementation of RNA sequencing derived predictive biomarkers of CSC response and selective Shh pathway inhibition as a strategy to invoke cycling of dormant LSC therapy sensitizing them to BCR-ABL inhibitors and obviating therapeutic resistance. This approach may also provide a viable strategy for CSC eradication in other refractory malignancies.
Methods
Patient Sample Preparation
Normal cord blood and adult peripheral blood samples were purchased from All Cells or obtained from the Cord Blood/Reproductive Sciences Core at UCLA. CML samples were obtained from consenting patients at the UC San Diego, Stanford University, and the University of Toronto Health Network according to Institutional Review Board approved protocols.
Transcriptome Analysis
The SOLiDTM total RNA-Seq kit (Applied Biosystems part #4445374) was used to prepare libraries from normal and blast crisis CML samples, which were sequenced on Solid v.3 plus instruments. Gene isoform models were developed by first combining the isoform models from June 2011 versions of REFSEQTM11, UCSC Known Genes12, and ENSEMBLTM13 and then creating a nonredundant set of models using the “CUFFCOMPARETM” program from version 0.9.3 of the CUFFLINKSTM software package14. Then, we mapped RNA-seq reads to the nucleotide sequences of the isoform models using BWA15 with default parameters and then translated the alignment coordinates in hg19/GRCh37 human genome reference sequence coordinates using a custom script.
Stromal Co-Culture and in vitro Drug Treatment
Normal or blast crisis CML CD34+ cells were plated on confluent mitomycin-C treated SL/M2 cells with different doses of PF-04449913, dasatinib, a combination of PF-04449913 and dasatinib, or vehicle for 14 days. After 1 week of culture, FACS was used to quantify human progenitors and progenitors were FACS sorted into hematopoietic progenitor assays. Colonies were scored after 2 weeks in culture.
Human Progenitor Primagrafts and Treatment
Equal numbers of primary normal or blast crisis CML CD34+ cells were transplanted intrahepatically into neonatal RAG2−/−γc−/− mice to form primagrafts, according to established methods1, 3. At 8-12 weeks post-transplant, mice were treated with PF-04449913, Dasatinib, a combination of PF-04449913 and Dasatinib, or drug vehicle for 14 days followed by FACS analysis of human hematopoietic engraftment in hematopoietic tissues.
Quantitative RT-PCR Analysis of GLI Family Gene Expression
Normal or BC CML cells from patient samples at the University of California San Diego, Stanford University, MD Anderson Cancer Center and the University of Toronto Health Network according to Institutional Review Board approved protocols, were CD34+ selected and FACS-sorted for analyses using a FACS Aria and FLOWJOTM software as described previously19, 20. Quantitative PCR (qRT-PCR) was performed in duplicate on an ICYCLERTM using SYBR Greener Super Mix (Invitrogen, Carlsbad, Calif.), GLI primers were purchased from ABSciences-Catalog number 4331182: GLI 1 (HS00171790_ml), GLI 2 (HS00257977_ml) and GLI 3 (HS00609233_ml). The following primers were used in reactions run with SYBR: BCR-ABL Forward: ctccagactgtccacagcat (SEQ ID NO:4), BCR-ABL Reverse: ccctgaggctcaaagtcaga (SEQ ID NO:5), HPRT Forward: cgtcttgctcgagatgtgatg (SEQ ID NO:6), HPRT Reverse: tttatagccccccttgagcac (SEQ ID NO:7). Relative levels of mRNA were determined according to standard curves. All values were then normalized to HPRT or RPL27 values from the same sample.
TAQMANTM primer/probe sets (ABI) for FoxM1 (Mm00514924_ml), Gli1 (Mm00494645_ml), Gli2 (Mm01293117_ml), Mycn (Mm00476449_ml), Ptch1 (Mm00436026_ml), Ptch2 (Mm00436047_ml), Sfrp1 (Mm00489161_ml), Smo (Mm01162710_ml) and mouse GAPDH (4352339E), on the ABI 7900HT instrument. Target gene expression levels were normalized to mouse GAPDH and calibrated to vehicle treated mice to yield the relative quantitation (RQ) value.
Confocal Fluorescence Microscopic Analyses
Spleens of xenografted mice that were subjected to 2 weeks of treatment were embedded in OCT freezing media (Sakura, Torrance, Calif.), frozen and sent off to histology core (UCSD Moores Cancer Center). For immunostaining, antibodies were used with MOM kit (Vector, Burlingame, Calif.). Primary antibodies used were anti-GLI2 (Abcam) and Alexa 647-conjugated anti-human CD45 (1:25, Serotec). Stained sections were mounted using PROLONG® Gold antifade with DAPI (Invitrogen). Confocal fluorescence images were acquired using Zeiss LSM510TM or Olympus FLUOVIEW FV10iTM microscopes and ADOBE PHOTOSHOP CS5TM software.
Nanoproteomic Immunoassay
Nanofluidic phospho-proteomic immunoassay (NPI) experiments were performed with the NANOPRO 1000TM instrument (Cell Biosciences) and all samples were run in triplicate at least. The FIREFLYTM system first performed a charge-based separation (isoelectric focusing). Predicted pIs were calculated with SCANSITETM. Each sample was run on a panel of different pH gradients (pH 5-8) to optimize the resolution of different peak patterns. After separation and photo-activated in-capillary immobilization, GLI-2 was detected using GLI2-specific antibody (Abcam). A β2-microglubulin-specific antibody (β2M; Upstate) was used to normalize the amount of loaded protein. The peaks were quantified by calculating the area under the curve (AUC).
Primagrafts, in vivo Drug Treatment, and Engraftment Analysis
