High-grade Serous Ovarian Cancer Cell Research Articles

Active Estrogen Receptor-alpha Signaling in Ovarian Cancer Models and Clinical Specimens.

Purpose: High-grade serous ovarian cancer (HGSOC) is an aggressive disease with few available targeted therapies. Despite high expression of estrogen receptor-alpha (ERα) in approximately 80% of HGSOC and some small but promising clinical trials of endocrine therapy, ERα has been understudied as a target in this disease. We sought to identify hormone-responsive, ERα-dependent HGSOC.Experimental Design: We characterized endocrine response in HGSOC cells across culture conditions [ two-dimensional (2D), three-dimensional (3D), forced suspension] and in patient-derived xenograft (PDX) explants, assessing proliferation and gene expression. Estrogen-regulated transcriptome data were overlapped with public datasets to develop a comprehensive panel of ERα target genes. Expression of this panel and ERα H-score were assessed in HGSOC samples from patients who received endocrine therapy. Time on endocrine therapy was used as a surrogate for clinical response.Results: Proliferation is ERα-regulated in HGSOC cells in vitro and in vivo, and is partly dependent on 3D context. Transcriptomic studies identified genes shared by cell lines and PDX explants as ERα targets. The selective ERα downregulator (SERD) fulvestrant is more effective than tamoxifen in blocking ERα action. ERα H-score is predictive of efficacy of endocrine therapy, and this prediction is further improved by inclusion of target gene expression, particularly IGFBP3Conclusions: Laboratory models corroborate intertumor heterogeneity of endocrine response in HGSOC but identify features associated with functional ERα and endocrine responsiveness. Assessing ERα function (e.g., IGFBP3 expression) in conjunction with H-score may help select patients who would benefit from endocrine therapy. Preclinical data suggest that SERDs might be more effective than tamoxifen. Clin Cancer Res; 23(14); 3802-12. ©2017 AACR.

Abstract 1428: Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer

Abstract Resistance to chemotherapy poses a major clinical problem in the treatment of ovarian cancer. The efficacy of platinum- based chemotherapy relies on defective DNA repair mechanisms, and increased expression of homologous recombination (HR)/DNA repair genes in primary tumor samples is associated with improved chemoresponse. However, the effects of neo-adjuvant chemotherapy (NACT) on the expression DNA repair genes in cancer cells are poorly understood. In addition, high-grade serous ovarian cancers (HGSOCs) display significant inter- and intratumoral heterogeneity, and the identification of mechanisms of drug resistance has been hampered by the lack of data at the single cell resolution. We collected 34 primary and 20 interval tumor samples from 15 patients treated for HGSOC at the Turku University Hospital, Finland. Tumor tissue total RNA was sequenced with Illumina HiSeq. For this study, we selected 363 genes selected due to their validated role in DNA repair pathways and compared their expression profiles between primary and interval samples. The treatment naïve primary tumor samples highly expressed genes related to HR, such as BRCA2, RAD54, PARPBP, FANCD2, and POLQ. Whereas after NACT the interval samples highly expressed genes regulating cell cycle, inflammatory response, and the drug efflux pump ABCB1. Moreover, higher expression of BRCA2 or POLQ in the interval samples correlated with poor primary therapy outcome (p<0.01). To reveal the transcriptomics landscapes of treatment naïve and NACT treated HGSOCs at the single cell level, we performed single cell mRNA sequencing of primary and interval samples using the Fluidigm C1 Platform. Unsupervised clustering revealed eight distinct cell subpopulations, of which two clusters were annotated HGSOC cells based on enriched expression of cancer specific genes. In global gene expression analysis of 98 HGSOC cells, the most differentially expressed genes in primary HGSOC cells were heat shock proteins reflecting active protein synthesis. By contrast, the cancer cells from interval samples highly expressed genes regulating the cell cycle and immune response. Of the DNA repair pathway genes, the interval samples expressed lower levels of HES1, a transcription factor that is known to promote cancer progression and stemness. The herein elucidated DNA repair pathway transcriptomics landscape at single-cell and whole tumor level provides novel targets for overcoming chemoresistance in HGSOC. Citation Format: Anniina Farkkila, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Rainer Lehtonen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2017-1428

Abstract 795: The capacity of high-grade serous ovarian cancer cells to form spontaneous multicellular structures (SMCS) in vitro predicts their in vivo tumorigenicity

Abstract High grade serous ovarian cancer (HGSOC) is the most frequent histopathological subtype among epithelial ovarian cancers (OC). While an increase in concentration of CA125 in the blood anticipates clinical remission following standard of care, we lack biomarker/s to diagnose early disease stage or predict progression speed. In this work we question whether the capacity of HGSOC cells to form Spontaneous Multi-Cellular Structures (SMCS) when incubated under culture conditions that promote, not prevent, adherence to a plastic surface, correlates with their degree of tumorigenicity. We studied 3 HGSOC cell lines developed from the same patient’s ascites: longitudinally along disease progression—established at platinum-sensitive relapse (PEO1); from further progressive disease 10 months (mo.) later (PEO4); and after failure to respond to high-dose cisplatin 3 mo. later (PEO6). We noticed all cell types developed an adherent phenotype and a differential capacity to form SMCS. This capacity was more evident in PEO6 cells having high SMCS forming ability, followed by PEO4 cells denoting some SMCS forming capacity, while PEO1 cells depicted no apparent capability to form SMCS. Next, 2x106 PEO1, PEO4 or PEO6 cells were implanted into the abdominal cavity of nude mice; the animals were sacrificed either after having met an end-of-wellness endpoint criterion, or after a maximum of 14 mo. if no such criteria were met. PEO6-injected mice reached the humane endpoint due to accumulation of ascites 6-7 mo. following injection, and presented discrete yet visible solid tumors in the omentum, pancreatic-spleen region, liver base and diaphragm. Animals injected with PEO4 or PEO1 cells, however, did not develop any apparent disease 14 mo. following injection. Yet, when a 10-fold higher load of PEO4 cells but not PEO1 cells (i.e., 20x106) were injected, animals met euthanasia criteria due to ascites accumulation 6 mo. later and displayed macroscopic disease similar to that caused by 1/10 of the load of their successors (PEO6 cells) within the same timeframe. Furthermore, the anatomical localization and histopathological aspect of the lesions generated with PEO6 cells were similar from that generated with PEO4 cells, suggesting that the more aggressive PEO6 cells were likely present, although in fewer quantities, within the less aggressive PEO4 cell population. Our data demonstrate, then, that the in vitro SMCS forming capacity of HGSOC cells has a positive correlation to their capacity to sicken the animals. Moreover, this study suggests that the in vitro SMCS forming capacity of epithelial OC cells obtained from ascites of patients diagnosed with HGSOC may be used to predict their aggressiveness and, consequently, guide prognosis. Citation Format: Alicia A. Goyeneche, Zu-hua Gao, Carlos M. Telleria. The capacity of high-grade serous ovarian cancer cells to form spontaneous multicellular structures (SMCS) in vitro predicts their in vivo tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 795. doi:10.1158/1538-7445.AM2017-795

