Clear Cell Ovarian Cancer Research Articles

Noncanonical IL6 Signaling-Mediated Activation of YAP Regulates Cell Migration and Invasion in Ovarian Clear Cell Cancer.

Ovarian clear cell adenocarcinoma (OCCA) is characterized by a particularly poor response to conventional chemotherapy and a short overall survival time in women with established disease. The development of targeted treatments for OCCA relies on a better understanding of its molecular characteristics. IL6 is strongly expressed in OCCA and may therefore provide a novel therapeutic target. Here we use CRISPR/Cas9 and conditional short hairpin interfering RNA to perform loss-of-function studies in human OCCA cell lines to explore the requirement for IL6 in vitro and in vivo. While reduction of IL6 expression exerted limited effects in vitro, its attenuation significantly impaired tumor growth and neovascularization in vivo. In contrast to typical signaling via STAT3, IL6 in OCCA signaled via a noncanonical pathway involving gp130, Src, and the Hippo pathway protein YAP. A high-throughput combination drug screen identified agents that enhanced cell killing following reduction of IL6 signaling. Intersection of screen hits obtained from two cell lines and orthogonal approaches to attenuation of IL6 yielded AKT and EGFR inhibitors as enhancers of the inhibitory monoclonal IL6 receptor antibody tocilizumab. This study defines for the first time the requirements for, and mechanisms of, signaling by IL6 in human OCCA cell lines and identifies potential combinatory therapeutic approaches. Given the molecular diversity of OCCA, further in vitro and in vivo studies are warranted to determine whether such approaches will overcome the limited efficacy of tocilizumab observed in ovarian cancer to date. SIGNIFICANCE: This study defines the requirements for and mechanisms of noncanonical signaling by IL6 in human ovarian clear cell adenocarcinoma cell lines and identifies combinatory therapeutic approaches to be explored clinically.

Ultrasound, macroscopic and histological features of endometrioid and clear cell ovarian cancer

The objective of [Video 1][1] is to present two cases of epithelial ovarian carcinomas, examined at the Gynecologic Oncology Unit of the Fondazione Policlinico Universitario Agostino Gemelli, IRCCS, in Rome,...

Superparamagnetic Iron Oxide Nanoparticles Induce Ferroptosis of Human Ovarian Cancer Stem Cells by Weakening Cellular Autophagy.

Human ovarian cancer stem cells (HuOCSCs) are the main source of ovarian cancer recurrence, metastasis, and drug resistance. Superparamagnetic iron oxide nanoparticles (SPIONs) are well-known nucleic acid or drug carriers owing to their controllable properties, superior stability, and easy modification. However, whether SPIONs can inhibit the activity of HuOCSCs by inducing ferroptosis remains unclear. In the present study, we isolated CD44+ /CD133+ HuOCSCs from tumours of four patients with clear cell ovarian cancer and added 0.2 mM SPIONs for mixed culture. Transmission electron microscopy showed that SPION-treated HuOCSCs contained multiple high-density electron clouds. Prussian blue staining showed high concentrations of iron ions in the cells. In vitro , SPIONs treatment of HuOCSCs inhibited cell proliferation, migration, and soft agar clone formation, weakened their resistance to multiple chemotherapeutics, and induced cell death. In vivo , SPIONs pretreatment of HuOCSCs significantly reduced their tumour-forming ability and induced angiogenesis in nude mice. Further, SPIONs induced the accumulation of reactive oxygen species in HuOCSCs and induced oxidative stress. qPCR analysis indicated that SPIONs-treated HuOCSCs had reduced expression of tumour stem cell markers (CD117, NANOG, CD133, and SOX2), cell proliferation factors (KI67, CCND), autophagy-related factors (ATG3, ATG5, MAP1ALC3a, MAP1ALC3b, and MAP1ALC3c), and certain negative regulators of ferroptosis, while the mRNA expression levels of cell death-related proteins (BAK1 and BID), and certain positive regulators of ferroptosis were significantly increased. Overall, our findings suggest that SPIONs induce oxidative stress and decrease autophagy activity in ovarian cancer stem cells, activate ferroptosis, and inhibit their proliferation, invasion, drug resistance, and tumorigenic ability.

