High-grade Serous Ovarian Tumors Research Articles

Abstract A34: Targeting genes which are amplified in ovarian cancer

Abstract Over the recent years significant knowledge has been accumulated on which drugs can be therapeutically beneficial for a given patient based on mutational profiling of their tumors using selected gene panels. Much less, however, is known under which conditions targeting of genes which are amplified could be beneficial. Apart from successfully targeting HER2-positive cancers, many of which frequently carry amplified ERBB2 gene, no other targeted therapy based on DNA amplifications or gains of the genes is offered to the patients due to lack of clinical response data. Using mate pair sequencing we have profiled 60 high grade serous ovarian tumors to compare genomic rearrangements and copy number changes. The analyses revealed that these tumors carry highly rearranged genome. Copy number changes amplifications, gains and losses were very common and included genes potentially therapeutically targetable. In contrast, only a few of targetable fusion genes were identified in a set of 60 cases. The tumors were propagated in immunocompromised mice (mouse avatars), sequenced and showed identical to original patient tumor landscape of rearrangements. Using these mouse avatar models we tested whether a therapy inhibiting selected based on genomic amplification targets provided a benefit compared to standard chemotherapy. Mice which harbored tumors showing amplifications of CDKs (CDK2, 4 and 9) and ERBB3 were treated with flavopiridol and pertuzumab respectively, and response was compared to that of standard for ovarian cancer chemotherapy (carboplatin and paclitaxel combination). We observed that application of either agent, significantly inhibited tumor growth in treated mice as compared to untreated. However, neither drug alone caused tumor regression. The treatment regimen then was modified to administer chemotherapy and targeted therapy together. A mouse avatars in which genomic analyses revealed amplification at loci involving AKT3 and RICTOR genes was treated with chemotherapy alone and in combination with MK2206 (AKT inhibitor) or MK8669 (mTOR inhibitor). The high level of both target proteins was confirmed by immunoblotting. Significant regression of tumor was observed after 6 weeks of treatment. Addition of either targeted drug provided an extra benefit in killing tumor cells compared to chemotherapy alone. The mice are followed to study tumor recurrence. These data indicated that genomic information obtained using next generation sequencing can aid therapy decisions by tailoring treatments to individual tumor makeup. Avatar models in conjunction with genomic analyses of the original tumors are invaluable for testing specific targeted therapies selected based on amplification/gains of putative genes-targets and for studying mechanisms of tumor recurrence and therapy resistance. Citation Format: Faye R. Harris, Lin Yang, Xiaonan Hou, Konstantinos Leventakos, Saravut J. Weroha, George Vasmatzis, Irina V. Kovtun. Targeting genes which are amplified in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A34.

NanoLC-MS coupling of liquid microjunction microextraction for on-tissue proteomic analysis

Mass spectrometry (MS)-based microproteomics on localized regions of tissue sections was achieved by direct coupling of liquid microjunction microextraction with a nanoscale liquid chromatography-tandem MS, resulting in the identification of >500 protein groups from a region as small as 250μm in diameter representing only a few hundred of cells. The method was applied on the examination of benign and tumor regions initially defined by imaging mass spectrometry (IMS) analysis of a consecutive high grade serous ovarian tumor tissue section. Results identified the higher abundance of eukaryotic translation initiation factors eIF4A, its isoform eIF4A2, and eIF5A and its isoform eIF5A2, and lower abundance of actin-binding proteins OBSCN, TAGLN and CNN3 on tumor regions, concomitant with previous findings. This demonstrates the use of the method for downstream characterization of distinct regions identified by IMS. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

InFlo: a novel systems biology framework identifies cAMP-CREB1 axis as a key modulator of platinum resistance in ovarian cancer