Immunocompromised RAG2−/−γc−/− mice were bred and maintained in the UC San Diego Moores Cancer Center vivarium. Neonatal mice were transplanted intrahepatically with normal progenitors or LSC from primary patient samples according to our previously published methods19. Upon detection of tumor or peripheral blood engraftment, mice were treated daily by oral gavage with vehicle (50% 1,2 Propandiol, 50% HBSS or methylcellulose), PF-04449913 (100 mg/kg dissolved in vehicle), Dasatinib (50 mg/kg dissolved in vehicle), combination of PF-04449913 (100 mg/kg) and dasatinib (50 mg/kg). After treatment, mice were euthanized and single cell suspensions of hematopoietic tissues were analyzed for human engraftment by FACS as described previously19, 20. Similarly, NOD.
Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ female mice at 7-10 weeks were irradiated sublethally and transplanted with 100K CD34+ human cord blood cells via retro-orbital injection. Eight weeks after transplantation of cord blood cells, the mice were treated for 14 days with either vehicle, or PF-04449913 via oral gavage.
Stromal Co-Culture and in vitro Drug Treatment
The mouse bone marrow stromal cells lines M2-10B4 (M2) and SL/SL (SL) were provided by StemCell Technologies on behalf of Dr. Donna Hogge in the Terry Fox Laboratory (Vancouver, British Columbia). One day prior to co-culture, the cell lines were treated with mitomycin-C (1 mg/ml) and plated in a 1:1 mixture in total concentration of 100,000/ml. 10,000-20,000 CD34+ blast crisis CML or normal cells were then plated on adherent SL/M2 stromal cells, cultured for 7 days, and analyzed by FACS as described previously19. To assess expansion of normal human HS/PCs, irradiated OP9 (M2 clone) stromal cells (20 Gray on a Saxon-Mark1 irradiator) were co-cultured with 50,000 human CD34+ cord blood. OP9M2 stroma was grown in AlphaMem from Gibco with 20% Hyclone FBS, 1% pen strep glutamine and supplemented with cytokines: 50 ug/ml SCF, 10 ug/ml thrombopoietin, and 10 ug/ml Flt3.
Transcriptome Splice Isoform Analysis
Four vehicle-treated and four PF-04449913-treated samples constituting two sets of four technical replicates. The reads from the four RNA sequencing experiments under each treatment regimen were then combined for analysis of effects of vehicle and PF-04449913-treatment containing a total of 65M and 64M reads, respectively and compared with normal FACS purified cord blood CD34+CD38+Lin− progenitors.
Cell Cycle FACS Analysis
Single cell suspensions of bone marrow cells from mice treated with PF-0449913 or vehicle were immunostained with Alexa647-conjugated anti-human CD45 (BioLegend) in 2% fetal bovine serum/PBS followed by live cell staining using the LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Surface stained cells were then fixed in 70% ethanol overnight. Fixed, surface stained cells were immunostained with FITC-conjugated anti-Ki-67 (Abcam, 1:100) in 0.15% saponin/2% fetal bovine serum/PBS, washed twice in saponin-containing staining media and incubated with 7-AAD (10 μg/mL in 0.1M sodium citrate/5 mM EDTA pH8.0/0.15M NaCl/0.5% BSA/0.02% saponin). Stained samples were analyzed using a FACSARIATM and FLOWJOTM21.
SMO Radioligand Competition Binding Assay
Membranes were prepared from a stable cell line created in HEK293FlpIn-TetR cells (Invitrogen) using Flp recombinase-mediated insertion of the pSecTag-FRT/V5-His vector containing a cDNA encoding amino acids 181-787 of human Smo fused to the murine Igk leader sequence to produce a cell surface expressed Smo 181-781 protein. LacZ-negative cells were analyzed for binding a well characterized cyclopamine-competitive tritiated Smo antagonist22. The tritiated ligand was prepared using Crabtree's catalyst and tritium gas. The labeled material was purified by RP HPLC (53.1 Ci/mmol specific activity at 99% purity). For the binding competition assay, 100 μl of assay buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MULTISCREEN-HTS-FBTM cat # MSFBN6B50). The plates were counted in a TOPCOUNTTM scintillation counter (Perkin Elmer). Data analysis uses EXCELTM for % Inhibition and GRAPHPAD PRISMTM for IC50 calculation.
Mouse Embryonic Fibroblast Gli-Luciferase Assay
Mouse Embryonic Fibroblasts expressing luciferase under control of an 8× Gli-response element (Gli-Luc MEFs)23 were obtained from the Pfizer transgenic core facility. Luciferase activity was quantified with an ENVISIONTM plate reader (Perkin Elmer). Graphpad Prism was used for data analysis and IC50 calculation.
Selective Shh Inhibition in a Mouse Medulloblastoma Allograft Model
Primary medulloblastoma tumors were harvested from Ptch+/−p53+/− or Ptch+/−p53−/− mice and propagated as allografts in SCID-bg mice 6-8 weeks of age (20 grams). Freshly isolated tumor fragments of approximately 50 mm3 were surgically implanted subcutaneously into the hind flank region. Body weights and tumor size (length and width) were measured at regular intervals using a caliper and tumor volume was calculated using the formula: length (mm)×width (mm)×width (mm)×0.4. For the tumor growth inhibition studies, cohorts of Ptch+/−p53+/− medulloblastoma allograft bearing mice with tumors ranging from 200 mm3 to 1000 mm3 were dosed daily by oral gavage with vehicle (30% PEG 400/70% PBS) or with PF-04449913 formulated in vehicle.
Assessment of Target Gene Expression in Mouse Model; Microarray Processing
Microarray data were RMA normalized using BIOCONDUCTOR AFFYTM package. Differentially expressed genes were identified based on joint thresholds of t-test pvalue <0.01 and fold change >2. Their human orthologs were mapped using HOMOLOGENE BUILD 62TM. They were compared with curated gene sets from a variety of pathway/signature databases and enrichment P-value was determined using hypergeometric statistics calculated with MATLABTM. More specifically, the probability of observing at least (k) genes from a gene set is given by