Enhanced anti-tumor effects of the PD-1 blockade combined with a highly absorptive form of curcumin targeting STAT3.

PD‐1/PD‐L1 immune checkpoint inhibitors are promising cancer immunotherapies however responses are still limited and the development of more effective combination immunotherapy is needed. We previously reported that STAT3 activation in cancer cells and immune cells was involved in immune‐resistant mechanisms. In this study, we evaluated the effect of highly absorptive forms of curcumin extracts and synthetic curcumin on anti‐tumor T cell responses. The curcumin po administration resulted in the significant augmentation of in vivo induction of tumor antigen‐specific T cells through restoration of dendritic cells (DCs) by inhibiting directly STAT3 in DCs and indirectly via reduced IL‐6 production from STAT3 activated cancer cells in 2 syngeneic MC38 and CT26 murine tumor models. Curcumin also showed direct DC enhancing activity and enhanced T cell induction for the immunized antigens in non‐tumor‐bearing mice and human hosts. Curcumin restored DC functions in xenogeneic nude mouse model implanted with high IL‐6‐producing human clear cell ovarian cancer cells. The combination of curcumin and PD‐1/PD‐L1 Abs demonstrated a synergistic anti‐tumor activity in MC38 murine tumor models. These results indicated that curcumin augments the induction of tumor antigen‐specific T cells by restoring the T cell stimulatory activity of DCs targeting activated STAT3 in both cancer cells and immune cells. Combination immunotherapy with curcumin and PD‐1/PD‐L1 Ab is an attractive strategy in the development of effective immunotherapy against various cancers.

Early-stage clear cell ovarian cancer compared to high-grade histological subtypes: An outcome exploratory analysis in two oncology centers

Objectiveadvanced stage clear cell ovarian cancer (CCOC) carries a higher risk of relapse and death compared to other histological subtypes. The prognosis of early-stage CCOC is controversial. MethodsEarly-stage high-grade OC patients from two Italian oncologic centers were included. Patients with early-stage CCOC were compared with those with high-grade endometrioid (HGE) and serous (HGS) OC in terms of relapse-free interval (RFI), cancer-specific survival (CSS) and post relapse cancer-specific survival (prCSS). The Cox proportional hazard model and the restricted mean survival time were used. ResultsBetween 1981 and 2012, 134 patients with CC, 152 with HGE and 160 with HGS were treated at two referral centers. Median follow-up was 11.5 years. Ten years RFI rates were 80.6%, 72.1%, 60.6%, and CSS rates were 84.3%, 82.6%, 81.7% respectively. Adjuvant chemotherapy significantly improved RFI (aHR 0.61, 95%CI 0.40 to 0.91, P = 0.015). In the multivariable analysis HGS histotype was associated with a shorter RFI compared to CC, (Hazard Ratio [HR]: 1.81; 95%CI: 1.12–2.93; P = 0.016), whereas CSS was not statistically different. prCSS was longer in HGS compared to CCOC (HR, 0.36; 95% CI, 0.17–0.74; P = 0.006).According to the stage, IA/IB/IC1 HGSOC had a shorter RFI (HR, 2.13; 95% CI, 1.14–3.99; P = 0.018) compared to IA/IB/IC1 CCOC, but similar CSS. For prCSS, CC compared to HGS conferred a worse prognosis regardless of the initial stage. ConclusionsEarly-stage CCOC is associated with a longer RFI, similar CSS and a shorter prCSS compared to HGSOC. No prognostic differences were observed between CC and HGE OC. The relapse risk was the lowest in IA/IB/IC1 CC compared to HGS, whereas CC displayed poor sensitivity to chemotherapy after relapse.

Cancer-associated protein Tetraspanin1 increases cell growth through AMPK in atypical endometriosis

Objective: Tetraspanin1 (TSPAN1), a member of transmembrane 4 superfamilies, has been demonstrated to be important in cell invasion and metastasis in various types of cancers. However, the role of TSPAN1 in clear cell ovarian cancer has not been fully elucidated. The aim of the present study was to determine the functions of TSPAN1 in progression of endometriosis to clear cell ovarian cancer.

