bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3560: MicroRNA-665 promotes metastasis of breast cancer by targeting nuclear receptor subfamily 4 group A member 3
Authors: Zhao Xinge
Published: August 2019

Background: It is the result of metastasis to the lymph nodes or other organs that leads breast cancer (BC) patients to death. Then the early detection of metastasis in BC is helpful to regulate progression of BC and then predict prognosis of BC patients. Since the abnormal level of miRNAs are bound up with BC and metastasis, of which may act as biomarkers to predict prognosis of BC, we find that expression level of miR-665 is ectopic up-regulated in BC patients and it may serve as an estimable biomarker of prognosis of BC patients. Methods: The expressions of miRNA were detected by our current microarray system and verified by quantitative real-time reverse transcription-PCR (qRT-PCR). In Student’s t-test and Kaplan-Meier analysis, the levels of miR-665 were positively correlated with poor survival in BC patients. Multivariate survival analysis identified either miR-665 expression or TNM stage as an independent prognostic factor of BC. Diverse roles of miR-665 played in various cellular functions were examined by loss-of-function and gain-of-function effects in vitro and in vivo. Dual-luciferase reporter gene experiment was then used to confirm the direct complementation between miR-665 and NR4A3, accompanied by activating the epithelial-mesenchymal transition (EMT). Furthermore, siRNAs targeting NR4A3 were used to rescue cell migration and invasion in miR-665 knockdown cells. Results: Expression of miR-665 was upregulated which predicted poor prognosis in BC patients. Since miR-665 may regulate key processes of tumorigenesis, targeting of NR4A3 was proved to promote cell migration and invasion by activating the EMT. Accordingly, either silencing of miR-665 or over-expressing of NR4A3 rescued the migration and invasion phenotype in vitro. Conclusions: Our study suggests that miR-665 plays an important part in progression of BC and promotes cell migration as well as invasion by down-regulation of NR4A3. The data collectively indicate that miR-665 is an onco-miRNA and may represents a potential predictor for diagnosis and prognosis of BC.

Citation Format: Xinge Zhao. MicroRNA-665 promotes metastasis of breast cancer by targeting nuclear receptor subfamily 4 group A member 3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3560.
Abstract 3566: SLCO4A1-AS1 promotes tumorigenesis of colorectal cancer by stabilizing SLCO4A1
Authors: Choi Yuri , Kwon Chae Hwa , Lee Seon Jin , Jeon Daye , Park Do Youn , Lee Sojeong , Kim Ahrong
Published: August 2019

Long noncoding RNAs (lncRNAs) constitute an important component of tumor biology, but the molecular mechanism through lncRNAs modulating colorectal cancer (CRC) development and progression remains unclear. Herein we analyzed RNA sequencing data of CRC patients (147 tumor and 47 matched normal tissues) to identify significant lncRNAs involved in CRC. We found 6032 lncRNAs (15%) and selected three up-expressed (ELFN1-AS1, SLCO4A1-AS1, and LINC02418) and six down-expressed (MIR22HG, BCYRN1, SATB2-AS1, LINC01752, CMAHP, and LINC01082) lncRNAs according to fold change and p-value. In Kaplan-Meier survival analysis, CRC patients with high expression of SLCO4A1-AS1 had poorer prognosis than the patients with low expression of SLCO4A1-AS1, and the other lncRNA expressions were not associated with prognosis. To concentrate on the biological function of SLCO4A1-AS1, we investigated the relationship between SLCO4A1-AS1 and SLCO4A1. They were validated in tumor and normal tissues by qRT-PCR and SLCO4A1 expression was also confirmed by immunohistochemistry, indicating their positive correlation. After knocking down of SLCO4A1-AS1, SLCO4A1 protein level was decreased and cell proliferation was suppressed in CRC cell lines. However, SLCO4A1-AS1 level was not changed by knock-down of SLCO4A1. It demonstrated that SLCO4A1-AS1 plays oncogenic role through influencing on SLCO4A1. Thus, SLCO4A1-AS1 might be useful biomarkers for CRC diagnosis and prognosis.

Note: This abstract was not presented at the meeting.

Citation Format: Yuri Choi, Chae Hwa Kwon, Seon Jin Lee, Daye Jeon, Do Youn Park, Sojeong Lee, Ahrong Kim. SLCO4A1-AS1 promotes tumorigenesis of colorectal cancer by stabilizing SLCO4A1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3566.
Abstract 3576: MALAT1 is essential for A-NHEJ pathway choice during B cell immunoglobulin class switch recombination
Authors: Zhao Jianjun , Hu Yi , Lin Jianhong , Fang Hua
Published: August 2019

Metastasis-associated lung adenocarcinoma transcript 1(MALAT1) has been found high expressed in multiple cancer type including B cell lymphoma and multiple myeloma. In human cancer murine models, deficiency of MALAT1 reduce the tumor burden and malignancy phynotype. We have identified MALAT1 in alternative-non-homozygous end joining(A-NHEJ) pathway by binding to PARP1 and LIG3 in multiple myeloma cells, two key components of the A-NHEJ protein complex. To investigate requirments for MALAT1 in A-NHEJ pathway, by using MALAT1 deficient mice, we found that the contribution of MALAT1 in the context of AID induced DNA breaks in CSR of B cells in mice. MALAT1 is required for A-NHEJ DNA repair pathway, but not the NHEJ pathway, which is the major DNA repair pathway for CSR. To further prove the hypothesis that MALAT1 promote A-NHEJ pathway in B cells, we have engineered mice that conditionally overexpress MALAT1 in B lymphocytes confers a strong A-NHEJ activity in CSR. More importantly, we also found IgH/c-myc translocations are promoted in MALAT1 B cell specific transgenic mice. Thus, we identified MALAT1 as a critical noncoding RNA in regulating A-NHEJ pathway during CSR.