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Statistical Analysis
Statistical analyses were performed with Microsoft Excel and Graphpad Prism software. Continuous variables for each comparison group were assessed for distribution through univariate statistics. If the assumption of normal distribution could be supported, then the Student's t test was performed for comparison of two samples with assessment of equality of variance with an F statistic. If the assumption of normal distribution was not supported, nonparametric testing was performed with the two samples Wilcoxon test using the t approximation for samples with N of less than 20.
Figure Legends
FIG. 8. Blast Crisis LSC Activate Sonic Hedgehog in Selective Niches: FIG. 8a. GLI1 and GLI2 transcripts were compared by TaqMan RT-PCR in FACS-purified human cord blood and peripheral blood CD34+CD38+Lin−PI− progenitor cells (n=9, black), chronic phase CML (n=7, blue) and in blast crisis CML (n=10, red) patient samples. Comparative qRT-PCR analysis of GLI3 transcript levels was performed on normal (n=7, black), chronic phase (n=6, blue) and blast crisis CML (n=7, red) cells. Values were normalized to RPL27 or HPRT housekeeping genes, and set to 1 for the normal progenitors. b. FACS analysis revealed a reduction in leukemic progenitor survival following 7 days of PF-04449913 (1 μM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures. c. Down regulation of GLI expression analyzed by TaqMan RT-PCR in human LSC engrafted bone marrow derived from PF-04449913 and vehicle treated mice. d. Spleen size in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n=16, blue) or PF-04449913 (n=6; 100 mg/kg daily, purple), e. Immunofluorescence analysis of splenic sections from no transplant or LSC engrafted mice treated with vehicle or PF-04449913. Photomicrographs of sections stained with DAPI (upper panel) and antibodies specific for human CD45 (upper middle panel), human GLI (lower middle panel) and the merged image (lower panel). f. Isoform-level transcriptome measurements of Shh pathway genes in vehicle-treated (blue) and PF-04449913-treated (purple) CML BC LSC and in normal CD34+CD38+Lin−FACS-purified progenitors (black). Of the 87/249 genes/isoforms in the Shh pathway, 50 isoforms of 18 genes were significantly differentially expressed in either PF-04449913-treated or normal cells when compared to vehicle-treated cells. In 37 of 50 instances (bold), the expression level of treated and normal isoforms followed the same directional change (concordant) when compared to vehicle-treated cells. g. A significant concordance was observed in the relative expression of the 50 isoforms in PF-0449913-treated and in normal cells relative to vehicle treated cells. h. Nanoproteomic (CB1000) traces of total GLI protein after vehicle (blue) and PF-04449113 (green) treatment. i. Quantification of GLI protein expression in splenic CD34+ cells derived from vehicle (n=3) or PF-04449913 (n=3) treated LSC engrafted mice. GLI expression was determined after normalizing the area under the curve (AUC) to a β2-microglobulin (β2M) loading control.
FIG. 9. Induction of Blast Crisis LSC Cycling with Sonic Hedgehog Inhibition: FIG. 9a. Frequency of CD45+ cells in hematopoietic tissues (liver n=4, spleen n=3, bone marrow n=6, tumor n=3) of blast crisis CML engrafted mice. b. FACS quantitation of common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP) populations within each hematopoietic tissue. c. Comparison of cell cycle status (G0, green; G1, light blue; G2/S, navy) of human CD45+ blast crisis CML progenitors engrafted mice in the bone marrow and spleen. More human CD45+ blast crisis cells in the marrow were in G0 than those in the splenic niche (n=8) d. Representative FACS plots comparing Ki67 and 7AAD in bone marrow engrafted viable human CD45+ cells after 14 days of vehicle or PF-04449913 treatment. e. Gene set enrichment analysis for the significantly down-regulated pathway “Regulators of Cell Cycle” (please see methods for details). The core enrichment subset shows the 18 most down regulated genes among the 41 genes in the pathway. f. Isoform-level transcriptome measurements of cell cycle genes in vehicle-treated (blue) and PF-04449913-treated (purple) blast crisis CML LSC and in normal CD34+CD38+Lin−FACS-purified progenitors (black). Of the 84/454 genes/isoforms in the cell cycle, 110 isoforms of 51 genes were significantly differentially expressed in either PF-04449913-treated or normal cells when compared to vehicle-treated cells. In 75 of 110 instances (bold), the expression level of treated and normal isoforms followed the same directional change (concordant) when compared to vehicle-treated cells. g. A significant concordance was observed in the relative expression of the 75 isoforms in PF-0449913-treated and in normal cells relative to vehicle treated cells.