883TiP MOONSTONE/GOG-3032: A phase II, open-label, single-arm study to evaluate the efficacy and safety of niraparib + dostarlimab in patients with platinum-resistant ovarian cancer

Niraparib, a poly(ADP-ribose) polymerase inhibitor (PARPi), is approved in the US and Europe for maintenance treatment of patients (pts) with recurrent ovarian (OC), fallopian tube, or primary peritoneal cancer following complete or partial response to platinum-based chemotherapy (chemo), and in the US for the treatment of pts with advanced OC who have been treated with ≥3 prior chemo regimens and whose cancer is BRCA mutated, regardless of platinum sensitivity, or associated with homologous recombination deficiency and platinum sensitivity. Pts with recurrent OC often develop platinum-resistant OC (PROC). In the US, approved treatments for PROC include bevacizumab + chemo. However, despite these therapies, improved outcomes for pts with PROC remain an unmet need. PARPi + anti–programmed death (PD)-1 combinations may have a synergistic antitumor effect. The objective of this currently enrolling study is to evaluate the safety and efficacy of niraparib + dostarlimab (an investigative anti–PD-1 humanized monoclonal antibody) in pts with advanced, relapsed, high-grade PROC without a known BRCA mutation that received and progressed on prior bevacizumab. Eligible pts must have recurrent high-grade serous, endometrioid, or clear cell ovarian, fallopian tube, or primary peritoneal cancer, received 1−3 lines of prior therapy (one of which included bevacizumab), and have disease progression <6 months from the last administered platinum-based chemo. Those having received a prior PARPi, anti–PD-1, or anti–programmed death-ligand 1 (PD-L1), or with a known or suspected deleterious BRCA mutation, are ineligible. Approximately 150 pts will be enrolled in the US and receive dostarlimab 500 mg IV Q3W for the first 4 doses and then 1000 mg IV Q6W for up to 3 years or until disease progression or toxicity. Pts with baseline bodyweight <77 kg or platelet count <150,000/μL will receive a 200-mg QD starting dose of niraparib, all other pts will receive a 300-mg QD starting dose. The primary endpoint is investigator-assessed objective response rate per RECIST v1.1 in the overall population and PD-L1+ subsets. NCT03955471. Writing and editorial support, funded by GlaxoSmithKline (Waltham, MA, USA) and coordinated by Hasan Jamal, MSc of GlaxoSmithKline, was provided by Eric Scocchera, PhD and Anne Cooper, MA of Ashfield Healthcare Communications (Middletown, CT, USA). GlaxoSmithKline, Waltham, MA, USA.

Arginine Depletion Therapy with ADI-PEG20 Limits Tumor Growth in Argininosuccinate Synthase-Deficient Ovarian Cancer, Including Small-Cell Carcinoma of the Ovary, Hypercalcemic Type.

Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT. Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo. Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Abstract 4125: Investigating determinants of sensitivity to ATR inhibition, as predictive biomarkers to single agent ATR inhibitor, VE-821, in ovarian cancer