Citation Format: Jianjun Zhao, Yi Hu, Jianhong Lin, Hua Fang. MALAT1 is essential for A-NHEJ pathway choice during B cell immunoglobulin class switch recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3576.
Abstract 3585: Amino acid transporter SLC6A14: A novel drug target for colorectal cancer
Authors: Sikder Mohd Omar Faruk , Sivaprakasam Sathish , Ganapathy Vadivel
Published: August 2019

SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including arginine, leucine (mTOR activators) and glutamine (necessary for nucleotide biosynthesis). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC). However, the relevance of this upregulation to disease progression remains unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function would suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. To test this postulate, we first used Slc6a14-null mice. With two different models of spontaneous CRC (Apcmin/- and DSS/AOM), we found the tumor incidence and tumor growth were much lower in mice with Slc6a14-null background than in mice with Slc6a14. To evaluate the impact of pharmacologic blockade of the transporter on tumor growth we used a syngeneic tumor mouse model with MC-38 cells (a mouse CRC cell line); blockade of Slc6a14 with alpha-methyl tryptophan (α-MT) markedly reduced tumor growth. We then determined the transcriptome profiles of colonic epithelial cells and colonic non-epithelial cells from wild type mice and Slc6a14-null mice by RNAseq. There were hundreds of genes that were either upregulated or downregulated in the null mice. Ingenuity pathway analysis (IPA) of these data revealed predictive activation of canonical AMPK signaling pathway in colonic epithelial of Slc6a14-null mice. AMPK has anticancer activities by modulating multiple pathways including negatively regulating mTOR signaling. Moreover, upstream analysis showed predictive inactivation of two important tumor growth and survival signaling pathways ERK and EGF in the colon of the null mice. Likewise, significant upregulation of APC downregulated 1 (APCDD1) in epithelial cells in null mice implies predictive suppression of canonical Wnt signaling pathway. We also found marked differences in fecal microbiota as a result of Slc6a14 deletion. There was an increase in the relative abundance of beneficial microbiota including those capable of generating short chain fatty acids and lactic acid in null mice. We conclude that deletion of Slc6a14 or its pharmacological blockade protects against CRC by inducing amino acid starvation in tumor cells, thereby causing changes in multiple signaling pathways and in colonic microbiome. These studies identify SLC6A14 as a novel drug target for the treatment of colorectal cancer.

Citation Format: Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy. Amino acid transporter SLC6A14: A novel drug target for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3585.
Abstract 3606: Blockade of the PPARα metabolic checkpoint with TPST-1120 suppresses tumor growth and stimulates anti-tumor immunity
Authors: Whiting Chan C. , Stock Nick , Messmer Davorka , Olafson Traci , Metzger Derek , Enstrom Amanda , McDevitt Jennifer , Spaner David , Prasit Peppi , Panigrahy Dipak , Laport Ginna
Published: August 2019

Tumors evolve to modulate metabolism to promote their own survival and to suppress tumor-specific immunity. Hypoxic conditions in the tumor microenvironment (TME) induce fatty acid oxidation (FAO), and diverse malignancies are reliant on this metabolic pathway. Additionally, suppressive immune cell populations including M2 macrophages, myeloid-derived suppressor cells and regulatory T cells preferentially utilize FAO. Peroxisome proliferator-activated receptor alpha (PPARα) is the principal transcription factor that regulates the expression of FAO genes, and this metabolic checkpoint is critical for tumor proliferation. TPST1120 is a first-in-class selective competitive antagonist of the human PPARα. To test the hypothesis that blocking FAO with TPST-1120 confers anti-tumor efficacy, we assessed TPST-1120 in multiple syngeneic and xenograft mouse models. Blockade of PPARα with TPST-1120 mediated potent anti-tumor immune responses and significant tumor regression in syngeneic models of breast, lung, colon, pancreatic and melanoma in addition to xenograft models of CLL, AML, pancreatic and melanoma cancers as a monotherapy or in combination with chemotherapy. In pancreatic and breast cancer models, TPST-1120 augmented regression of tumor growth in combination with chemotherapy. In combination with anti-PD1, TPST-1120 treatment resulted in significant reduction of tumor growth in ovarian orthotopic (ID8) and colon (MC38) models; cured mice were completely protected against autologous tumor challenge, strongly suggesting immunological T cell memory against the primary tumor. Studies in genetic knock-out mice indicated that macrophages and antigen cross-presenting dendritic cells are required for TPST-1120 activity, mediated through thrombospondin-1(TSP-1) and stimulator of interferon genes (STING). Consistent with prior reports, inhibition of PPARα with TPST-1120 skewed macrophages in vivo toward an M1 effector phenotype. These results provide the rationale for evaluating TPST-1120 in patients with advanced malignancies. A Phase 1/1b open-label, dose-escalation and dose-expansion study of TPST-1120 as a single agent or in combination with systemic anti-cancer therapies is planned in early 2019.