Abstract 4347: Epigenetic targeting of DNMT1 in adipocytes inhibits high-grade serous ovarian cancer cell migration and invasion through TIMP3 upregulation

Abstract Ovarian cancer frequently metastasizes to the omentum and adipocytes play a significant role in tumor progression. As methylation levels in obese adipose tissue are increased due to increased DNMT1 levels and activity, it was of interest to test the hypothesis that inhibiting DNMT1 would reverse adipocyte methylation, alter adipokine secretion, and decrease migration and invasion of ovarian cancer cells towards adipocytes. Human adipocytes were seeded in a 24-well plate and treated with low-dose guadecitabine (100nM daily for 3 days). Ovarian cancer cells (SKOV3, Kuramochi, OVCAR4, OVCAR8) were seeded in the Boyden chamber and allowed to migrate or invade toward adipocytes for 8 and 16h, respectively. Expression of epithelial-mesenchymal transiton (EMT) markers (SLUG, FN1, TWIST1) were assessed by qRT-PCR. Adipocyte-conditioned media was used to culture ovarian cancer cells in clonogenicity assay and a human adipokine array (R&D Systems) was used to assess changes in cytokine secretion. DNMT1 protein levels in adipocytes were determined by western blot. To determine a possible mechanism, DNA and RNA from guadecitabine-treated adipocytes were subjected to methylcapture-sequencing (MBD-seq) and RNA-seq, respectively. Guadecitabine treatment of adipocytes decreased (P<0.05) migration of OVCAR4 and OVCAR8 (35% and 40%, respectively, compared to control), and a 50% decrease (P<0.05) in invasion towards adipocytes after guadecitabine treatment was observed for OVCAR4, OVCAR8, and Kuramochi cells. Expression of EMT markers SLUG, FN1 and TWIST1 decreased (P<0.05) after guadecitabine treatment. Conditioned media from guadecitabine-treated adipocytes decreased (P<0.05) clonogenic survival by 18% compared to control. Adipokine array results revealed increased secretion of LIF (lipoprotein lipase inhibitor) and TIMP3 (metalloproteinase inhibitor) after guadecitabine treatment (1.6- and 1.8-fold increase, respectively; verified by qRT-PCR). Treatment with recombinant TIMP3 (50nM) decreased invasion of OVCAR8 (63%, P<0.001) and OVCAR4 cells (73%, P<0.05). DNMT1 protein levels in adipocytes decreased in the presence of guadecitabine, despite the presence of a DNA synthesis inhibitor. Ingenuity Pathway Analysis (IPA) of MBD-seq data showed significant changes in cell-to-cell signaling and interaction pathways (FC >10; FDR <0.05). RNA-seq demonstrated increased expression of matrix metalloprotease inhibitors (THSB2, TFPI2, and NDRG4) and IPA analysis revealed a significant change in regulation of EMT pathway (FC > 1.5; FDR <0.05). Guadecitabine treatment of adipocytes alters adipokine secretion resulting in decreased cancer cell migration and invasion. In addition to direct effects on ovarian cancer cells, hypomethylating agents may impact the tumor microenvironment to alter adipokine secretion leading to decreased metastasis. Citation Format: Jessica Tang, Fang Fang, Aaron Buechlein, Pietro Taverna, Kenneth P. Nephew. Epigenetic targeting of DNMT1 in adipocytes inhibits high-grade serous ovarian cancer cell migration and invasion through TIMP3 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4347. doi:10.1158/1538-7445.AM2017-4347

Abstract 4675: Novel combination therapy of DNA methyltransferase inhibitor guadecitabine (SGI-110) and PARP inhibitor talazoparib (BMN-673) for BRCA-proficient high-grade serous ovarian cancer