Characterizing the complex interplay of cellular processes in cancer would enable the discovery of key mechanisms underlying its development and progression. Published approaches to decipher driver mechanisms do not explicitly model tissue-specific changes in pathway networks and the regulatory disruptions related to genomic aberrations in cancers. We therefore developed InFlo, a novel systems biology approach for characterizing complex biological processes using a unique multidimensional framework integrating transcriptomic, genomic and/or epigenomic profiles for any given cancer sample. We show that InFlo robustly characterizes tissue-specific differences in activities of signalling networks on a genome scale using unique probabilistic models of molecular interactions on a per-sample basis. Using large-scale multi-omics cancer datasets, we show that InFlo exhibits higher sensitivity and specificity in detecting pathway networks associated with specific disease states when compared to published pathway network modelling approaches. Furthermore, InFlo’s ability to infer the activity of unmeasured signalling network components was also validated using orthogonal gene expression signatures. We then evaluated multi-omics profiles of primary high-grade serous ovarian cancer tumours (N=357) to delineate mechanisms underlying resistance to frontline platinum-based chemotherapy. InFlo was the only algorithm to identify hyperactivation of the cAMP-CREB1 axis as a key mechanism associated with resistance to platinum-based therapy, a finding that we subsequently experimentally validated. We confirmed that inhibition of CREB1 phosphorylation potently sensitized resistant cells to platinum therapy and was effective in killing ovarian cancer stem cells that contribute to both platinum-resistance and tumour recurrence. Thus, we propose InFlo to be a scalable and widely applicable and robust integrative network modelling framework for the discovery of evidence-based biomarkers and therapeutic targets.

Ezrin Is Associated with Disease Progression in Ovarian Carcinoma.

ObjectiveEzrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC).MethodsOC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression.ResultsHigher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome.ConclusionsThe 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The in vitro results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas.

Abstract 4083: Tumor associated macrophages mediates the oncogenic effect of FGF18 in ovarian cancer

Abstract Epithelial ovarian cancer is the fifth leading cause of cancer-related death among women and has the highest case-fatality rate among gynecologic cancers. Effective management of this deadly disease still remains scanty due to the inadequate understanding of the molecular mechanisms associated with the tumor progression and prognosis. We have previously reported the tumor-promoting role of Fibroblast growth factor 18 (FGF18), which have been identified through genomic analysis of ovarian tumors. Initial studies suggested FGF18 promotes ovarian tumor progression through NF-κB pathway mediated elevation of proinflammatory cytokines, which mediate the increased tumor burden, angiogenesis and infiltration of macrophages in FGF18 expressing xenografts. Based on these findings, the present study aims to delineate the mechanism by which FGF18 modulates the phenotype of tumor associated macrophages (TAMs) as well as the reciprocal tumor-promoting feedback from the TAMs which potentially contributes FGF18 mediated ovarian tumorigenesis. Immunohistochemical staining of a tissue array comprising 216 archived high-grade advanced stage serous ovarian tumor specimens revealed a significant correlation between the FGF18 expression and infiltration of M2-polarized macrophages (by CD163). In addition, the density of intratumoral M2 TAMs inversely associated with patients’ overall survival as a negative prognostic factor independent to age, tumor grade and debulking status. These findings suggest intratumoral TAMs may play an important role in ovarian tumorigenesis. In vitro co-culture studies further demonstrated that human monocyte cell line THP-1 or murine peritoneal macrophage cell line IC-21 could significantly activate the NF-κB pathway and increase the production of various proinflammatory cytokines in A224 ovarian cancer cells. Co-culture also increased the migratory potential of A224 cells. Addition of an IKKβ inhibitor into the co-culture system nullified the cytokine production and migration of tumor cells promoted by monocytes/macrophages, implicating the involvement of canonical NF-κB signaling in the crosstalk. Conversely, liposome clodronate mediated in vivo macrophage depletion significantly shrunk the tumor burden of the intraperitoneal xenograft derived from FGF18 overexpressing SKOV3 cells, while minimal effect was observed over the control RFP overexpressing xenograft. Besides, macrophage depletion also markedly decreased the intratumoral microvessel density induced by FGF18 overexpression. Taken together, our data potentially suggest TAMs may mediate the FGF18 related ovarian tumorigenesis by augmenting the proinflammatory status of the tumors. Citation Format: Wei Wei, Michael Birrer. Tumor associated macrophages mediates the oncogenic effect of FGF18 in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4083.

Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer.

BackgroundOvarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis.MethodsThe expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice.ResultsWe report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts.ConclusionThe expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

High-grade serous ovarian cancer primary tumor BRCA1 mRNA expression is a candidate biomarker for patient selection for intraperitoneal chemotherapy

High-grade serous ovarian cancer primary tumor BRCA1 mRNA expression is a candidate biomarker for patient selection for intraperitoneal chemotherapy

A proteomic signature of ovarian cancer tumor fluid identified by highthroughput and verified by targeted proteomics

Tumor fluid samples have emerged as a rich source for the identification of ovarian cancer in the context of proteomics studies. To uncover differences among benign and malignant ovarian samples, we performed a quantitative proteomic study consisting of albumin immunodepletion, isotope labeling with acrylamide and in-depth proteomic profiling by LC-MS/MS in a pool of 10 samples of each histological type. 1135 proteins were identified, corresponding to 505 gene products. 223 proteins presented associated quantification and the comparative analysis of histological types revealed 75 differentially abundant proteins. Based on this, we developed a panel for targeted proteomic analysis using the multiple reaction monitoring (MRM) method for validation of 51 proteins in individual samples of high-grade serous ovarian tumor fluids (malignant) and benign serous cystadenoma tumor fluids. This analysis showed concordant results in terms of average amounts of proteins, and APOE, SERPINF2, SERPING1, ADAM17, CD44 and OVGP1 were statistically significant between benign and malignant group. The results observed in the MRM for APOE were confirmed by western blotting, where APOE was more abundant in malignant samples. This molecular signature can contribute to improve tumor stratification and shall be investigated in combination with current biomarkers in larger cohorts to improve ovarian cancer diagnosis. Biological significanceDespite advances in cancer research, ovarian cancer has a high mortality and remains a major challenge due to a number of particularities of the disease, especially late diagnosis caused by vague clinical symptoms, the cellular and molecular heterogeneity of tumors, and the lack of effective treatment. Thus, efforts are directed to better understand this neoplasia, its origin, development and, particularly the identification and validation of biomarkers for early detection of the disease in asymptomatic stage. In the present work, we confirmed by MRM method in individual ovarian tumor fluid samples the regulation of 27 proteins out of 33 identified in a highthroughput study. We speculate that the presence and/or differential abundance observed in tumor fluid is a cooperation primarily of high rates of secretion of such tumor proteins to extra tumor environment that will at the end accumulate in plasma, and also the accumulation of acute-phase proteins throughout the entire body. On top of that, consideration of physiological influences in the interpretation of expression observed, including age, menopause status, route-of-elimination kinetics and metabolism of the tumor marker, coexisting disease, hormonal imbalances, life-style influences (smoking, alcoholism, obesity), among others, are mandatory to enable the selection of good protein tumor marker candidates for extensive validation.

Insights from HuR biology point to potential improvement for second-line ovarian cancer therapy.

This retrospective study aimed to investigate the role that an RNA-binding protein, HuR, plays in the response of high-grade serous ovarian tumors to chemotherapeutics. We immunohistochemically stained sections of 31 surgically-debulked chemo-naïve ovarian tumors for HuR and scored the degree of HuR cytoplasmic staining. We found no correlation between HuR intracellular localization in tumor sections and progression free survival (PFS) of these patients, 29 of whom underwent second-line gemcitabine/platin combination therapy for recurrent disease. Ribonucleoprotein immunoprecipitation (RNP-IP) analysis of ovarian cancer cells in culture showed that cytoplasmic HuR increases deoxycytidine kinase (dCK), a metabolic enzyme that activates gemcitabine. The effects of carboplatin treatment on HuR and WEE1 (a mitotic inhibitor) expression, and on cell cycle kinetics, were also examined. Treatment of ovarian cancer cells with carboplatin results in increased HuR cytoplasmic expression and elevated WEE1 expression, arresting cell cycle G2/M transition. This may explain why HuR cytoplasmic localization in chemo-naïve tumors is not predictive of therapeutic response and PFS following second-line gemcitabine/platin combination therapy. These results suggest treatment of recurrent ovarian tumors with a combination of gemcitabine, carboplatin, and a WEE1 inhibitor may be potentially advantageous as compared to current clinical practices.

Abstract A15: The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP ribose polymerase inhibitors via E2F1 downregulation.