Citation Format: Chan C. Whiting, Nick Stock, Davorka Messmer, Traci Olafson, Derek Metzger, Amanda Enstrom, Jennifer McDevitt, David Spaner, Peppi Prasit, Dipak Panigrahy, Ginna Laport. Blockade of the PPARα metabolic checkpoint with TPST-1120 suppresses tumor growth and stimulates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3606.
Abstract 3635: RNA-based nanostructures for therapeutic siRNA delivery
Authors: Ke Yonggang , Shin DongMoon , Chen Georgia
Published: August 2019

Short interfering RNA (siRNA) has emerged as a promising molecular therapeutic tool for targeted cancer treatment. However, systemically administered siRNA has demonstrated only limited success, due to limited delivery to cancer cells. Therefore, the lack of a robust and versatile siRNA delivery system is a critical issue in translating this therapeutic tool for cancer treatment. Recent developments in DNA nanotechnology have made programmable DNA nanoparticles (DNPs) a potent drug delivery platform. This study focuses on the development of a novel RNA-based siRNA delivery system to knockdown Bcl2 gene, as a targeted cancer therapeutic. By eliminating the use of DNA, the RNA-based nanodelivery can potentially create more robust siRNA delivery vehicles for targeted cancer therapeutics.

Citation Format: Yonggang Ke, DongMoon Shin, Georgia Chen. RNA-based nanostructures for therapeutic siRNA delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3635.
Abstract 3642: Combination nanotherapy using the PARP inhibitor talazoparib and cyclin dependent kinase inhibitor dinaciclib
Authors: Baldwin Paige , Orriols Adrienne , Sridhar Srinivas
Published: August 2019

Introduction: PARP inhibitors exploit defects in DNA repair pathways to selectively target cancerous cells. As such, Talazoparib (TLZ), a potent PARP inhibitor, offers a way to target the biology of a number of cancers with DNA repair defects until these tumors develop resistance. PARP inhibitors must be used in combination with other inhibitors or chemotherapeutics to reverse resistance and sensitize non-responsive tumors. Dinaciclib, a potent cyclin dependent kinase (CDK) inhibitor, has been shown to sensitize both BRCA wild-type tumors and PARP inhibitor resistant tumors to PARP inhibition through disruption of homologous recombination. In clinical trials, Talazoparib and Dinaciclib have both demonstrated hematologic toxicities, suggesting a combination of these drugs would result in compounded toxicity, leading to dose reduction and an ineffective combination. Nanoparticle delivery systems offer a means to modify the toxicity profiles of these drugs and enhance the therapeutic window, therefore allowing for effective combination treatment.

Methods: Separate nanoformulations of Talazoparib (NanoTLZ) and Dinaciclib (NanoDCB) were optimized, and pharmacokinetics and pharmacodynamics assessed. Nanoformulations were tested alone and in combination in vitro to ensure NanoDCB could sensitize a model with no known DNA repair defects to NanoTLZ. The combination of the two nanoformulations was then assessed for efficacy and toxicity in orthotopic MDA-MB-231 xenografts.

Results: Robust formulations of NanoTLZ and NanoDCB were developed. Each nanoformulation extended the half-life of the drug it encapsulates. A constant low dose of Dinaciclib sensitized MDA-MB-231 cells to Talazoparib, significantly lowering the IC50 value. As a single agent NanoDCB was more effective in vitro than free Dinaciclib. In vivo, the combination of the two nanoformulations was more effective than either single nanoformulation or the combination of the two free drugs. Assessments of hematologic toxicities are underway, but thus far, there were no signs of gross toxicity in the combination therapy group.

Conclusions: The combination of NanoDCB and NanoTLZ has provided an effective method for sensitizing tumors to PARP inhibition that are otherwise nonresponsive to this therapy. The development of two separate nanoformulations has allowed for tailored dosing. These long-circulating nanoformulations have proven more effective than the free drugs in stabilizing tumor growth and were well tolerated. This work was supported by ARMY/W81XWH-16-1-0731.

Citation Format: Paige Baldwin, Adrienne Orriols, Srinivas Sridhar. Combination nanotherapy using the PARP inhibitor talazoparib and cyclin dependent kinase inhibitor dinaciclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3642.
Abstract 3655: Mapping the osteogenic lineage and identification of the cell of origin of osteosarcoma through high-dimensional analysis
Authors: Wang Yifei , Kannan Sankaranarayanan , Zhang Zhongting , Zhang Wendong , Roth Michael , Gill Jonathan B. , Xu Zhaohui , Tian Xiangjun , Wang Jing , Gorlick Richard
Published: August 2019