Abstract Ovarian cancer recurrence has been shown to be associated with increased DNA damage response (DDR) mediated by poly-(ADP)-ribose polymerase 1/2 (PARP1/2), which can be therapeutically targeted by PARP inhibitors (PARPi). PARPi are indicated for platinum-responsive, BRCA-mutated high-grade serous ovarian cancer, but most ovarian cancer patients have BRCA-proficient disease. Our previous studies support a role for DNA methylation in chemoresistant ovarian cancer mediated by the enzyme DNA methyltransferase 1 (DNMT1), and report on a functional role for DNMT1 in DNA damage repair. We therefore hypothesized that combining a DNMTi and PARPi will impair BRCA-mediated DDR, resulting in cytotoxicity in ovarian cancer cells. A panel of ovarian cancer cell lines (A2780, platinum sensitive, BRCA1/2-wild type; A2780-cp and HeyC2, platinum resistant, BRCA1/2-wild type; high-grade serous Kuramochi, platinum resistant, BRCA2 mutant) was examined for cell growth using colony formation assays after treatment with DNMTi guadecitabine (low dose, 20-100nm) and PARPi talazoparib (1-10nm), alone or in combination. While talazoparib alone only marginally reduced colony formation in all cell lines (dose-dependent effect), combining guadecitabine with talazoparib further decreased (P<0.05) survival at all doses examined. To focus more specifically on BRCA status, we utilized two high-grade serous ovarian cancer cell lines (“PEO”) derived from the same patient but harboring a mutant (PEO1) or wild type (PEO4) BRCA2 gene (Langdon et al, 1988; Sakai et al, 2009). Treatment with low-dose guadecitabine (20 nm, 3 days) increased (P<0.05) PARP levels (western blot analysis) as well as enzymatic activity (P<0.05; ELISA analysis), while talazoparib treatment alone increased (P<0.05) DNMT1 levels and decreased (P<0.05) PARP enzymatic activity. Treatment with guadecitabine or talazoparib alone had no effect on cell proliferation; however, combining the two drugs inhibited (>80%; P<0.05) PEO1 and PEO4 proliferation and increased (3-fold; P<0.05) apoptosis (caspase 3 cleavage) in both cell lines. Furthermore, co-administration guadecitabine (0.5mg/kg) and talazoparib (0.25mg/kg) to mice harboring BRCA2-wild type ovarian tumor xenografts decreased (p<0.05) tumor volume (>60%) and tumor weight (~70%) compared to control, respectively. In summary, combining a hypomethylating agent with a PARP inhibitor results in enhanced cytotoxicity in high-grade serous ovarian cancer cell lines harboring either wild type- or mutant-BRCA, indicating that the talazoparib-guadecitabine drug combination is effective regardless of BRCA-mediated DDR and may represent an effective treatment regimen for BRCA-related cancers. Citation Format: Nicholas Pulliam, Pietro Taverna, John Lyons, Kenneth P. Nephew. Novel combination therapy of DNA methyltransferase inhibitor guadecitabine (SGI-110) and PARP inhibitor talazoparib (BMN-673) for BRCA-proficient high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2017-4675

Abstract 1197: Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer

Abstract The widespread activation of YAP and TAZ transcriptional regulators has been shown to promote tumor progression and resistance to anti-cancer therapies. Extensive studies have identified different upstream regulators of YAP/TAZ, including G-protein coupled receptor (GPCR). However how GPCR signals regulate YAP/TAZ puzzles have remained elusive. Among the GPCR, we reveal that the endothelin-1 (ET-1) receptor activates YAP/TAZ pathway in high-grade serous ovarian cancer (HG-SOC) sensitive and resistant cell lines and primary cultures. In HG-SOC cells, the aberrant activation of ET-1 receptor (ET-1R), promotes an enhanced nuclear shuttling of dephosphorylated YAP and TAZ in a time-dependent manner, through the scaffold protein β-arrestin1 (β-arr1). Interestingly, ET-1-induced YAP/TAZ nuclear accumulation is further improved in platinum-resistant cells and is prevented by expressing a mutant β-arr1 incapable of nuclear distribution. Moreover, the ET-1/ETAR axis activation induces the co-localization of β-arr1, YAP and TAZ in the nucleus. Mechanistically, in sensitive and to a greater extent in resistant cells, ET-1 induces the formation of a complex β-arr1/Trio, a Rho-Guanine nucleotide exchange factor family member, which dephosphorylates YAP, inducing its nuclear accumulation and the transcription of YAP/TAZ target genes. β-arr1, RhoA or Trio depletion, as well as the treatment with the dual ETAR/ETBR antagonist macitentan, significantly reduces RhoA GTPase activity and YAP nuclear translocation, suggesting that ET-1/ETAR axis regulates YAP/TAZ activity by driving β-arr1/Trio/RhoA pathway. According with these results, the ET-1-induced upregulation of YAP/TEAD target genes, such as CYR61, CTGF, ANKRD1 and EDN1, as well as TEAD transcriptional activity and cell invasion, are inhibited upon β-arr1, Trio, or YAP silencing, or macitentan treatment, demonstrating that β-arr1-mediated intersections are required for ET-1-induced YAP signaling. At the chromatin level, the activation of ET-1 axis promotes the recruitment of YAP, TEAD and β-arr1 on TEAD binding sites of CTGF, ANKRD1 and EDN1 promoters, an effect inhibited by macitentan treatment. These results indicate that β-arr1 can act as a transcription co-activator that bind TEAD thereby activating transcription of EDN1, which in turn, can sustain persistent YAP/TAZ activity through an autocrine loop. In murine orthotopic model of patient-derived xenograft (PDX) of HG-SOC, ET-1R blockade by macitentan, inhibits tumor growth, enhances the sensitivity to chemotherapy and reduces YAP and TEAD transcriptional activity. Altogether our results establish for the first time YAP/TAZ as critical downstream effectors of ET-1R/β-arr1 signaling, providing mechanistic insights that targeting ET-1R signaling overcomes YAP/TAZ driven platinum-based therapy failure in HG-SOC. Citation Format: Piera Tocci, Roberta Cianfrocca, Laura Rosanò, Rosanna Sestito, Valeriana Di Castro, Giovanni Blandino, Anna Bagnato. Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2017-1197