Abstract Background: The lack of treatment options for chemoresistant ovarian cancer, the deadliest gynecologic malignancy, is a central clinical problem. Approximately 50% of high-grade serous ovarian tumors with defects in DNA repair pathways including germline BRCA1 and 2 mutations (the so-called BRCAness phenotype), and are sensitive to DNA damaging agents such as cisplatin and poly ADP ribose polymerase inhibitors (PARPi). In contrast, non-BRCAness tumors such as those exhibiting CCNE1 (cyclin E) amplification are chemoresistant, show upregulated DNA repair processes and cell cycle progression via activation of the CDK2/cyclin E/E2F1 axis, and have poor clinical outcomes. Targeting the cyclin E axis with CDK inhibitors has been unsuccessful in part due to chemoresistance through upregulation of oncogenic E2F1. An area of urgent need is to develop new treatments for these intractable subtypes of non-BRCAness ovarian cancer. To that end, our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to various chemotherapeutic drugs, including PARPi, through reduction of expression of DNA repair genes. Aims: The objective of this study was to (i) determine expression patterns of cyclin E axis components in publically available databases of high-grade serous ovarian tumors and established cell lines; and (ii) test an epigenetic drug strategy using HDACi to convert non-BRCAness ovarian cancer cells to a BRCAness phenotype as a means of sensitizing chemoresistant cells to PARPi. Methods: RNA-seq V2 RSEM expression levels of cyclin E, CDK2, BRCA1 and E2F1 in 265 ovarian tumors were extracted from The Cancer Genome Atlas (TCGA) via cbioportal.org. mRNA expression of these genes in ovarian cancer cell lines was extracted from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Preliminary western blot studies indicated that of the FDA-approved HDACi (vorinostat, romidepsin, panobinostat), panobinostat was most effective at reducing protein expression of cyclin E axis components (cyclin E, BRCA1 and E2F1). We therefore treated cultured cyclin E overexpressing ovarian cancer cells via CCNE1 amplification (OVCAR-3) or high-level gain (OVCAR-4) with panobinostat alone and in combination with the PARPi, olaparib. The following endpoints were examined: cell viability in sulforhodamine B (SRB) assays, with drug interactions tested for synergism by isobologram analysis; DNA damage by immunofluorescence analysis of the double-strand DNA break marker, pH2AX(ser139); apoptosis by immunofluorescence and western blot analysis of cleaved PARP and cleaved caspase-3. Two-tailed Student's t tests were used for measuring differences between groups in drug treatment experiments. Results: There was significant correlation (Spearman) between cyclin E expression and that of CDK2, E2F1 and BRCA1 in ovarian tumors and in serous-like ovarian cancer cell lines. These results are consistent with the relationship between cyclin E overexpression and enhanced E2F1-dependent transcription of established E2F1 targets, including cyclin E itself, and DNA repair genes such as BRCA1. In SRB assays, the combination of panobinostat and olaparib synergized in both OVCAR-3 and OVCAR-4 cells. When combined, the drugs also induced co-operative effects on expression of pH2AX(ser139) and apoptosis markers. Enhanced cytotoxicity of the combination was accompanied by a panobinostat-induced reduction in expression of cyclin E, E2F1 and BRCA1. A key role for E2F1 downregulation in mediating this BRCAness shift was suggested by the observation that E2F1 knockdown with siRNA synergized with panobinostat in reducing BRCA1 expression and inducing apoptosis. Conclusions: The development of a new PARPi combination therapy with panobinostat has immediate prospects for rapid translation to the clinic and great potential for improving clinical outcomes for non-BRCAness, chemoresistant ovarian cancer. Citation Format: Andrew J. Wilson, Jeanette Saskowski, Dineo Khabele. The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP ribose polymerase inhibitors via E2F1 downregulation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A15.

Abstract B81: The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to the cyclin-dependent kinase 2 inhibitor dinaciclib.