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Neo-adjuvant chemotherapy combined with wide resection increased the 5-year survival rate to 60%-70%. However, treatment and outcome of OS have not changed in several decades. OS is histologically defined by the presence of malignant osteoid, suggesting it could be derived from anywhere in the osteogenic lineage between mesenchymal stem cells (MSC) and mature osteoblasts, but the cell of origin of OS is still undefined. Tracing the cells from which most cancers formed are characterized by its multipotent and/or unipotent stem cell nature. Studies on the cellular origin of tumors from phenotypic lineage-tracking and its derived progeny can reveal tissue-specific tumor-initiating cells. Investigating cell of origin of OS and osteogenic differentiation using developmentally relevant model using MSC to OB differentiation is promising. Here we developed a high-dimensional analytic pipeline using single-cell mass cytometry (CyTOF) and RNA sequencing to discover the unrecognized progenitor-osteoblast populations in the osteogenic lineage. For the data analysis, the population clustering using Flowsom and ConsensusClusterPlus on arcsinh transformed FCS data combined with t-SNE plot visualization shows that combination of CD49f, CD95, CD56, CD117 expression enables the identification and future isolation of the progenitor populations. Also, when comparing OS with the populations from MSC to OB, there are progenitor -osteoblast / mature osteoblast-like populations in tumor cells, but no MSC-like component is found. The expression level of CD56 and CD117 are significantly higher in OS compare to MSC and OB, which warrants future investigation of new therapeutic strategy. Isolation of the progenitor-osteoblast cells, gene expression comparison between OS and these progenitors, validation of their differentiation potential and tumor-initiating capacity is still underway. In this study, we developed a CyTOF based pipeline to discover the progenitor populations in the osteogenic lineage. By comparison of the OS and these progenitor cells, we may be able to unveil the cell of origin and new targeted treatment strategy of OS.

Citation Format: Yifei Wang, Sankaranarayanan Kannan, Zhongting Zhang, Wendong Zhang, Michael Roth, Jonathan B. Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Mapping the osteogenic lineage and identification of the cell of origin of osteosarcoma through high-dimensional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3655.
Abstract 3675: Elucidating the role and function of APOBEC3 DNA deaminases in myeloproliferative neoplasms
Authors: Isquith Jane , Jiang Qingfei , Diep Raymond , Pham Jessica , Holm Frida , Jamieson Catriona
Published: August 2019

The prevalence of upregulated apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) transcripts as well as a distinct mutational signature has been identified and presented extensively in numerous cancer types. Previously, this family of cytidine deaminases has been studied in innate immunity, as they are active in the conversion of cytosine to uracil to restrict retroviral replication. Recent studies employing whole exome sequencing of multiple cancer types have shown a significant increase in both APOBEC3 transcript level as well as an increase in mutations indicated as a result of APOBEC3 driven mutagenesis. However, the expression and role of these enzymes in cancer initiation and progression as well as the mechanisms by which the APOBEC3 enzymes elicit a response in the cancer microenvironment remains unknown, especially in hematopoietic malignancies. Here, we elucidate the APOBEC3 mutation phenotype in Myeloproliferative Neoplasms (MPNs) as well as the naïve cell populations of CD34+ cord blood and normal aged samples. Through RNA sequencing we have found a cell type and context specific nature of these enzymes, notably the upregulation of APOBEC3G (A3G) in the Myelofibrosis (MF) stem and progenitor cell population as compared to normal aged counterparts. There is also significant differential expression of APOBEC3C (A3C), APOBEC3D (A3D), and APOBEC3F (A3F) in MF disease states compared to normal controls. By cloning the APOBEC3 enzymes into lentiviral expression vectors, we can now study the physiological effects of changes in APOBEC3 transcript level in relation to the known changes in expression seen in many cancers, focusing on the upregulation of A3G through lentiviral overexpression. Using these techniques, we will also elucidate the mutation signature of APOBEC3G in CD34+ cord blood as compared to the known mutagenesis pattern derived from the editing signature of APOBEC3B. Upon APOBEC3G overexpression we can connect the prevalence and proposed activity of the enzymes to the physiological deregulation present in hematopoietic malignancies. We have found that A3G induces a proliferative burst in normal CD34+ cord blood, which leads us to study the effects of APOBEC3 upregulation in regards to proliferation, differentiation and self-renewal in normal and malignant cells as well as their potential function in disease initiation and progression in MPNs.

Citation Format: Jane Isquith, Qingfei Jiang, Raymond Diep, Jessica Pham, Frida Holm, Catriona Jamieson. Elucidating the role and function of APOBEC3 DNA deaminases in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3675.
Abstract 369: Antitumor activity of M4344, a potent and selective ATR inhibitor, in monotherapy and combination therapy
Authors: Zenke Frank T. , Zimmermann Astrid , Dahmen Heike , Elenbaas Brian , Pollard John , Reaper Philip , Bagrodia S , Spilker M E. , Amendt C , Blaukat Andree
Published: August 2019