FIG. 10. Shh Inhibition Spares Normal Human Hematopoietic Stem and Progenitor cells: FIG. 10a. Left, Differentiation into CFU-Mix (black), BFU-E (red), CFU-G (orange), CFU-M (yellow), CFU-GM (blue) of normal cord blood HSPC was assessed in hematopoietic progenitor assays (n=3) after PF-04449913 (1 M) or vehicle treatment for 12 days. Right, Representative photomicrographs of cord blood colonies after 12 days of treatment with vehicle (DMSO) or PF-04449913 (1 M). b. Human cord blood (n=3), CD34+38+Lin−PI− cells, plated on SL/M2 stroma and treated with vehicle (DMSO) or PF-04449913 (1 μM) for 7 days followed by FACS analysis. c. Representative FACS plots depicting HSPC, myeloid and lymphoid differentiation in human cord blood engrafted mice after 14 days of treatment with vehicle (n=3) or PF-04449913 100 mg/kg (n=4). d. FACS analysis was used to determine the total human CD45+, HSPC, myeloid and lymphoid cell count in bone marrow after 14 days of treatment with vehicle (n=3, green) or 100 mg/kg of PF-04449913 (n=4, purple). e. FACS quantification of G0 (green), G1 (light blue) and G2/S (navy) human CD45+ cells in cord blood engrafted marrow after 14 days of treatment with vehicle (n=3) or PF-04449913 100 mg/kg (n=4).
FIG. 11. Combined BCR-ABL and Shh Inhibition Reduces LSC Survival in the Niche: FIG. 11a. Schematic of in vivo experiments. RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ cells within 48 hours of birth. After 8 to 10 weeks, blast crisis CML engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or combination (PF-04449913 100 mg/kg and Dasatinib 50 mg/kg). Hematopoietic tissues were FACS analyzed for leukemia engraftment and qRT-PCR for BCR-ABL1 transcripts. b. Myeloid sarcoma count of blast crisis CML engrafted mice in each treatment group vehicle (n=13, green), PF-04449913 (n=7, purple), dasatinib (n=6, red) and combination (n=3, black) after 14 days of treatment. c. BCR-ABL1 transcripts in the spleens of blast crisis CML engrafted mice after 14 days of treatment with vehicle (green, n=9), PF-04449913 (purple, n=11), dasatinib (n=8, red) or combination (n=5, black) d. Shh gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n=3, green), PF-04449913 (n=4, purple) dasatinib (n=4, maroon), combination (n=3, dark grey) was analyzed via qPCR array (SA Biosciences). Expression levels of 5 Shh regulatory genes (NUMB, PRKABC, CTNNB1, FKBP8, CSNK1A1, CSNK1D and STK36) were significantly upregulated by the synergistic effects of PF-04449913 and dasatinib treatment after performing limma test. e. FACS analysis of percentage of marrow engrafted blast crisis LSC (n=3 patients) after 14-day treatment with vehicle (n=31, green), PF-04449913 (n=25, purple), dasatinib (n=27, maroon) and combination (n=27, grey). f. Myeloid sarcoma counts in mice serially transplanted with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n=8, maroon) or combination (n=3, grey) treated human progenitors.
FIG. 12 (Supplementary FIG. 1): PF-04449913 structure and chemical properties: FIG. 12a. Chemical structure of PF-04449913, a selective smoothened (SMO) antagonist. b. Competition-binding assay using a characterized cyclopamine-competitive SMO antagonist, PF-04449913 competes with the radiolabeled SMO antagonist for binding to human SMO (amino acids 181-787) with an IC50 of 4 nM (4.3 nM+/−5.2 nM, N=5). c. Inhibition of Shh stimulated luciferase expression using mouse embryonic fibroblasts expressing luciferase under control of an 8× Gli-response element (Gli-Luc MEFs). d. Dose dependent inhibition by PF-04449913 in the Gli-Luc MEF reporter assay; PF-04449913 inhibits Shh stimulated reporter activity with an IC50 of 6.8 nM (n=5).
FIG. 13 (Supplementary FIG. 2): PF-04449913 Inhibits Shh Signaling in Ptch+/−p53+/− Tumor model: FIG. 13a. Anti-tumor activity of PF-04449913 against Ptch+/−p53+/− medulloblastoma. Allograft ( ̃700 mm3) bearing SCID-bg female mice were dosed orally once a day for six days with 100 mg/kg of PF-04449913 or vehicle. Tumor size (length and width) was measured using a caliper at regular intervals and tumor volume was calculated by standard procedure. Results are mean+/−standard error of the mean (n=3 animals per group). b. Dose dependent anti-tumor efficacy of PF-04449913 against Ptch+/−p53+/− medulloblastoma allografts. Cohorts of allograft ( ̃100 mm3 to 1000 mm3) bearing SCID-bg female mice were dosed orally once a day for six days with different dose levels of PF-04449913. Tumor size (length and width) was measured using a caliper at regular intervals and tumor volume was calculated by standard procedure. The percent change of an individual tumor volume was calculated from the tumor volume on the first day of dosing to the sixth day. Results are expressed as mean+/−standard deviation (n=3 to 12 animals per group; p<0.01 for 1 mg/kg group and p<0.001 for all other groups). c. Hh pathway inhibition in PF-04449913 treated Ptch+/−p53+/− medulloblastoma allografts. Ptch+/−p53+/− allograft bearing SCID-bg female mice were dosed orally once a day for six days with 100 mg/kg of PF-04449913 or vehicle. Expression levels of FoxM1, Gli1, Gli2, Mycn, Ptch1, Ptch2, Sfrp1 and Smo were determined by real time PCR. The vehicle-treated levels for each gene were normalized to 100%. d. Genes significantly down-regulated by PF-04449913 treatment in Ptch+/−p53−/− mice. Hierarchical clustering was performed on Z-score transformed data using correlation similarity metric and centroid linkage method. e. Gene signatures enriched for within the top 31 PF-04449913-downregulated genes in Ptch+/−p53−/− mice. Statistical significance of over-representation and observed/expected ratio were calculated as described in supplementary methods.
FIG. 14 (Supplementary FIG. 3): LSC Responses to Shh Inhibition are Niche Dependent: FIG. 14a. Percentage of weight changes in each treatment group of mice over the course of 14 days of treatment. b. Representative photographs of spleens from no transplant, blast crisis CML engrafted mice treated with vehicle or PF-04449913. c. Representative FACS plots of blast crisis CML engrafted mice treated with vehicle, PF-04449913, dasatinib, or combination. d. Total blast LSC count in bone marrow, spleen and liver after 14 days of treatment with vehicle (n=15) or PF-04449913 (n=14).