Abstract Background: The lack of treatment options for chemoresistant ovarian cancer, the deadliest gynecologic malignancy, is a central clinical problem. Approximately 50% of high-grade serous ovarian tumors harbor defects in DNA repair pathways including germline BRCA1 and 2 mutations (a BRCAness phenotype), and are sensitive to DNA damaging agents such as cisplatin and poly ADP ribose polymerase inhibitors (PARPi). In contrast, non-BRCAness tumors such as those exhibiting CCNE1 (cyclin E) amplification are chemoresistant, show upregulated DNA repair processes and cell cycle progression via activation of the CDK2/cyclin E/E2F1 axis, and have poor clinical outcomes. Targeting the cyclin E axis with CDK2 inhibitors such as dinaciclib is a promising clinical strategy. However, their efficacy in cyclin E-overexpressing ovarian cancer cells is limited by acquired chemoresistance mediated in part through upregulation of CDK2, cyclin E and E2F1, and toxicity. Therefore, new strategies are needed to improve the efficacy of CDK2-targeting therapy in ovarian cancer. Our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to multiple chemotherapeutic drugs. Aims: The objectives of this study were to (i) determine expression patterns of cyclin E axis components in publically available databases of high-grade serous ovarian tumors and established cell lines; and (ii) test an epigenetic drug strategy using HDACi to convert non-BRCAness ovarian cancer cells to a BRCAness phenotype as a means of sensitizing chemoresistant cells to dinaciclib. Methods: RNA-seq V2 RSEM expression levels of cyclin E, CDK2, BRCA1 and E2F1 in 265 ovarian tumors were extracted from The Cancer Genome Atlas (TCGA) via cbioportal.org. mRNA expression of these genes in ovarian cancer cell lines was extracted from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Preliminary western blot studies indicated that of the FDA-approved HDACi (vorinostat, romidepsin, panobinostat), panobinostat was most effective at reducing protein expression of cyclin E axis components (cyclin E, BRCA1 and E2F1). We therefore treated cultured cyclin E overexpressing ovarian cancer cells via CCNE1 amplification (OVCAR-3) or high-level gain (OVCAR-4) with panobinostat alone and in combination with the CDK2 inhibitor, dinaciclib. The following endpoints were examined: cell viability in sulforhodamine B (SRB) assays; apoptosis by western blot analysis of cleaved PARP; and protein expression of cyclin E, E2F1 and BRCA1 by western blot. Two-tailed Student's t tests were used for measuring differences between groups in drug treatment experiments. Results: There was significant correlation (Spearman) between cyclin E expression and that of CDK2, E2F1 and BRCA1 in ovarian tumors and in serous-like ovarian cancer cell lines. These results are consistent with the relationship between cyclin E overexpression and enhanced E2F1-dependent transcription of established E2F1 targets, including cyclin E itself, and DNA repair genes such as BRCA1. In SRB assays, the combination of panobinostat and dinaciclib synergized in both OVCAR-3 and OVCAR-4 cells. When combined, the drugs also induced co-operative effects on expression of apoptosis markers. Enhanced cytotoxicity of the combination was accompanied by panobinostat-induced reduction in expression of cyclin E, E2F1 and BRCA1. Conclusions: The development of a new CDK2 inhibitor combination therapy with panobinostat has immediate prospects for rapid translation to the clinic and great potential for improving clinical outcomes for non-BRCAness, chemoresistant ovarian cancer. Citation Format: Andrew J. Wilson, Jeanette Saskowski, Dineo Khabele. The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to the cyclin-dependent kinase 2 inhibitor dinaciclib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B81.

Abstract B37: The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP ribose polymerase inhibitors via E2F1 downregulation.

Abstract Background: The lack of treatment options for chemoresistant ovarian cancer, the deadliest gynecologic malignancy, is a central clinical problem. Approximately 50% of high-grade serous ovarian tumors with defects in DNA repair pathways including germline BRCA1 and 2 mutations (the so-called BRCAness phenotype), and are sensitive to DNA damaging agents such as cisplatin and poly ADP ribose polymerase inhibitors (PARPi). In contrast, non-BRCAness tumors such as those exhibiting CCNE1 (cyclin E) amplification are chemoresistant, show upregulated DNA repair processes and cell cycle progression via activation of the CDK2/cyclin E/E2F1 axis, and have poor clinical outcomes. Targeting the cyclin E axis with CDK inhibitors has been unsuccessful in part due to chemoresistance through upregulation of oncogenic E2F1. An area of urgent need is to develop new treatments for these intractable subtypes of non-BRCAness ovarian cancer. To that end, our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to various chemotherapeutic drugs, including PARPi, through reduction of expression of DNA repair genes. Aims: The objective of this study was to (i) determine expression patterns of cyclin E axis components in publically available databases of high-grade serous ovarian tumors and established cell lines; and (ii) test an epigenetic drug strategy using HDACi to convert non-BRCAness ovarian cancer cells to a BRCAness phenotype as a means of sensitizing chemoresistant cells to PARPi. Methods: RNA-seq V2 RSEM expression levels of cyclin E, CDK2, BRCA1 and E2F1 in 265 ovarian tumors were extracted from The Cancer Genome Atlas (TCGA) via cbioportal.org. mRNA expression of these genes in ovarian cancer cell lines was extracted from the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). Preliminary western blot studies indicated that of the FDA-approved HDACi (vorinostat, romidepsin, panobinostat), panobinostat was most effective at reducing protein expression of cyclin E axis components (cyclin E, BRCA1 and E2F1). We therefore treated cultured cyclin E overexpressing ovarian cancer cells via CCNE1 amplification (OVCAR-3) or high-level gain (OVCAR-4) with panobinostat alone and in combination with the PARPi, olaparib. The following endpoints were examined: cell viability in sulforhodamine B (SRB) assays, with drug interactions tested for synergism by isobologram analysis; DNA damage by immunofluorescence analysis of the double-strand DNA break marker, pH2AX(ser139); apoptosis by immunofluorescence and western blot analysis of cleaved PARP and cleaved caspase-3. Two-tailed Student's t tests were used for measuring differences between groups in drug treatment experiments. Results: There was significant correlation (Spearman) between cyclin E expression and that of CDK2, E2F1 and BRCA1 in ovarian tumors and in serous-like ovarian cancer cell lines. These results are consistent with the relationship between cyclin E overexpression and enhanced E2F1-dependent transcription of established E2F1 targets, including cyclin E itself, and DNA repair genes such as BRCA1. In SRB assays, the combination of panobinostat and olaparib synergized in both OVCAR-3 and OVCAR-4 cells. When combined, the drugs also induced co-operative effects on expression of pH2AX(ser139) and apoptosis markers. Enhanced cytotoxicity of the combination was accompanied by a panobinostat-induced reduction in expression of cyclin E, E2F1 and BRCA1. A key role for E2F1 downregulation in mediating this BRCAness shift was suggested by the observation that E2F1 knockdown with siRNA synergized with panobinostat in reducing BRCA1 expression and inducing apoptosis. Conclusions: The development of a new PARPi combination therapy with panobinostat has immediate prospects for rapid translation to the clinic and great potential for improving clinical outcomes for non-BRCAness, chemoresistant ovarian cancer. Citation Format: Andrew J. Wilson, Jeanette Saskowski, Dineo Khabele. The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP ribose polymerase inhibitors via E2F1 downregulation.. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B37.