Abstract 88: The role of RBPMS in cisplatin resistant ovarian cancer

Abstract Epithelial ovarian cancer is the fifth leading cause of cancer-associated deaths among women in the United States. High-grade serous ovarian cancer (HGSOC) is the most common type of epithelial ovarian cancer and accounts for 70-80% of ovarian cancer patient deaths. RNA-Binding Protein With Multiple Splicing (RBPMS) is a member of a family of proteins that bind to the nascent RNA transcripts and regulate their processing, including the pre-mRNA splicing and the transport, localization, and stability of the RNA molecules. However, little is known about the biological function of RBPMS family in ovarian cancer. We hypothesized that decreasing the expression of RBPMS in high-grade serous ovarian cancer increases ovarian cancer progression and platinum-based drug resistance. Quantitative real-time PCR (qPCR) and Western blot (WB) analysis were conducted to determine the basal mRNA and protein expression of RBPMS in ovarian cancer cells. The invasion ability of the ovarian cancer cells after targeting RBPMS with silencing RNA (siRNA) was determined by invasion assays. The proliferation rate of the ovarian cancer cells overexpressing RBPMS was determine by colony formation assay. RBPMS is highly expressed in A2780 cells when compared to the cisplatin resistance ovarian cancer cells and HGSOC cancer cells. Targeting RBPMS increased the invasion ability of the A2780 cells. Cisplatin resistant, A2780CP20 cells overexpressing RBPMS showed a decreased in cell proliferation when compared to the A2780CP20 empty vector clones. We described here for the first time the mRNA and protein expression profile of RBPMS in HGSOC and epithelial ovarian cancer cells. These studies will help to better understand the role of RBPMS in ovarian cancer and may support the use of RBPMS as an adjuvant treatment to overcome the platinum-based chemotherapy resistance characteristic of the high-grade serous ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Perla M. Báez-Vega, Fatma Valiyeva, Ginette Santiago, Pablo Vivas-Mejia. The role of RBPMS in cisplatin resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 88. doi:10.1158/1538-7445.AM2017-88

Abstract 5708: HOXA4 and HOXB3 gene expression signature as a biomarker of tumor recurrence in patients with high-grade serous ovarian cancer (HGSOC) following primary cytoreductive surgery and first-line adjuvant chemotherapy

Abstract Background: Aberrant HOX gene expression has been observed in various malignancies; however, their role as a biomarker for recurrence in patients with HGSOC is unknown. We tested the hypothesis that a HOX gene signature is a biomarker for disease-free survival (DFS) in HGSOC patients following primary cytoreductive surgery and adjuvant platinum-based chemotherapy. Methods: A panel of HOX genes associated with DFS in a discovery cohort of 19 HGSOC patients with available RNA-Seq data, and/or previously reported as associated with survival endpoints in the literature, were selected for testing by qPCR using the Fluidigm platform in an independent training cohort of primary tumors from 73 HGSOC patients. Cox proportional hazards model was used to identify HOX genes significantly associated with DFS. A prognostic gene signature was developed based on the combined linear predictor from the final multivariable Cox model. Patients were stratified into risk groups using the optimal cutoff of the linear predictor risk score, whereby the optimal cutoff was defined as the value that maximized the log-rank test statistic. Risk group stratification was further tested for association with DFS in a larger HGSOC dataset (N=414) with similar clinical characteristics from The Cancer Genome Atlas (TCGA). The role of the identified HOX genes in influencing drug sensitivity was examined by overexpressing these genes in a HGSOC cell line, PEO1. Results: Of 23 HOX genes selected for testing in the training cohort, overexpression of HOXA4 (HR=1.21, 95% CI=1.08-1.35, P=0.001) and HOXB3 (HR=1.09, 95% CI=1.02-1.17, P=0.016) were significantly associated with decreased DFS in multivariate analysis. The median DFS in patients with a HOXA4/HOXB3 risk score ≤ the optimal cutoff was > 80 months (not yet reached), whereas the median DFS in patients with a HOXA4/HOXB3 risk score > the optimal cutoff was 16.9 months (HR=7.85, 95% CI=4.19-14.69, P<0.001). The HOXA4/HOXB3 risk score remained significantly associated with DFS in the HGSOC dataset from the TCGA (P=0.02). In vitro studies demonstrated that PEO1 cells overexpressing either HOXA4 or HOXB3 were significantly (P<0.05) more resistant to cisplatin and carboplatin, but not to paclitaxel, as compared to empty vector transfected cells. Conclusion: The HOXA4/HOXB3 risk score was significantly associated with DFS in a training cohort of HGSOC patients, and remained significantly associated in a larger independent HGSOC dataset from the TCGA. Further, in vitro studies suggest that HOXA4 and HOXB3 overexpression is associated with platinum resistance. Thus, overexpression of HOXA4 and HOXB3 in primary HGSOC may serve as a potential biomarker for recurrence and the risk score model developed warrants prospective validation in HGSOC patients. Citation Format: Jai N. Patel, Katherine Miller, James Symanowski, Jalid Sehouli, Chad Michener, Ioana Braicu, Darla Destephanis, Ashley P. Sutker, Eric J. Norris, David Tait, Wendell Jones, Qing Zhang, Chad Livasy, Charles Biscotti, Ram N. Ganapathi, Mahrukh K. Ganapathi. HOXA4 and HOXB3 gene expression signature as a biomarker of tumor recurrence in patients with high-grade serous ovarian cancer (HGSOC) following primary cytoreductive surgery and first-line adjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5708. doi:10.1158/1538-7445.AM2017-5708