FIG. 15 (Supplementary FIG. 4): Decreased Cell Cycle Regulator Expression Enhances LSC Cycling: FIG. 15a. Heat-map of normalized expression values on log 2 scale for 10,573 highly expressed genes in FACS-purified primary blast crisis CML CD34+CD38−Lin− and CD34+CD38+Lin− (SOLiD RNAseq gene count per million reads >1 in all the 6 samples); RAG2−/−g-c−/− marrow engrafted blast crisis CML CD34+CD38−Lin− and CD34+CD38+Lin−; and normal cord blood CD34+CD38−Lin− and CD34+CD38+Lin− samples. Hierarchical cluster analysis based on Euclidean distance measure was performed to check the similarities between the samples. b. Gene set enrichment analysis (GSEA) summary table obtained from SOLiD RNAseq data comparing PF-04449913 treated mice (n=4) to control (n=4) (average 24.7-58.0 million mapped reads/sample). In this analysis, 13,850 protein-coding genes with >10 reads in at least one sample were included. Read counts were normalized16 and genes ranked by Significance Analysis of Microarrays (SAM)17. Eight a priori cell cycle pathways were considered in the GSEA analysis18, with significance assessed by gene-wise permutation. The “Regulation of Cell Cycle” pathway was significantly down-regulated in human progenitors from PF-04449913 treated mice; 7 of 8 investigated pathways decreased. Table columns are pathway name, number of genes (pathway size), nominal p-value; FDR adjusted q-value and adjusted p-value controlling for the family-wise error rate. c. GSEA enrichment plot for the significantly down-regulated pathway (Regulation of Cell Cycle). The horizontal heatmap shows SAM score in descending order for all 13,850 genes. The GSEA enrichment score for the pathway (genes indicated as vertical bars) is indicated as the maximal excursion of the green line. Because the pathway is down regulated, the core enrichment subset consists of the right-most 18 among the 41 genes in the pathway. d. Cell cycle analysis of bone marrow from blast crisis CML engrafted mice after 14 days of vehicle (n=8) or PF-04449913 (n=8), *p<0.05 for both G0 and G1 population compared with vehicle treatment.
FIG. 16: SHH Pathway Gene Expression Pattern Portends BC Transformation. Using the SHH pathway allowed us to distinguish CML CP from BC and normal counterparts, demonstrating that this pathway is activated with disease progression.
a. Heatmap from unsupervised agglomerative hierarchical clustering of sonic hedgehog (SHH) pathway genes using RNA Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients, 3 normal cord blood (CB) and 3 normal peripheral blood (NPB) sample. Red indicates over- and green, under-expression relative to the median RPKM (log 2 scale). Grey represent not expressed (RPKM=0). b. Principal components plots derived from RNA Seq data for 41 genes in the SHH pathway, from 8 chronic phase (CP; black triangles) and 9 blast crisis (BC; red circles) subjects, as well as 3 cord blood normal samples (CB; blue diamonds) and 3 normal peripheral blood (NPB; blue circles). c. Box plots for GLI2 expression of 7 chronic phase (CP) and 6 blast crisis (BC) non-treated subjects, as well as 3 cord blood normal samples (CB) and 3 normal peripheral blood (NPB). Two-sided Jonckheere-Terpstra trend test: p=0.014. d. GLI1 and GLI2 transcripts were compared using quantitative RT-PCR in FACS-purified human cord blood and normal peripheral blood CD34+CD38+Lin−PI− progenitor cells (n=9, black), chronic phase CML (n=7, blue) and in blast crisis CML (n=10, red) patient samples. Values were normalized to RPL27 or HPRT housekeeping genes, and set to 1 for the normal progenitors. (Student's t-test *p<0.05).
FIG. 17: FIG. 2. Selective SHH Inhibition Reduces BC LSC Burden in Selective Niches. In our preclinical model that utilized human samples, we tested a SHH pathway inhibitor on AML and CML samples and observed a decrease in survival after treatment. We also identified GLI2 as a biomarker of response.
a. Chemical structure of PF-04449913, a selective smoothened (SMO) antagonist. b. FACS analysis revealed a significant (Student's t-test, *p=0.047) reduction in blast crisis leukemic progenitor survival (n=4 patients) following 7 days of PF-04449913 (1 mM, purple) compared with vehicle (DMSO, blue) treatment in SL/M2 co-cultures. c. Cord blood (n=3) or AML (n=4 patients) CD34+ cells were plated on SL/M2 co-cultures and treated with vehicle (DMSO) or PF-04449913 (1 uM) for 7 days. Colony forming unit (CFU) survival was determined and compared to vehicle treatment. (Student's t-test, **p=0.001). d. Spleen weight in blast crisis CML LSC engrafted mice after 14 days of treatment with vehicle (n=16, blue) or PF-04449913 (n=12; 100 mg/kg daily, purple). A significant (Student t-test, *p=0.006) reduction is observed after PF-044449913 treatment. e. Nanoproteomic (CB1000) traces of total GLI2 protein after vehicle (blue) and PF-04449913 (green) treatment. f. Quantification of GLI2 protein expression in sorted progenitors derived from vehicle (n=3) or PF-04449913 (n=3) treated LSC engrafted mice. GLI2 expression was determined after normalizing the area under the curve (AUC) to a b2-microglobulin (b2M) loading control (Student's t-test *p=0.001) g. Confocal fluorescence microscopic analysis of spleen sections from no transplant or LSC engrafted mice treated with vehicle of PF-04449913. Photomicrographs of sections stained with DAPI and antibodies specific for human CD45, human GLI2 and the merged image.