The protein kinase ataxia telangiectasia mutated and Rad3-related ATR is one of the key mediators of the DNA damage response. ATR is recruited to regions of single-stranded DNA, which most commonly arise during replication stress (RS). RS occurs during S-phase when the cell’s DNA replication machinery encounters problems such as unresolved DNA lesions. In addition, treatment of cells with DNA-damaging agents can lead to RS as cells progress to S-phase without resolving damage incurred by such agents. Elevated levels of RS are evident in some cancer cells, even in the absence of a DNA-damaging agent resulting from expression of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways. RS in cancer cells can drive reliance on ATR for survival and, accordingly, ATR inhibitors may have benefit as monotherapy. M4344 was determined to be an adenosine triphosphate (ATP)-competitive, highly potent, and tight-binding inhibitor of ATR with a Ki of < 150 pM. Minimal inhibitory activity was observed against a large panel of unrelated protein kinases, with 308 of 312 kinases tested having a measured Ki corresponding to more than 100-fold selectivity. M4344 potently inhibits ATR-driven phosphorylated checkpoint kinase-1 (P-Chk1) phosphorylation with an IC50 of 8 nM. Profiling on a selected set of cancer cell lines showed synergy with several types of DNA damaging chemotherapeutics as well as PARP1/2 and CHK1 inhibitors. In monotherapy efficacy studies M4344 showed tumor stasis to regression in tumor models with alternative lengthening of telomeres (ALT). In combination with PARP inhibitors, tumor regression could be observed in triple-negative breast cancer xenograft models. A dose-escalation phase 1 study in patients with advanced solid tumors is currently ongoing.

Citation Format: Frank T. Zenke, Astrid Zimmermann, Heike Dahmen, Brian Elenbaas, John Pollard, Philip Reaper, S Bagrodia, M E. Spilker, C Amendt, Andree Blaukat. Antitumor activity of M4344, a potent and selective ATR inhibitor, in monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 369.
Abstract 3693: GLIS1 can replace MYC to generate induced pluripotent stem cells from ovarian cancer cells
Authors: Bindhya S , Sidhanth C , Krishnapriya S , Nagare R P. , Garg M , Ganesan T S.
Published: August 2019

Ovarian cancer is the most common cause of mortality among gynecological cancers, despite advances in treatment. Recurrence is common and is due to development of drug resistance. One of the reasons for drug resistance is the persistence of cancer stem cells. To understand the role of CSCs, it is essential to capture and propagate cells continuously in culture. Reprogramming cancer cells to induced pluripotent stem cells (iPSCs) is an approach to achieve this. An ovarian cancer cell line, PEO4 (high grade serous adenocarcinoma), was initially reprogrammed into iPSCs using the classical four factors OCT4, SOX2, KLF4 and MYC (OSKM) using STEMCCA by lentivirus transduction. Embryonic stem cell (ESC)-like bodies appeared between 8 to 15 days post-transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. Two individual clones were further characterized. The reprogrammed PEO4-OSKM-iPSCs expressed alkaline phosphatase and pluripotency markers, NANOG, OCT4, SSEA4, TRA-1-60 and TRA-1-81 by immunofluorescence. Further, reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed expression of the pluripotency markers NANOG, SOX2, OCT4, TERT, NESTIN, DMNT, DPPA4 in PEO4-OSKM-iPSC which were absent in the parental PEO4 cells. PEO4-OSKM-iPSC cells could be differentiated in vitro with appropriate growth factors into ectodermal, mesodermal and endodermal lineages. MYC was replaced with GLIS1 in the lentiviral cassette and PEO4 cells were able to be transformed to iPSCs. The transfection efficiency was two fold better with OCT4-SOX2-KLF4-GLIS1 with larger colonies. Individual iPSC colonies expressed all pluripotency markers and were able to differentiate into all 3 lineages. Characterization of iPSC cells for expression of cell surface markers specific for serous adenocarcinoma, showed that CD133, EPHA1, CD44 and LGR5 were expressed. Cell viability assays demonstrated that IC50 of cisplatin in parental PEO4 cells (15uM) was less as compared to iPSC cells (32uM) (p<0.03) and similarly, IC50 of paclitaxel was less in parental PEO4 (17 uM) as compared to iPSC cells (27 uM) (p<0.02). These results demonstrate for the first time that an ovarian cancer cell line derived from a patient with high grade serous adenocarcinoma can be reprogrammed. Further, GLIS1 can successfully replace MYC as a transcription factor to generate induced pluripotent stem cells.

Citation Format: S Bindhya, C Sidhanth, S Krishnapriya, R P. Nagare, M Garg, T S. Ganesan. GLIS1 can replace MYC to generate induced pluripotent stem cells from ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3693.
Abstract 370: The role of DLX1 as a biomarker and novel target in poor prognostic prostate cancer
Authors: Forsythe Nicholas , McKinney Cathal , McCabe Nuala , Kennedy Richard D.
Published: August 2019

Introduction: Previous work from our lab has identified a molecularly distinct subgroup of primary prostate cancers with biology similar to metastatic prostate cancers. These “met-like” cancers can be identified by a 70 gene classifier and are statistically more likely to result in distant metastases than their “non-met like” counterparts. This study aimed to identify exploitable biology common to both “met-like” primary tumours and metastatic prostate cancer.

Experimental procedures: Differential gene expression and Ingenuity pathway analysis (IPA) was used to identify nodal genes upregulated in both “met-like” primary prostate cancers and metastatic prostate cancers. siRNA screening was performed on 40 nodal genes in a panel of normal, primary and metastatic prostate cancers. Cell viability was assessed using cell titer-glo luminescent Assay. Relative levels of apoptosis were assessed using PARP, caspase-8 and caspase-3 cleavage Western Blots. siRNA mediated knockdowns were confirmed by Western Blot and rtPCR.