Abstract C53: Homologous recombination deficiency (HRD) of high grade serous ovarian tumors from the NOVA Phase III clinical study

Abstract Background: Genome-wide analysis was conducted on tumors obtained from patients enrolled in the NOVA study, a Phase 3 clinical trial evaluating the PARP inhibitor niraparib as a maintenance treatment in patients with platinum-sensitive ovarian cancer. Homologous recombination deficiency (HRD) and mutations in DNA damage repair genes were evaluated. Material and methods: DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue and used to create libraries that were hybridized to a custom Agilent SureSelect capture array carrying probes for 54,091 single nucleotide polymorphism sites distributed across the human genome, as well as probes targeting 43 genes involved in DNA repair, including BRCA1 and BRCA2. The captured and enriched DNA was sequenced on an Illumina HiSeq 2500 sequencer. Sequences covering SNP positions were used to generate allelic imbalance profiles. Measures of genomic instability, including determination of an HRD score (integer value of 0-100), were calculated using allelic imbalance profiles and determination of loss of heterozygosity (LOH) by allele-specific copy number (ASCN). A previously identified HRD threshold score of 42 was used to define HRD positivity in the absence of a BRCA mutation. Results: The NOVA study is a Phase 3, multicenter, randomized, double-blind, placebo-controlled study of niraparib as maintenance therapy in ovarian cancer patients who have either gBRCAmut or a tumor with high-grade serous histology and who have responded to their most recent chemotherapy containing a platinum agent. Tumor BRCA mutational status, HRD score and genomic sequencing of 43 DNA repair genes were obtained from tumor samples from both gBRCAmut and non-gBRCAmut cohorts. In the gBRCAmut cohort, HRD analysis of the tumor confirmed the presence of a deleterious or suspected deleterious mutation in all cases. In addition, an HRD score ≥ 42 and the presence of a deleterious mutation in TP53 with loss of heterozygosity (LOH) were observed in nearly all tumors. In the non-gBRCA cohort, somatic BRCA mutations were observed in approximately 13% of tumors, and approximately half of tumors with no evidence of a BRCA mutation had a high HRD score. In both cohorts, the use of three scoring algorithms (LOH, telomeric allelic imbalance [TAI], large-scale state transitions [LST]), was more predictive of BRCA mutational status than LOH alone. Additional genomic sequencing identified deleterious mutations with LOH in DNA repair genes, such as BRIP1, CDK12, RAD51C, PTEN, and RAD51D, with many tumors exhibiting multiple deleterious mutations. Conclusions: High grade serous ovarian cancer is characterized by a high degree of genomic instability. Genomic analysis in the clinical setting is able to identify patients with both germline and somatic BRCA mutations, in addition to BRCAwt tumors with other genetic defects that may be sensitive to agents exploiting deficiencies in HR. Citation Format: Keith Wilcoxen, Christopher Neff, Victor Abkevich, Joshua Timothy Jones, Kathryn Kolquist, Michael Mirza, Jerry Lanchbury, Keith Mikule, Shefali Agarwal, Anne-Renee Hartman, Alexander Gutin, Kirsten Timms. Homologous recombination deficiency (HRD) of high grade serous ovarian tumors from the NOVA Phase III clinical study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C53.