FIG. 18: SHH PATHWAY Inhibition Induces Cycling of Dormant BC LSC.
a. Heatmap from unsupervised agglomerative hierarchical clustering of cell cycle pathway genes using RNA Seq data from FACS-purified progenitors (CD34+CD38+lin−PI−) from 8 chronic phase (CP) and 9 blast crisis (BC) patients sample. Red indicates over- and green, under-expression relative to the median RPKM (log 2 scale). Grey represent not expressed (RPKM=0). b. Network analysis performed on differentially expressed genes between BC and CP revealed CDKN1A as a key hub for cell cycle difference. c. Representative FACS plots comparing Ki67 and 7AAD in bone marrow engrafted viable human CD45+ cells after 14 days of vehicle or PF-04449913 treatment. d. Cell cycle analysis of bone marrow from blast crisis CML engrafted mice after 14 days of vehicle (n=8) or PF-04449913 (n=8). Student's t-test *p<0.05 for both G0 and G1 population compared with vehicle treatment. e. GSEA enrichment plot for the significantly down-regulated pathway (Regulation of Cell Cycle). The horizontal heatmap shows SAM score in descending order for all 13,850 genes (SHH pathway genes indicated as vertical black bars). The GSEA enrichment score for the pathway (0-45) is indicated as the maximal excursion of the green line. f. Normalized gene expression values for the 18 genes in the core enrichment subset from the “Regulation of Cell Cycle” pathway. All the genes had a negative SAM score and are sorted in order of descending SAM score along the x-axis. This order agrees with the order in the GSEA enrichment plot, where expression levels for these genes are significantly reduced in the PF-04449913 treated mice.
FIG. 19: SHH Inhibition in clinical samples. We tested the effects of SHH pathway inhibition in a clinical study and observed similar results to what we observed in our preclinical model.
a. Characteristics of patients enrolled in clinical trial NCT01546038. b. Clinical response to PF-04449913 in the bone marrow of AML patient samples. c. Representative FACS cell cycle plots of Ki67 and 7AAD staining of human CD34+CD38− and CD34+CD38+ cells derived from primary patient samples after 4 weeks (C1D28) of treatment with PF-04449913 (40 mg) on the Phase I clinical trial. d. Cell cycle analysis (peripheral blood-CD45+PI−) from a secondary AML patient (AML-4, Supplementary table 1) that was treated with PF-04449913 (40 mg) for 4 weeks on the Phase I clinical trial. Student's t-test *p<0.05 for both G0 and G1 population compared with pre-treatment. e. Characteristics of patient samples analyzed for their cell cycle study. Patients in red represent clinical responders.
FIG. 20: SHH inhibitor Induced cell cycle activation Enhances BC LSC TKI Sensitivity. Using our preclinical model, we demonstrated that using SHH pathway inhibitor in combination with current therapy is the best possible route for curative treatment.
a. Schematic of in vivo experiments. RAG2−/−γc−/− pups were transplanted intrahepatically with 50,000 CD34+ cells within 48 hours of birth. After 8 to 10 weeks, blast crisis CML engrafted mice were treated daily for 14 days by oral gavage with vehicle, PF-04449913 (100 mg/kg), Dasatinib (50 mg/kg) or combination (PF-04449913 100 mg/kg and Dasatinib 50 mg/kg). Hematopoietic tissues were FACS analyzed for human leukemic engraftment and qRT-PCR for BCR-ABL1 transcripts. b. Myeloid sarcoma count in blast crisis CML engrafted mice in each treatment group vehicle (n=13, blue), PF-04449913 (n=7, purple), dasatinib (n=6, red) and combination (n=3, black) after 14 days of treatment. Graph shows mean+/−SEM; *p<0.05 and *p<0.01 by ANOVA and Tukey post-hoc analysis c. FACS analysis of percentage of marrow engrafted blast crisis progenitor LSC (n=3 patients) after 14-day treatment with vehicle (n=31, blue), PF-04449913 (n=25, purple), dasatinib (n=27, maroon) and combination (n=27, grey). Graph shows percentage of CD34+CD38+lin− cells in the bone marrow; *p<0.05 by ANOVA and Tukey post-hoc analysis d. BCR-ABL transcripts in the blast crisis CML engrafted marrow mice after 14 days of treatment with vehicle (blue, n=9), PF-04449913 (purple, n=11), dasatinib (n=8, red) or combination (n=5, black). Graph shows normalized BCR-ABL expression (HPRT)+/−SEM; *p<0.05 by ANOVA and Tukey post-hoc analysis e. Hedgehog pathway gene expression in FACS purified human progenitor cells from blast crisis LSC engrafted mouse marrow treated with vehicle (n=3, blue), PF-04449913 (n=4, purple) dasatinib (n=4, maroon), combination (n=3, dark grey) was analyzed by hedgehog (SHH) qPCR array (SAbiosciences). The limma method was used to test for main effects of PF-04449913 and Dasatinib, and their synergistic interaction among 41 genes. Null hypotheses were rejected at p=0.05 significance level without adjusting for multiple comparisons. Expression levels of seven genes were significantly altered by synergistic effect of PF-04449913 and Dasatinib (NUMB, PRKACB, CTNNB1, FKBP8, CSNK1A1, CSNK1D and STK36), where five represent SHH regulatory genes (graphed). f. Mice serially transplanted with FACS purified human progenitors from LSC engrafted mice treated with vehicle (n=12, green), PF-04449913 (n=12, purple), dasatinib (n=8, maroon) or combination (n=7, grey) were examined for myeloid sarcomas. Graph shows mean myeloid sarcoma count+/−SEM; *p<0.05 and *p<0.01 by ANOVA and Tukey post-hoc analysis.