Abstract Introduction: ATR is a key kinase in the DNA damage response (DDR) that signals replication stress to the S and G2/M checkpoints. Loss of G1 checkpoint control is almost ubiquitous in cancer cells, which, coupled with frequent activation of oncogenes makes them more reliant on their S and G2/M checkpoints. Therefore, ATR is an attractive anti-cancer target. Several determinants of sensitivity to ATR inhibitors (ATRi) have been identified in the literature, such as ATM, TP53, cyclin E and ARID1A. In high grade serous ovarian cancer, almost 100% of all tumours have lost TP53 function, around 20% have activation of cyclin E, and ARID1A mutations are found in nearly 50% of all clear cell ovarian cancers. Therefore, ATR inhibition may be a successful treatment strategy in ovarian cancer. Experimental Procedures: ATRi, VE-821, the preclinical candidate of VX-970 now known as M6620, was used throughout these experiments. The inhibition of ATR activity and the cytotoxicity of VE-821 was assessed in 13 ovarian cancer cell lines and related to the protein and mRNA expression of DDR proteins, oncogenes and other known determinants of sensitivity to ATRi. Growth inhibition by VE-821 was determined in patient derived primary cultures of malignant ovarian cancer ascites cells. Results: Ovarian cancer cell lines and patient samples display a range of sensitivities to VE-821. UWB1.289 cells lacking BRCA1 were the most sensitive to VE-821 (LC50 0.64 µM ± 0.06) and 1.5 times more sensitive than its BRCA1 corrected derivative, confirming previous reports of BRCA1 determining sensitivity to ATRi. NIH-OVCAR3 cells were the least sensitive to VE-821 (LC50 17.87 ± 1.86 µM) and CAOV3 were the second most sensitive to VE-821 (LC50 0.78 ± 0.55 µM), which did not correlate with ATR inhibition or expression of DDR proteins, oncogenes or other known determinants of sensitivity. There was a range of sensitivities to VE-821 in the 7 patient samples analysed to date, with the GI50 ranging from 0.78 µM to 29.08 µM, with no correlation between the growth rates of the sample to its sensitivity. Conclusions: These data show that determinants of sensitivity to ATRi, identified in isogenic cell line pairs, may not translate to the more complex molecular pathology of cancer cells where several determinants of sensitivity and resistance may be present simultaneously. More complex pathway analysis or functional readouts e.g. of replication stress, may be needed as predictive biomarkers for ATRi sensitivity. Citation Format: Alice Mary Bradbury, Frank T. Zenke, Yvette Drew, Nicola J. Curtin. Investigating determinants of sensitivity to ATR inhibition, as predictive biomarkers to single agent ATR inhibitor, VE-821, in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4125.

Serum D-dimer, albumin and systemic inflammatory response markers in ovarian clear cell carcinoma and their prognostic implications

BackgroundThis study attempts to evaluate whether preoperative systemic inflammatory response (SIR) markers or other hematological variables, such as albumin, D-dimer, and carbohydrate antigen 125, play roles in predicting chemotherapy response and survival outcome in patients with ovarian clear cell carcinoma (OCCC).MethodsPreoperative leukocyte differential counts, as well as platelet, serum albumin, plasma D-dimer and CA-125 levels, were measured in patients with FIGO IC-IV ovarian clear cell cancer. The correlations of these hematological biomarkers with clinicopathological features, chemotherapy response, and survival outcomes were further analyzed. Survival time was estimated using the Kaplan-Meier model, whereas Cox regression was conducted for multivariate analysis.ResultsAmong the 84 patients, 28.6% were classified as platinum resistant, and 69.0% were platinum sensitive. Preoperative CA125, albumin, and D-dimer levels; neutrophil to lymphocyte ratios (NLR); and monocyte to lymphocyte ratios were significantly correlated with FIGO stage, residual tumor, and platinum response. Platelet to lymphocyte ratio was not related to platinum response (P = 0.060). The median follow-up time was 28 months (range, 1 to 128 months). Preoperative CA125, albumin, and D-dimer levels were significant prognostic factors for overall survival (OS) and progression-free survival (PFS). In the univariate analysis, only NLR exhibited prognostic significance for PFS (P = 0.007). Multivariate analysis indicated that D-dimer > 3.27 (P = 0.001 for OS; P = 0.040 for PFS) and albumin < 39.6 (P = 0.005 for OS and P = 0.041 for PFS) retained significance.ConclusionsPreoperative NLR has some predictive value for platinum resistance in patients with IC-IV stage OCCC but has little predictive effect on prognosis. Elevated D-dimer and reduced albumin might be potential biomarkers for worse response to first-line platinum-based chemotherapy and poor clinical outcomes.

Expanding Our Understanding of Ovarian Cancer Risk: The Role of Incomplete Pregnancies.

Parity is associated with decreased risk of invasive ovarian cancer; however, the relationship between incomplete pregnancies and invasive ovarian cancer risk is unclear. This relationship was examined using 15 case-control studies from the Ovarian Cancer Association Consortium (OCAC). Histotype-specific associations, which have not been examined previously with large sample sizes, were also evaluated. A pooled analysis of 10 470 invasive epithelial ovarian cancer cases and 16 942 controls was conducted. Odds ratios (ORs) and 95% confidence intervals (CIs) for the association between incomplete pregnancies and invasive epithelial ovarian cancer were estimated using logistic regression. All models were conditioned on OCAC study, race and ethnicity, age, and education level and adjusted for number of complete pregnancies, oral contraceptive use, and history of breastfeeding. The same approach was used for histotype-specific analyses. Ever having an incomplete pregnancy was associated with a 16% reduction in ovarian cancer risk (OR = 0.84, 95% CI = 0.79 to 0.89). There was a trend of decreasing risk with increasing number of incomplete pregnancies (2-sided Ptrend < .001). An inverse association was observed for all major histotypes; it was strongest for clear cell ovarian cancer. Incomplete pregnancies are associated with a reduced risk of invasive epithelial ovarian cancer. Pregnancy, including incomplete pregnancy, was associated with a greater reduction in risk of clear cell ovarian cancer, but the result was broadly consistent across histotypes. Future work should focus on understanding the mechanisms underlying this reduced risk.