Abstract 2787: Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing

Abstract Epithelial ovarian cancer is a complex disease and patients vary considerably in response to treatment. A subset of patients responds well to endocrine therapy, however hormone receptor positivity does not predict response and there are no biomarkers in clinical use to help select patients that would benefit from endocrine agents. We aimed to identify markers of endocrine response in high-grade serous ovarian cancer (HGSOC) by analysis of genes that are differentially expressed in tumors from patients that are endocrine responders compared with non-responders, combined with analysis of estrogen (E2) regulated genes that differ between E2 sensitive and E2 insensitive HGSOC cell lines. Methods: Among women recruited to the Australian Ovarian Cancer Study, we identified 10 HGSOC patients treated with endocrine therapy (5 responders and 5 non-responders, based on GCIG CA125 response criteria) and performed RNAseq on cryopreserved tumor tissue. We also utilized paired E2 receptor (ER) positive HGSOC cell lines, derived from the same patient, with differential sensitivity to the growth effect of E2, PEO1 (E2 insensitive) and PEO4 (E2 sensitive). RNA was extracted following treatment with 0.1 nM E2 (or vehicle) for 24 hrs and gene expression changes in response to E2 were determined using RNAseq. Expression of selected ER responsive genes was validated using PCR array. Differential gene expression was determined using EdgeR. Pathway and Gene Ontology enrichment analysis of differentially expressed genes were performed using Metacore. Results: Between endocrine responders and non-responders, 27 genes were significantly differentially expressed (False Discovery Rate (FDR)<0.05, absolute log2 Fold-Change (FC)≥1). In the paired HGSOC cell lines, significantly more genes were regulated by E2 in E2-sensitive PEO4, compared with E2-insensitive PEO1 (896 genes compared to 56 genes, respectively, FDR<0.05, absolute log2FC≥1). In response to E2, up-regulation of NRP1 in PEO1, up-regulation of LTBP1, MYC, PDZK1 and down-regulation of S100A6 and CYP1A1 in PEO4 were validated by PCR array profiling of ER-related genes. Genes regulated by E2 in cell lines and also differentially expressed between endocrine responders and non-responders included anoctamin 1 (ANO1) and NOTCH-Regulated Ankyrin Repeat Protein (NRARP). Both were up-regulated in endocrine responders and also up-regulated by E2 in PEO4. Conclusion: The use of RNAseq in both tumors and cell line models was able to identify plausible sets of genes with prior evidence of association with endocrine sensitivity. In breast cancer, ANO1 has been associated with good prognosis following tamoxifen treatment, while NOTCH signaling, implicated in endocrine resistance, is negatively regulated by NRARP. Further validation is required to test the utility of these genes as predictive biomarkers that may aid in identifying HGSOC patients likely to respond to endocrine treatment. Citation Format: Cristina Mapagu, Sian Fereday, Australian Ovarian Cancer Study Group, David D. Bowtell, Paul R. Harnett, Anna deFazio. Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2787. doi:10.1158/1538-7445.AM2017-2787

Abstract 776: Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry

Abstract Introduction: Platinum resistant High Grade Serous Ovarian Cancer (HGSOC) has a poor outcome with limited treatment options. Angiogenesis is a key pathological feature of ovarian cancer and anti-angiogenics have dominated the field of drug development in EOC, particularly in the second-line setting (Marachini et al 2013). In this study we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents. Method: A review of phase III anti-angiogenic clinical trials was used to investigate the association between platinum resistance and response to anti-angiogenic agents. To investigate the effect of chemotherapy on predefined ovarian cancer molecular subgroups (Gourley, et al. J Clin Oncol 32:5s, 2014), we analysed 35 matched pre- and post-chemotherapy samples by gene expression. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the mechanisms of cisplatin section pressure and shift to an angiogenic phenotype. This was further validated in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy. Results: Critical review of 22 phase III anti-angiogenic trials suggested that there was a better response to anti-angiogenics following previous platinum-based chemotherapy. Our analysis demonstrated that 67% of treatment naïve tumours that were initially classified as non-angiogenic shifted to an angiogenic biology, which was associated with platinum resistance. Additionally we found that cisplatin resistant cancer cell lines demonstrated hallmarks of vascular mimicry and an associated increase in vessel density using an angiogenesis Matrigel plug assay in Athymic nude mice (p-value=<0.0001). In addition, cell lines established from platinum-resistant patients as well as cell lines made platinum resistant in-vitro, demonstrated overexpression of VEGFa which would be expected to stimulate angiogenesis. Conclusion: We have demonstrated that platinum-resistance in HGSOC is associated with angiogenic biology supporting the use of anti-angiogenic agents in this setting. Citation Format: Aya El Helali, Nuala McCabe, Naomi Dickson, Lara Dura Perez, Denis P Harkin, Richard Wilson, Richard Kennedy. Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 776. doi:10.1158/1538-7445.AM2017-776

Targeting the ATR/CHK1 Axis with PARP Inhibition Results in Tumor Regression in BRCA-Mutant Ovarian Cancer Models.

Purpose: PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA-mutant (BRCAMUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression.Experimental Design: Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776), or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell cycle, genome instability, and apoptosis were evaluated in BRCA1/2MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2MUT patient-derived xenograft (PDX) model.Results: PARPi monotherapy resulted in a decrease in BRCAMUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum tolerated dose (MTD) in a BRCA2MUT PDX. PARPi treatment increased pATR and pCHK1, indicating activation of the ATR-CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared with single-agent treatments in BRCAMUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA2MUT PDX with the PARPi-ATRi combination inducing tumor regression and in most cases, complete remission.Conclusions: PARPi causes increased reliance on ATR/CHK1 for genome stability, and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCAMUT models. Clin Cancer Res; 23(12); 3097-108. ©2016 AACR.

Abstract MIP-044: FOXM1 INDUCED REPLICATION STRESS IS MITIGATED BY ITS BIDIRECTIONAL GENE PARTNER, RHNO1, IN HIGH GRADE SEROUS OVARIAN CANCER