Connective tissue growth factor as a novel therapeutic target in high grade serous ovarian cancer.

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.

BCAT1 expression associates with ovarian cancer progression: possible implications in altered disease metabolism.

Previously, we have identified the branched chain amino-acid transaminase 1 (BCAT1) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Knockdown of the BCAT1 expression in epithelial ovarian cancer (EOC) cells led to sharp decrease of cell proliferation, migration and invasion and inhibited cell cycle progression. BCAT1 silencing was associated with the suppression of numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, and the induction of some tumor suppressor genes (TSGs). Moreover, BCAT1 suppression resulted in downregulation of numerous genes implicated in lipid production and protein synthesis, suggesting its important role in controlling EOC metabolism. Further metabolomic analyses were indicative for significant depletion of most amino acids and different phospho- and sphingolipids following BCAT1 knockdown. Finally, BCAT1 suppression led to significantly prolonged survival time in xenograft model of advanced peritoneal EOC. Taken together, our findings provide new insights about the functional role of BCAT1 in ovarian carcinogenesis and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target.

458 DNA repair deficiencies in ovarian cancer: Genomic analysis of high grade serous ovarian tumors from the NOVA study

458 DNA repair deficiencies in ovarian cancer: Genomic analysis of high grade serous ovarian tumors from the NOVA study

Abstract POSTER-BIOL-1331: Nucleotide excision repair mutations confer sensitivity to platinum but not PARP-inhibitors in epithelial ovarian cancer

Abstract The marked platinum sensitivity of epithelial ovarian cancer (EOC) is thought to be related to underlying defects in the homologous recombination (HR) DNA repair pathway, which are present in approximately 50% of tumors with high-grade serous histology. However, repair of platinum-induced DNA damage does not always involve the HR pathway. More than 90% of platinum-DNA lesions are intrastrand cross-links which are repaired by the nucleotide excision repair (NER) pathway. We curated the TCGA ovarian cancer dataset and identified 22 tumors (7%) with nonsynonymous or splice site NER gene mutations among the 316 sequenced high grade serous ovarian tumors. All NER mutations were mutually exclusive and 8 (36%) were accompanied by heterozygous loss of the respective NER gene. In 12 (55%) of the 22 cases, there were no concomitant HR alterations identified. In vitro functional evaluation of two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in patients with advanced stage tumors and extreme response to first-line platinum therapy, showed that both mutations conferred platinum, but not PARPi, or doxorubicin sensitivity, and did not affect HR activity. Interestingly, ERCC6-Q524* was not associated with heterozygous loss but sensitized cells to platinum in a dominant negative fashion. Our findings suggest a novel mechanism of platinum sensitivity in EOC that involves NER gene mutations, which may occur independently of HR alterations. Unlike tumors with defective HR, tumors harboring only NER mutations may be sensitive to platinum but not PARP inhibition. Dissociation of platinum and PARPi sensitivity in this setting may have important implications for PARPi trials for both HR-proficient and HR-deficient EOCs. Citation Format: Raphael Ceccaldi, Kevin O’Connor, Kent W. Mouw, Adam Y. Li, Ursula A. Matulonis, Alan D. D’Andrea, Panagiotis A. Konstantinopoulos. Nucleotide excision repair mutations confer sensitivity to platinum but not PARP-inhibitors in epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1331.