FIG. 21 (supplementary FIG. 1): PF-04449913 chemical properties and Inhibition of Shh Signaling in a Ptch+/−p53+/− Tumor model. This figure summarizes the effect of inhibiting the SHH pathway in a different preclinical mouse model.
a. Competition-binding assay using a characterized cyclopamine-competitive SMO antagonist. PF-04449913 competes with the radiolabeled SMO antagonist for binding to human SMO (amino acids 181-787) with an IC50 of 4 nM (4.3 nM+/−5.2 nM, N=5). b. Inhibition of Shh stimulated luciferase expression using mouse embryonic fibroblasts expressing luciferase under control of an 8× GLI-response element (GLI-LUC MEFs). c. Dose dependent inhibition by PF-04449913 in the GLI-Luc MEF reporter assay; PF-04449913 inhibits Shh stimulated reporter activity with an IC50 of 6.8 nM (n=5). d. Anti-tumor activity of PF-04449913 against Ptch+/−p53+/− medulloblastoma. Allograft ( ̃700 mm3) bearing SCID-bg female mice were dosed orally once a day for six days with 100 mg/kg of PF-04449913 or vehicle. Tumor size (length and width) was measured using a caliper at regular intervals and tumor volume was calculated by standard procedure. Results are mean+/−standard error of the mean (n=3 animals per group). e. Dose dependent anti-tumor efficacy of PF-04449913 against Ptch+/−p53+/− medulloblastoma allografts. Cohorts of allograft ( ̃200 mm3 to 1000 mm3) bearing SCID-bg female mice were dosed orally once a day for six days with different dose levels of PF-04449913. Tumor size (length and width) was measured using a caliper at regular intervals and tumor volume was calculated by standard procedure. The percent change of an individual tumor volume was calculated from the tumor volume on the first day of dosing to the sixth day. Results are expressed as mean+/−standard deviation (n=3 to 12 animals per group; p<0.01 for 1 mg/kg group and p<0.001 for all other groups). f. Genes significantly down-regulated by PF-04449913 treatment in Ptch+/−p53−/− mice. Hierarchical clustering was performed on Z-score transformed data using correlation similarity metric and centroid linkage method. g. Hh pathway inhibition in PF-04449913 treated Ptch+/−p53+/− medulloblastoma allografts. Ptch+/−p53+/− allograft bearing SCID-bg female mice were dosed orally once a day for six days with 100 mg/kg of PF-04449913 or vehicle. Expression levels of FoxM1, Gli1, Gli2, Mycn, Ptch1, Ptch2, Sfrp1 and Smo were determined by qRT-PCR. The vehicle-treated levels for each gene were normalized to 100%. h. Gene signatures enriched for within the top 31 PF-0449913-downregulated genes in Ptch+/−p53−/− mice. Statistical significance of over-representation and observed/expected ratio were calculated as described in supplementary methods.
FIG. 22 (supplementary FIG. 2): Cell cycle analysis in clinical samples—this data supports the effect of these clinical study.
a. GSEA analysis summary table obtained from RNA sequencing data comparing PF-04449913 treated engrafted mice (n=4) to control (n=4) (average 24.7-58.0 million mapped reads/sample). In total, 13,850 protein-coding genes with >10 reads in at least one sample were included. Read counts were normalized16 and genes ranked by Significance Analysis of Microarrays (SAM).17 Eight cell cycle pathways were considered in the GSEA analysis18, with significance assessed by gene-wise permutation. The “Regulation of Cell Cycle” pathway was significantly down-regulated in PF-04449913 purified human progenitors derived from treated mice (family-wise p value=0.02). Table columns show pathway name, number of genes (pathway size), nominal p-value, FDR adjusted q-value. b. Characteristics of patients enrolled and sequenced using the gene expression profile by Affymetrix GeneChip 1.0 ST after PF-04449913 treatment for 28 days (C1D28), Clinical trial Gov.NTC00953758. c. GSEA analysis summary table obtained from patients (n=8) sequenced after PF-04449913 treatment (C1D28). GSEA was performed at FDR=5% and data compared pre-treatment (screening) v/s, PF-04449913 treated. Table columns show pathway name, number of genes (ES), nominal p-value and FDR adjusted q-value.
FIG. 23 (supplementary FIG. 3): SHH Inhibition Spares Normal Human Hematopoietic Progenitors. We did not observe a toxic effect on the normal counterparts analyzed; thus, a clear therapeutic index for the use of SHH inhibition was demonstrated.
a. Differentiation into CFU-Mix (purple), BFU-E (red), CFU-G (orange), CFU-M (yellow), CFU-GM (blue) of normal cord blood progenitors was assessed in hematopoietic progenitor assays (n=3) after PF-04449913 (1 mM) or vehicle treatment for 14 days. b. FACS analysis was used to determine the total human CD45+, hematopoietic stem and progenitor cell (HSPC), myeloid and lymphoid cell count in bone marrow after 14 days of treatment with vehicle (n=3, green) or 100 mg/kg of PF-04449913 (n=4, purple). c. FACS quantification of G0 (green), G1 (light blue) and G2/S (navy) human CD45+ cell in cord blood engrafted marrow after 14 days of treatment with vehicle (n=3) or PF-04449913, 100 mg/kg (n=4). d. Representative FACS plots depicting HSPC, myeloid and lymphoid differentiation (panel B) in human cord blood engrafted mice after 14 days of treatment with vehicle or 100 mg/kg of PF-04449913.
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A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