Abstract PURPOSE: FOXM1 is located at 12p13.33, a significantly amplified locus in high-grade serous ovarian cancer (HGSC). The 12p13.33 amplicon contains ~33 genes, which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 expression is associated with cancer genomic instability, it is unclear whether FOXM1 causes this phenotype and, if so, by what mechanism. We hypothesized that FOXM1 induces replication stress, which is the slowing or stalling of replication forks. We further noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 signaling, is contained within the 12p13.33 amplicon, and arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1-RHNO1 bidirectional promoter, FOXM1 and RHNO1 expression patterns in HGSC, and determined the relationship between FOXM1 and RHNO1 in replication stress. METHODS: We used 5' RACE and a luciferase reporter construct to characterize the FOXM1-RHNO1 bidirectional promoter. We analyzed copy number, mRNA and protein expression datasets in TCGA HGSC and additional TCGA cancers. We measured FOXM1 and RHNO1 expression in HGSC cell lines and primary HGSC tissues. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to replication stress. We used RHNO1 knockdown to dissect its functional contribution to ATR-CHK1 signaling in HGSC cells. RESULTS: FOXM1 and RHNO1 were co-amplified in ~12% of HGSC and their mRNA expressions were highly correlated. The FOXM1-RHNO1 bidirectional promoter showed similar activity in each direction in HGSC cells, and correlated with mRNA expression. Analysis of TCGA HGSC data revealed that FOXM1 associates with markers of DNA replication and CHK1-Ser345 phosphorylation, a canonical marker of replication stress. FOXM1 overexpression in immortalized FTE cells induced CHK1-Ser345 phosphorylation. RHNO1 knockdown attenuated CHK1-Ser345 phosphorylation in response to replication stress, and, importantly, reduced clonogenic growth in FOXM1 overexpressing HGSC cells. CONCLUSIONS: Our data reveal that FOXM1 and RHNO1 share a bidirectional promoter resulting in their frequent co-expression. FOXM1 induces replication stress whereas RHNO1 is necessary for efficient ATR-CHK1 signaling. We hypothesize that balanced FOXM1 and RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1 and RHNO1 on replication stress and genomic instability. Finally, our findings suggest ATR-CHK1 signaling as a potential therapeutic vulnerability in FOXM1 activated HGSC. Citation Format: Carter J Barger, Linda Chee, Ronny Drapkin, Kunle Odunsi, Adam R. Karpf. FOXM1 INDUCED REPLICATION STRESS IS MITIGATED BY ITS BIDIRECTIONAL GENE PARTNER, RHNO1, IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-044.

Abstract NTOC-081: PHARMACOLOGIC ANALYSIS OF HIGH–GRADE SEROUS OVARIAN CANCER TCGA DATASET IDENTIFIES A NOVEL CHEMO–ADJUVANT ROLE FOR NON–STEROIDAL ANTI–INFLAMMATORY DRUGS (NSAIDS)

Abstract Computation-based drug repositioning approaches that automatically search vast amounts of genomic-chemical-phenotypic data for tens of thousands of drugs and diseases can greatly speed up the traditional drug-discovery process. To date, systematic and comprehensive computation-based approaches to identify and validate drug repositioning candidates for High-grade serous ovarian cancer (HGSOC) have not been undertaken. Using a novel computational drug-repositioning platform (Drug-Predict) with HGSOC gene expression dataset from The Cancer Genome Atlas (TCGA) as the input, we have uncovered that Non-steroidal anti-inflammatory drugs (NSAIDs) could be potential candidates for Drug repositioning in HGSOC. Given that numerous epidemiological studies have shown that regular intake of NSAIDs in women is associated with decreased incidence of ovarian cancer, we assessed whether NSAIDs could have chemo-adjuvant applications in HGSOC and have identified Indomethacin as a novel chemo-adjuvant in HGSOC. Indomethacin decreased survival of primary HGSOC tumor cells and interestingly, cisplatin resistant ovarian tumor cells (derived from primary HGSOC PDX models) exhibited significantly higher cell death upon Indomethacin treatment suggesting that Indomethacin could exert chemo-adjuvant effects in HGSOC. Accordingly, Indomethacin treatment induced chemo-sensitivity in cisplatin resistant HGSOC tumor cells and combo treatment with Indomethacin and cisplatin exerted synergistic cell death as compared to individual drugs alone. Furthermore, Indomethacin decreased stem-like properties and induced chemo-sensitivity in ALDHhigh cisplatin-resistant tumor-initiating cells (TICs) thus suggesting that the chemo-adjuvant effect of the drug is mediated by decrease in TIC properties in HGSOC cells. Mechanistically, we found that Indomethacin inhibits Wnt/β-catenin signaling by degrading β-catenin and β-catenin modulation inversely affected Indomethacin functioning in HGSOC. Our study is the first report describing functional effects of an NSAID in patient-derived HGSOC models and demonstrates that combining novel computational predictions with experimental validation has potential in identifying viable drug candidates and moving them into patient trials efficiently and cost-effectively. Since NSAIDs are in routine clinical use in gynecological settings and have acceptable safety profile, this discovery provides with a potentially rapid and cost-effective translational opportunity for testing NSAIDs as chemo-adjuvants in patient trials in HGSOC. Citation Format: Anil Belur Nagaraj, QuanQiu Wang, Olga Kovalenko, Peronne Joseph, Yang Chen, Rong Xu and Analisa DiFeo. PHARMACOLOGIC ANALYSIS OF HIGH–GRADE SEROUS OVARIAN CANCER TCGA DATASET IDENTIFIES A NOVEL CHEMO–ADJUVANT ROLE FOR NON–STEROIDAL ANTI–INFLAMMATORY DRUGS (NSAIDS) [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-081.

Abstract NTOC-119: VCP INHIBITORS INDUCE ENDOPLASMIC RETICULUM (ER) STRESS AND SYNERGISTICALLY KILL OVARIAN CANCER CELLS IN COMBINATION WITH SALUBRINAL OR MIFEPRISTONE