Abstract 2068: Cyclooxygenase-1 gene expression contributes to multiple oncogenic pathways in high grade serous ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. Most women diagnosed with high grade serous ovarian cancer present with advanced disease characterized by widespread peritoneal carcinomatosis. Recent epidemiological results indicate that cyclooxygenase (COX) inhibitors aspirin and other non-steroidal anti-inflammatory drugs are a promising preventive strategy to reduce the risk of developing ovarian cancer. COX-1 and COX-2 catalyze the rate-limiting step in the biosynthesis of prostaglandins and thromboxanes, which contribute to tumorigenesis by promoting proliferation, escape from apoptosis, and angiogenesis. Emerging evidence from our group and others suggests that COX-1 is an important molecular target in ovarian cancer, distinguishing ovarian cancer from most other solid tumors, where COX-2 is the dominant isoform. However, the precise roles of COX-1 and COX-2 in human ovarian cancer remain controversial. Therefore, the aim of this study was to gain additional insight into COX-1 gene expression and function in human ovarian cancer. First, we extracted RNA-seq V2 mRNA expression data for COX-1 and COX-2 in the PANCAN12 tumor panel of The Cancer Genome Atlas. COX-1 mRNA levels were significantly higher than those of COX-2 in high grade serous ovarian tumors, and higher than COX-1 expression in 11 other solid tumor types. We confirmed these findings in a tissue microarray generated from 209 ovarian cancer patients. COX-1 protein was highly expressed in a large subset of serous tumors, with significantly lower COX-2 expression present. In contrast, there was markedly lower COX-1 expression in endometrioid, mucinous and clear cell ovarian tumors compared to COX-2. Second, stable COX-1 knockdown in serous-representative OVCAR-3 cells significantly reduced cell viability compared to control cells. Pathway analysis from RNA-seq data revealed that COX-1 knockdown induced a coordinated transcriptional response consistent with reduced cell proliferation (eg. reduced cyclin D1 and increased p21 expression), cell migration/invasion (eg. reduced TGF-β signaling and expression of MMPs), angiogenesis (eg. reduced VEGF expression), epithelial-mesenchymal transition (eg. increased E-cadherin expression and reduced BMP signaling), and NF-κB signaling. Gene expression changes were validated by western blot. In SKOV-3 ovarian cancer cells stably overexpressing COX-1, opposite effects on cell viability and on these genes/pathways were observed. Our findings confirm that COX-1 is over-expressed in serous ovarian cancer, and likely plays a key role in tumor progression. Exploiting high COX-1 expression in ovarian cancer with COX-1-targeted molecules has the potential to improve the treatment and detection of this deadly disease. Citation Format: Andrew J. Wilson, Brian D. Lehmann, Jeanette Saskowski, MD Jashim Uddin, Cristina Daniel, Brenda C. Crews, Qi Liu, Shilin Zhao, Deok-Soo Son, Jennifer A. Pietenpol, Lawrence J. Marnett, Dineo Khabele. Cyclooxygenase-1 gene expression contributes to multiple oncogenic pathways in high grade serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2068. doi:10.1158/1538-7445.AM2015-2068

Systematic transcriptome analysis reveals tumor-specific isoforms for ovarian cancer diagnosis and therapy

Tumor-specific molecules are needed across diverse areas of oncology for use in early detection, diagnosis, prognosis and therapy. Large and growing public databases of transcriptome sequencing data (RNA-seq) derived from tumors and normal tissues hold the potential of yielding tumor-specific molecules, but because the data are new they have not been fully explored for this purpose. We have developed custom bioinformatic algorithms and used them with 296 high-grade serous ovarian (HGS-OvCa) tumor and 1,839 normal RNA-seq datasets to identify mRNA isoforms with tumor-specific expression. We rank prioritized isoforms by likelihood of being expressed in HGS-OvCa tumors and not in normal tissues and analyzed 671 top-ranked isoforms by high-throughput RT-qPCR. Six of these isoforms were expressed in a majority of the 12 tumors examined but not in 18 normal tissues. An additional 11 were expressed in most tumors and only one normal tissue, which in most cases was fallopian or colon. Of the 671 isoforms, the topmost 5% (n = 33) ranked based on having tumor-specific or highly restricted normal tissue expression by RT-qPCR analysis are enriched for oncogenic, stem cell/cancer stem cell, and early development loci--including ETV4, FOXM1, LSR, CD9, RAB11FIP4, and FGFRL1. Many of the 33 isoforms are predicted to encode proteins with unique amino acid sequences, which would allow them to be specifically targeted for one or more therapeutic strategies--including monoclonal antibodies and T-cell-based vaccines. The systematic process described herein is readily and rapidly applicable to the more than 30 additional tumor types for which sufficient amounts of RNA-seq already exist.