Results: We have identified upregulation of the homeobox transcription factor DLX1 in “met-like” primary prostate cancers and metastatic prostate cancers. High levels of DLX1 expression were found to be independently predicative of biochemical recurrence and metastatic disease in primary prostate cancers. DLX1 knockdown resulted in significantly decreased cell viability in primary and metastatic prostate cancer cell lines with minimal reduction of viability in normal prostate cells. Additionally DLX1 knockdown was shown to result in increased PARP, caspase-8 and caspase-3 cleavage providing evidence of apoptotic cell death. Furthermore knockdown of DLX1 resulted in significantly increased transcript levels of the pro-apoptotic BH3 protein BIK, suggesting its involvement in the observed apoptotic cell death.

Conclusion: DLX1 has previously been identified as a marker of prostate cancer metastasis with increased expression being observed in both primary and metastatic prostate cancers. However we have observed increased DLX1 expression in a subset of primary prostate cancers that developed metastatic diseased compared to primary tumors that did not metastasise. The elucidation of apoptotic mechanisms following DLX1 knockdown may uncover potential exploitable biology that may be used for the treatment of prostate cancer.

Citation Format: Nicholas Forsythe, Cathal McKinney, Nuala McCabe, Richard D. Kennedy. The role of DLX1 as a biomarker and novel target in poor prognostic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 370.
Abstract 3706: The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues
Authors: Berthelsen Martin F. , Riedel Maria , Vendelbo Mikkel H. , Alstrup Aage K. , Dagnæs-Hansen Frederik , Luo Yonglun , Møller Simone S. , Callesen Henrik , Jakobsen Jannik E. , Thomsen Martin K.
Published: August 2019

The aim of the project is to generate a toolbox pig model. This is being done because the pig probably is the best non-primate model in translational medicine. Unfortunately, pig models have proven difficult to produce. The two main complications have been the cloning of viable pigs and that just a few genes are altered, which often is not sufficient to induce the desired disease. Especially, models based on multiple genetic modifications of the pig genome, e.g. cancer, are challenging to generate. This was changed with the development of the CRISPR-Cas9 technology, which made it possible to perform multiple in vivo gene editing.

We have cloned an inducible Cas9 expressing minipig and have obtained healthy F1 offspring. Activation of the Cas9 expression is attained by transduction with adeno-associated virus that delivers both the recombinase and the guide RNA to the designated tissue. This design provides spatial and temporal control of the activation. The Cas9 expression is mediated by a ubiquitous promotor, which also co-expresses fluorescent proteins for expression analysis. Transgenic Cas9 expression is observed in all major organs with strong expression in the epithelia, muscles and hepatocytes. Successful in vitro gene editing has been conducted in primary keratinocytes and fibroblast obtained from the transgenic pigs, which confirms the genetic design.

By delivery of our construct by adeno-associated virus to the skin of the pigs, In vivo activation of the transgenic construct has been achieved. To validate the model, lung cancer has been induced in nine pigs by alteration a panel of cancer related genes including STK11, TP53, PTEN, and KRAS, all key drivers of human lung cancer. To obtain the constitutive active KRAS-G12D isoform of the KRAS oncogene a repair template was supplied. The pigs are being followed by PET/CT scanning in combination with 18F-FDG as a tracer. Preliminary data indicates abnormalities/cancer initiation in the pigs eight months after induction.

In conclusion, an inducible Cas9 expressing minipig allows rapid gene editing to study different scenarios of human cancer in a pig model. The development of the Cas9 pig will extend the variety of pig disease models, both to generate a model of lung cancer as proposed but also models for other cancers.

Citation Format: Martin F. Berthelsen, Maria Riedel, Mikkel H. Vendelbo, Aage K. Alstrup, Frederik Dagnæs-Hansen, Yonglun Luo, Simone S. Møller, Henrik Callesen, Jannik E. Jakobsen, Martin K. Thomsen. The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3706.
Abstract 3713: Strong expression of EZH2 and H3K27me3 is associated with poor survival time in small cell lung cancer
Authors: Fernandes Ana Paula , Alves Rita deCassia S , Watte Guilherme , Kunze Philipp , Roehe Adriana Vial , Schneider-Stock Regine
Published: August 2019

BACKGROUND: Small-cell lung cancer (SCLC) is a highly aggressive neoplasm, characterized by early development of metastasis and very poor clinical outcome. The therapeutic possibilities are limited and poorly changed over the past three decades. Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb repressive complex 2. It is involved in epigenetic gene silencing through the trimethylation of H3K27 and it has been identified as a biomarker of aggressive and highly proliferating tumors, emerging as a potential target for cancer therapy.

OBJECTIVE: We aimed to correlate the expression profile of the EZH2 and H3K27me3 proteins in tumor tissues of non-treated SCLC patients with patient's outcome.

METHODS: EZH2 and H3K27me3 expression were analyzed by immunohistochemistry in 48 SCLC tumor biopsies. The expression of the markers was quantified in a semi quantitative way and correlated with clinic pathological factors and patients overall survival. To mimic an EZH2 loss, H209 SCLC cells were treated with an EZH2 inhibitor for 48h and the in vivo growth pattern was investigated in the chorioallantoic membrane (CAM) assay.