## Claims

1. A method for treating, ameliorating or preventing diseases and conditions responsive to the inhibition or slowing of cell differentiation, or self-renewal or self-renewal capacity of a dysfunctional cell, a cancer cell, or a hematopoietic stem cell, comprising,
(a) providing a composition that inhibits or slows the expression of or the activity of: an ADAR1 gene (adenosine deaminase acting on RNA 1) comprising a sequence as set forth in (SEQ ID NO:1) and/or an ADAR1 transcript comprising a sequence as set forth in (SEQ ID NO:2)
and the composition that inhibits or slows the expression of the ADAR1 gene or the ADAR1 transcript comprises an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript; and
(b) administering a sufficient amount of the composition to an individual in need thereof, wherein a sufficient amount comprises the inhibition or slowing of cell differentiation, or self-renewal or self-renewal capacity of the dysfunctional cell the cancer cell, or the hematopoietic stem cell.

2. The method of claim 1, wherein the method treats, ameliorates or prevents conditions responsive to the inhibition or slowing of the self-renewal or self-renewal capacity of a hematopoietic stem cell.

3. The method of claim 1, wherein the inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript comprises: a ribozyme; an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (sRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA).

4. The method of claim 1, wherein the inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript comprises a single or doublestranded and/or sense or antisense sequence or subsequence comprising or complementary to SEQ ID NO:1 or SEQ ID NO:2.

5. The method of claim 1, wherein the method treats, ameliorates or prevents diseases and conditions responsive to the inhibition or slowing of cell differentiation.

6. The method of claim 1, wherein the method treats, ameliorates or prevents conditions responsive to the inhibition or slowing of the self-renewal or self-renewal capacity of a dysfunctional cell or a cancer cell.

7. The method of claim 6, wherein the cancer cell is a cancer stem cell.

8. The method of claim 7, wherein the cancer stem cell is a leukemia stem cell or a Chronic Myeloid Leukemia (CML) stem cell.

9. The method of claim 6, wherein the cancer cell is a leukemia cell.

10. The method of claim 9, wherein the leukemia cell is a Chronic Myeloid Leukemia (CML) cell.

11. The method of claim 1, wherein the method treats or ameliorates diseases and conditions responsive to the inhibition or slowing of cell differentiation.

12. The method of claim 1, wherein the method treats or ameliorates diseases and conditions responsive to the self-renewal or self-renewal capacity of dysfunctional cells, cancer cells.

13. The method of claim 1, wherein the method treats or ameliorates diseases and conditions responsive to the self-renewal or self-renewal capacity of hematopoietic stem cells.

14. The method of claim 1, wherein the cancer cell is a leukemia cell.

15. The method of claim 1, wherein the composition formulated as a pharmaceutical composition, optionally a lipid-based formulation.

16. The method of claim 1, wherein the inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript is contained in an expression construct, or optionally a vector or a plasmid.

17. A method for treating or ameliorating a cancer, comprising:
administering a sufficient amount of a composition to an individual in need thereof,
wherein a sufficient amount of the composition inhibits or slows the self-renewal or self-renewal capacity of a cancer cell or a cancer stem cell,
and the composition inhibits or slows the expression of or the activity of: an ADAR1 gene (adenosine deaminase acting on RNA 1) comprising a sequence as set forth in (SEQ ID NO:1) and/or an ADAR1 transcript comprising a sequence as set forth in (SEQ ID NO:2),
and the composition that inhibits or slows the expression of the ADAR1 gene or the ADAR1 transcript comprises an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of the ADAR1 gene or ADAR1 gene transcript.

18. The method of claim 17, wherein the cancer stem cell is a leukemia stem cell or a Chronic Myeloid Leukemia (CML) stem cell.

19. The method of claim 17, wherein the cancer cell is a leukemia cell.

20. The method of claim 17, wherein the leukemia cell is a Chronic Myeloid Leukemia (CML) cell.