Abstract Valosin-containing protein (VCP) or p97, a member of AAA-ATPase protein family, has been linked to several cellular functions including endoplasmic reticulum associated degradation (ERAD), cell division, golgi-membrane reassembly and autophagy. Recent studies have indicated VCP as one of the vulnerabilities in ovarian cancer. Here, we show that treatment with CB-5083, an orally bioavailable compound derived from previously identified quinazoline scaffold-based VCP inhibitor ML240, contributes to cytotoxicity in high-grade serous or clear cell ovarian cancer cell lines. CB-5083 treatment results in increased unfolded protein response (UPR), accumulation of cell cycle proteins as a result of inhibition of proteosomal degradation, cell cycle arrest at G1, and subsequent cell death mediated by both intrinsic and extrinsic apoptotic pathways. These results support an emerging concept that ER stress pathway can be therapeutically targeted in ovarian cancer. Since CB-5083 induces CHOP-dependent apoptosis, we tested the extent to which VCP inhibitors act synergistically with salubrinal, a compound that inhibits GADD34 and enhances eIF2α phosphorylation. Results from Sulforadamine B and clonogenic assays indicate that VCP inhibitors and salubrinal act synergistically across various concentrations of drug combination. Based on recent studies indicating that mifepristone, a steroidal antagonist to progesterone and glucocorticoid, induce ER stress and subsequent cell death, we also tested the potential drug synergies between VCP inhibitors and mifepristone. Our results indicate these drugs act synergistically across various concentrations in ovarian cancer cells. Analysis of TCGA RNA-sequencing datasets indicate VCP expression is down-regulated in cisplatin-resistant tumor samples compared to cisplatin-sensitive cancer. This data is consistent with the fact that tumors with lower expression of VCP shows significant association with poor progression-free survival and overall survivor. Finally, our results indicate that cells with lower expression of VCP are more sensitive to VCP inhibitors than those with higher expression. Considering that cisplatin-resistant tumor samples have lower expression of VCP, VCP inhibitors may be a good therapeutic candidate to treat cisplatin-resistant ovarian cancer. Collectively, our results provide evidence that VCP inhibitors can be used as a single agent and can be synergized with compounds that prolong or induce ER stress in ovarian cancer. Citation Format: Prabhakar Bastola, Frank J. Schoenen, Jeremy Chien. VCP INHIBITORS INDUCE ENDOPLASMIC RETICULUM (ER) STRESS AND SYNERGISTICALLY KILL OVARIAN CANCER CELLS IN COMBINATION WITH SALUBRINAL OR MIFEPRISTONE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-119.

Abstract MIP-055: IDENTIFICATION OF A MOLECULAR SUBTYPE OF HIGH GRADE SEROUS OVARIAN CANCER REPRESENTING MAPK PATHWAY ACTIVATION AND PLATINUM RESISTANCE

Abstract BACKGROUND: We previously defined 3 molecular subgroups of High Grade Serous Ovarian Cancer (HGSOC), using gene expression data from 265 FFPE samples obtained from treatment naive patients, who received platinum based treatment following surgical resection. The 3 molecular subgroups were Angio: characterised by upregulation of angiogenesis genes; Immune: characterised by upregulation of immune genes and AngioImmune: characterised by upregulation of angiogenesis and immune genes. Patients within these 3 subgroups respond differently to standard of care treatment The Immune subgroup have the best prognosis and the Angio and AngioImmune subgroups have similar worse prognosis. A weighted gene signature to identify each of the molecular subgroups was developed. This dataset was used as a reference to investigate the effect of chemotherapy on molecular subgroup designation. METHODS: To investigate the effect of chemotherapy on predefined molecular subgroups, we analysed 35 matched pre- and post- chemotherapy samples by gene expression. The molecular subgroup assignment for each of the paired samples was determined using the gene expression signatures for each subgroup. Novel cisplatin resistant HGSOC cell lines were generated to study the mechanisms of acquired cisplatin resistance. RESULTS: 40% of the treatment naive samples that were aligned with the AngioImmune subgroup and this increased to 67.5% post-chemotherapy. 10/15 (67%) treatment naïve tumours that were initially assigned to the good prognostic Immune molecular subgroup shifted to the bad prognostic AngioImmune molecular subgroup post chemotherapy. Hence platinum chemotherapy selects for the AngioImmune subgroup, suggesting that this subgroup represents tumours which are innately platinum resistant but also provides a mechanism of acquired resistance. Additionally we demonstrate that the AngioImmune subgroup is driven by activation of the MAPK pathway and shows that cisplatin resistant HGSOC cell lines are specifically sensitive to MEK inhibitors. CONCLUSIONS: The MAPK pathway is a mechanism of innate and acquired platinum resistance in HGSOC. Furthermore the data suggests that original pre-treatment surgical/biopsy samples may fall within a different molecular subgroup to samples taken post-platinum therapy. Citation Format: Aya El-Helali, Nuala McCabe, Charlie Gourley, Andrena McCavigan, Caroline O. Michie, Bethanie Price, Niamh McGivern, Michael Churchman, Aya El-Helai, Eamonn J. O'Brien, Laura Hill, Timothy S Davison, Alistair Williams, W Glenn McCluggage, Katherine E Keating, Denis P Harkin, and Richard Kennedy. IDENTIFICATION OF A MOLECULAR SUBTYPE OF HIGH GRADE SEROUS OVARIAN CANCER REPRESENTING MAPK PATHWAY ACTIVATION AND PLATINUM RESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-055.

Uncovering the role of nuclear Lysyl oxidase (LOX) in advanced high grade serous ovarian cancer

ObjectiveLysyl oxidase (LOX) is an enzyme that catalyzes the cross-linking of collagen and elastin in the extracellular matrix, thus controlling the tensile strength of tissues. Along with this primary function, there are evidences supporting a role for LOX in many critical biological functions, including gene expression regulation, cell growth, adhesion and migration. Accordingly, recent studies have supported a pivotal role for LOX in cancer progression and metastasis. The current study aimed at investigating the prognostic significance and the functional role of intracellular LOX in ovarian cancer. MethodsTo this end, we analyzed LOX expression by immunohistochemistry in archived tumor material from advanced high grade serous ovarian cancer (HGSOC) patients (n=70) and correlated data with clinicopathological parameters and with response to chemotherapy. In vitro experiments were also used to investigate the functional consequences of LOX expression on behavioral aspects of HGSOC cells. ResultsOur results showed that nuclear LOX expression is associated with unfavorable outcome in advanced HGSOC, being an independent prognostic factor for disease recurrence. Besides, high nuclear levels were seen to be associated with resistance to first-line chemotherapy. Through gene expression modulation experiments in HGSOC cell lines, we demonstrate that LOX positively regulates cell proliferation, migration and anchorage-independent growth. ConclusionsCollectively, our data suggest that LOX functions as a tumor promoter in HGSOC and positively regulates several aspects of the metastatic cascade.