RESULTS: All patients died within one year after diagnosis. There was a heterogeneous expression pattern for EZH2 and H3K27me3. Strong expression of EZH2 and H3K27me3 was observed in appr. 50% of the patients. The Kaplan Meier curve showed a significant association of high expression pattern of EZH2 and shorter overall patient survival time after 12 and 25 weeks of follow up, respectively (p=0.030; p=0.014) Different markers of tumor aggressiveness after EZH2 inhibition were investigated in the CAM model.

CONCLUSION: Strong EZH2 and H3K27me3 expression in non-treated SCLC patients was related to worse prognosis. EZH2 inhibitors should be considered in future trials for SCLC.

Citation Format: Ana Paula Fernandes, Rita deCassia S Alves, Guilherme Watte, Philipp Kunze, Adriana Vial Roehe, Regine Schneider-Stock. Strong expression of EZH2 and H3K27me3 is associated with poor survival time in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3713.
Abstract 3735: An anti-tumor immune response is evoked by partial-volume single dose radiation
Authors: Haimovitz-Friedman Adriana , Humm Johnn , Deasy Joseph O. , Wolchok Jedd , Merghoub Taha , Russell James , Li Hongyan , Samstein Robert , Budhu Sadna , Powell Simon , Bodden Chloe , Markovsky Ela , Chen Qing
Published: August 2019

Purpose: To study tumor growth delay resulting from partial irradiation in preclinical mouse models.

Methods and Materials: We investigated 67NR murine orthotopic breast tumors in both immunocompetent and nude mice. Treatment was delivered to 50% or 100% of the tumor using a 2x2 cm collimator on a micro-irradiator. Radiation response was modulated by treating with anti-CD8 and anti-ICAM antibodies. Similar experiments were performed using the less immunogenic Lewis Lung Carcinoma (LLC) mouse model. Tumor growth delay and γH2AX phosphorylation were measured and immune response was assessed by immunofluorescence and flow cytometry at 1 and 7 days post-radiotherapy (RT). Tumor expression of cellular adhesion molecules was also measured at different times post-RT.

Results: Partial irradiation led to tumor responses similar to fully exposed tumors in immunocompetent mice, but not in nude mice. After a single dose of 10Gy, infiltration of CD8+ T cells was observed, along with increased expression of ICAM. The response to 10Gy in hemi-irradiated tumors was abrogated by treatment with either anti-CD8 or anti-ICAM antibodies. Similar responses were obtained in the less immunogenic LLC mouse model delivering 15Gy to half the tumor volume. Treatment with FTY720, a compound that inhibits T cell egress from lymph nodes, did not affect tumor response at the time of CD8+ T cells infiltration in the non-irradiated area of the tumor, indicating that the most likely source of these cells is the irradiated portion of the hemi-irradiated tumors. In addition, a significant abscopal effect was observed after partial irradiation with a single dose of 10Gy in the 67NR model.

Conclusions: In these models, radiation controls tumor growth both directly through cell killing and indirectly through immune activation. This raises the possibility that this effect could be induced in the clinic.

Citation Format: Adriana Haimovitz-Friedman, Johnn Humm, Joseph O. Deasy, Jedd Wolchok, Taha Merghoub, James Russell, Hongyan Li, Robert Samstein, Sadna Budhu, Simon Powell, Chloe Bodden, Ela Markovsky, Qing Chen. An anti-tumor immune response is evoked by partial-volume single dose radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3735.
Abstract 3769: Detection of allele specific loss of heterozygosity in 70,000 patients with ctDNA
Authors: Zhao Jing , Fairclough Stephen R. , Chudova Darya , Lanman Richard , Talasaz AmirAli , Hardin Aaron
Published: August 2019

Background: Loss of Heterozygosity (LoH) is a common feature of tumor biology. LoH frequently arises from defects in Homologous Recombination Repair (HRR) and results in uni-parental deletions that manifest as (LoH). In the absence of a driving force, the likelihood of allelic loss is equal, so in a population, the rate of retention and loss of a given allele is equal, but allele specific loss (or retention) is known to occur.

Methods: Using a novel analysis method incorporating observed allele frequency and copy number variation, we investigated the Guardant Health database of variants detected from 70,000+ samples from whole blood samples from patients with advanced solid tumors that were tested using a 73-gene cell-free DNA next-generation sequencing panel (Guardant360, Guardant Health, Redwood City, CA) to identify allele specific loss.

Results: Analysis of the Guardant Health database revealed that LoH frequently manifests as allele imbalance with the observed mutation allele frequency (MAF) of the retained allele exceeding an observed allele frequency of 50% and the lost allele having observed mutation allele frequencies below 50% in an individual sample. This imbalance occurs because allele frequency is a relative measurement and loss of one allele causes the relative abundance of the remaining allele to increase in a proportional amount. Population analysis reveals that the majority of of alleles are lost without preference, but certain alleles may be more prone to retention or loss.