Hyper-phosphorylation of Sequestosome-1 Distinguishes Resistance to Cisplatin in Patient Derived High Grade Serous Ovarian Cancer Cells.

Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by Western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with Western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper-phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down-regulation of apoptosis.

Abstract A24: A genome-wide RNAi screen identifies synthetic lethality of CX-5461 with homologous recombination repair deficiency in ovarian cancer

Abstract Cancer is characterized by deregulated cell growth and proliferation, both of which are associated with hyperactivation of ribosome biogenesis. Inhibition of ribosome biogenesis using CX-5461, a specific inhibitor of RNA polymerase I-dependent transcription, has shown therapeutic efficacy in a MYC driven B-cell lymphoma mouse model, which is enhanced when used in combination with the mTORC1 inhibitor Everolimus. However, the therapeutic potential of CX-5461 in solid cancers is yet to be determined. Our preliminary data utilizing a panel of 36 ovarian cancer (OVCA) cell lines suggest that acute CX-5461 treatment results in cell cycle arrest and does not induce apoptosis. We hypothesize that the identification of genes that can be targeted to cooperate with CX-5461 will define novel drug combinations for the improved treatment of OVCA. Therefore, we performed a genome-wide RNAi screen to identify synthetic lethal genes with CX-5461 in the high-grade serous ovarian cancer (HGSOC) cell line OVCAR4. Pathway enrichment analysis of the candidate hits showed significant enrichment in the homologous recombination DNA repair (HR) pathway. Synergy with CX-5461 was validated in multiple HGSOC cell lines by both genetic and pharmacological inhibition of HR pathway components. We are currently investigating the mechanism of this synergy and will further assess efficacy in vivo. As HR deficiency is observed in 20% of OVCA patients, we suggest that future application of our studies will lead to new therapeutic options to improve the survival of this cohort of patients. Citation Format: Shunfei Yan, Keefe T. Chan, Kaylene J. Simpson, Elaine Sanij, Karen E. Sheppard, Katherine M. Hannan, Ross D. Hannan, Richard B. Pearson. A genome-wide RNAi screen identifies synthetic lethality of CX-5461 with homologous recombination repair deficiency in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A24.

Selective Targeting of Cyclin E1-Amplified High-Grade Serous Ovarian Cancer by Cyclin-Dependent Kinase 2 and AKT Inhibition.

Purpose: Cyclin E1 (CCNE1) amplification is associated with primary treatment resistance and poor outcome in high-grade serous ovarian cancer (HGSC). Here, we explore approaches to target CCNE1-amplified cancers and potential strategies to overcome resistance to targeted agents.Experimental Design: To examine dependency on CDK2 in CCNE1-amplified HGSC, we utilized siRNA and conditional shRNA gene suppression, and chemical inhibition using dinaciclib, a small-molecule CDK2 inhibitor. High-throughput compound screening was used to identify selective synergistic drug combinations, as well as combinations that may overcome drug resistance. An observed relationship between CCNE1 and the AKT pathway was further explored in genomic data from primary tumors, and functional studies in fallopian tube secretory cells.Results: We validate CDK2 as a therapeutic target by demonstrating selective sensitivity to gene suppression. However, we found that dinaciclib did not trigger amplicon-dependent sensitivity in a panel of HGSC cell lines. A high-throughput compound screen identified synergistic combinations in CCNE1-amplified HGSC, including dinaciclib and AKT inhibitors. Analysis of genomic data from TCGA demonstrated coamplification of CCNE1 and AKT2 Overexpression of Cyclin E1 and AKT isoforms, in addition to mutant TP53, imparted malignant characteristics in untransformed fallopian tube secretory cells, the dominant site of origin of HGSC.Conclusions: These findings suggest a specific dependency of CCNE1-amplified tumors for AKT activity, and point to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1-amplified HGSC. Clin Cancer Res; 23(7); 1862-74. ©2016 AACR.

OCT4 controls mitotic stability and inactivates the RB tumor suppressor pathway to enhance ovarian cancer aggressiveness.

OCT4 (Octamer-binding transcription factor 4) is essential for embryonic stem cell self-renewal. Here we show that OCT4 increases the aggressiveness of high-grade serous ovarian cancer (HG-SOC) by inactivating the Retinoblastoma tumor suppressor pathway and enhancing mitotic stability in cancer cells. OCT4 drives the expression of Nuclear Inhibitor of Protein Phosphatase type 1 (NIPP1) and Cyclin F (CCNF) that together inhibit Protein Phosphatase 1 (PP1). This results in pRB hyper-phosphorylation, accelerated cell proliferation and increased in vitro tumorigenicity of ovarian cancer cells. In parallel, OCT4 and NIPP1/CCNF drive the expression of the central Chromosomal Passenger Complex (CPC) components, Borealin, Survivin and the mitotic kinase Aurora B, promoting the clustering of supernumerary centrosomes to increase mitotic stability. Loss of OCT4 or NIPP1/CCNF results in severe mitotic defects, multipolar spindles and supernumerary centrosomes, finally leading to the induction of apoptosis. These phenotypes were recapitulated in different cancer models indicating general relevance for human cancer. Importantly, activation of these parallel pathways leads to dramatically reduced overall survival of HG-SOC patients. Altogether, our data highlights an unprecedented role for OCT4 as central regulator of mitotic fidelity and RB tumor suppressor pathway activity. Disrupting this pathway represents a promising strategy to target an aggressive subpopulation of HG-SOC cells.