Conclusions: The detection of allele specific loss from ctDNA provides insight into the underlying tumor biology and the selective pressures shaping tumor evolution over the course of treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Jing Zhao, Stephen R. Fairclough, Darya Chudova, Richard Lanman, AmirAli Talasaz, Aaron Hardin. Detection of allele specific loss of heterozygosity in 70,000 patients with ctDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3769.
Abstract 3800: HDAC8 regulates plasticity and escape from therapy in BRAF mutant melanoma
Authors: Emmons Michael , Flores Fernanda , Koomen John , Seto Edward , Messina Jane , Lau Eric , Licht Jonathan , Smalley Keiran
Published: August 2019

Melanoma cells are highly plastic and have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. We here show that exposure to melanoma cell lines and patient specimens to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia and UV-irradiation leads to an increase in HDAC8 expression/activity, and in turn, the adoption of a drug-resistant, invasive phenotype. Systems level analyses using mass spectrometry-based phosphoproteomics and RNA-Seq demonstrated HDAC8 to be involved in pathways that regulated cell cycle entry, ribosome function, RNA binding, regulation of the cytoskeleton and MAPK pathway signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance in vitro and in in vivo xenograft models. The HDAC8-mediated BRAF inhibitor resistance was mediated through upstream receptor tyrosine kinase (RTK) activation, Ras/CRAF/MEK/ERK signaling and the suppression of apoptosis through increased stabilization of Mcl-1 and the inhibition of pro-apoptotic BIM. Among the multiple RTKs increased by HDAC8, EGFR emerged as a direct transcriptional target, and it was found that EGFR inhibition could overcome HDAC8-mediated tolerance to BRAF inhibition. Although it is known that HDACs primarily function at the histone level, they can also regulate signaling through the modulation of cytoplasmic protein acteylation. In line with this, we observed that HDAC8 introduction decreased the acetylation of c-JUN, leading to an increase in its transcriptional activity and the enrichment for an AP-1 gene signature. Mutation of putative acetylation sites in c-JUN (K268R, K271R, K273R) reduced the transcriptional activation of c-JUN in melanoma cells and conveyed resistance to BRAF inhibition through increased MAPK pathway activity. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with either a pan-HDAC inhibitor (panobinostat) or an HDAC8 inhibitor (PCI-34051) enhancing the durability of response to BRAF inhibitor therapy. We have thus identified HDAC8 as a key driver of phenotype switching in melanoma that regulates the responses to multiple cellular stresses and conveys resistance to BRAF inhibition. Our studies demonstrate that isoform-specific HDAC8 inhibitors could be an excellent strategy to limit the adaptation of melanoma cells to multiple stresses and therapeutic interventions, including the BRAF-MEK inhibitor combination.

Citation Format: Michael Emmons, Fernanda Flores, John Koomen, Edward Seto, Jane Messina, Eric Lau, Jonathan Licht, Keiran Smalley. HDAC8 regulates plasticity and escape from therapy in BRAF mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3800.
Abstract 3815: Overcoming acquired resistance of Lapatinib in breast cancer with Palbociclib
Authors: Jeong Da Eun , Min Ahrum , Kim Seongyeong , Kim So Hyeon , Park Yoonjung , Yang Yaewon , Lee Kyung-Hun , Im Seock-Ah
Published: August 2019

Background : Lapatinib is a dual tyrosine kinase inhibitor that interrupts the ErbB family pathways in many solid tumors. However, the vast majority of patients who initially respond to the treatment will eventually relapse due to acquired resistance. The defects in cell cycle regulation via increased levels of cyclin D-CDK4/6 complex is well known mechanism of the acquired resistance to lapatinib. Thus, CDK4/6 inhibition can be considered as an attractive strategy to overcome lapatinib resistance. In this study, we investigated whether palbociclib, a palbociclib can overcome the resistance to lapatinib by modulating cell cycle regulation.

Methods : Acquired resistant SK-BR-3-cells was established by treating lapatinib consistently. The features of lapatinib resistance (LR) cell lines were evaluated by MTT, FACS, Western blotting, qPCR, methylation assay and WES. The antitumor effects of palbociclib in LR cell lines were evaluated through changes in cell cycle progression, proliferative ability and signaling transduction.

Results : In LR cell lines, the change of the molecules that were related to G1/S transition of cell cycle was prominent. It was confirmed that cell cycle progression and proliferation were significantly faster in LR cells compared to parental cells. Upregulation of cyclin D1, CDK4, CDK6 was shown in LR cells but cyclin-dependent kinase inhibitor, p16 was downregulated. The increase of Cyclin D1, CDK4, and CDK6 occur at the transcription level, and the epigenetic alteration of CDKN2A was observed. We identified that the palbociclib inhibited the cell cycle progression and reduced the cell viability significantly in LR cells by cell cycle arrest via blocking hyperphosphorylation of Rb. It was also confirmed that these changes resulted in apoptosis.

Conclusion : The changes of cell cycle molecules that were related to G1/S transition was observed in LR cells. Upregulation of cyclin D1, CDK4, CDK6, and deficiency in CDKN2A led to the increasing cell cycle progression and prolfieration in LR cells. Therefore, inhibition of these molecules by palbociclib could be an effective strategies for overcoming lapaitnib resistance.

Citation Format: Da Eun Jeong, Ahrum Min, Seongyeong Kim, So Hyeon Kim, Yoonjung Park, Yaewon Yang, Kyung-Hun Lee, Seock-Ah Im. Overcoming acquired resistance of Lapatinib in breast cancer with Palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3815.