Patient-derived ovarian cancer organoids capture the genomic profiles of primary tumours applicable for drug sensitivity and resistance testing

The use of primary patient-derived organoids for drug sensitivity and resistance testing could play an important role in precision cancer medicine. We developed expandable ovarian cancer organoids in < 3 weeks; these organoids captured the characteristics of histological cancer subtypes and replicated the mutational landscape of the primary tumours. Seven pairs of organoids (3 high-grade serous, 1 clear cell, 3 endometrioid) and original tumours shared 59.5% (36.1–73.1%) of the variants identified. Copy number variations were also similar among organoids and primary tumours. The organoid that harboured the BRCA1 pathogenic variant (p.L63*) showed a higher sensitivity to PARP inhibitor, olaparib, as well as to platinum drugs compared to the other organoids, whereas an organoid derived from clear cell ovarian cancer was resistant to conventional drugs for ovarian cancer, namely platinum drugs, paclitaxel, and olaparib. The overall success rate of primary organoid culture, including those of various histological subtypes, was 80% (28/35). Our data show that patient-derived organoids are suitable physiological ex vivo cancer models that can be used to screen effective personalised ovarian cancer drugs.

Abstract PR11: Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear-cell ovarian cancers

Abstract Purpose: Clear-cell ovarian cancer (CCOC) is often resistant to standard chemotherapy and has limited treatment options in the advanced or recurrent setting. ARID1A is the most prevalent mutation, with approximately 50% of all CCOC harboring this mutation. ARID1A, a member of the SWI/SNF family, regulates transcription and has a major role in the repair of DNA lesions, directly facilitating DNA accessibility on the chromatin or indirectly by facilitating the functions of DNA repair proteins. We propose that CCOCs unique genomic alterations (e.g., ARID1A frameshift or nonsense mutations) will increase dependency on chromatin remodeling and DNA repair (e.g., ATR/CHK1/WEE1) pathways for survival. We hypothesize that combination of low-dose small-molecule inhibitors of the BET family (BRD4i) and the DNA damage repair pathway (ATR/CHK1/WEE1), will especially target ARID1A mutant cancer cells promoting mitotic catastrophe, apoptosis, and tumor regression, sparing normal cells. Method: We compared the sensitivity of BRD4i + ATRi/WEE1i combinations in ARID1A knockdown and knockout cells compared with isogenic parental lines. The effects on transcription, kinase signaling, cell cycle, apoptosis, and DNA damage were evaluated. RNAseq/ATACseq and Reverse Phase Protein Array (RPPA) were also performed. A CCOC preclinical drug development platform was established and BRD4i combinations were evaluated in ARID1A mutant and ARID1A wild-type (ARID1AMUT and ARID1AWT) PDX models. Results: Low-dose BRD4i in combination with DNA damage repair inhibitors, ATRi/WEE1i (BDR4i-ATRi or BRD4i-WEE1i), were synergistic in decreasing survival and colony formation with an increase in apoptosis in ARID1AMUT cells compared to ARID1AWT. BRD4i combinations caused a robust G1 arrest with a decrease pCDC6 levels along with an increase in DNA double-strand breaks in the ARID1AMUT or knockout cells. Combination BRD4i-ATRi showed significant tumor regression and increased overall survival compared to standard chemotherapy or monotherapy in several ARID1AMUT PDX models but not in an ARID1AWT PDX. Among BRD4i-DDRi combinations, BRD4i-ATRi is superior to BRD4i-WEE1i in terms of antitumor effect and drug tolerability. Conclusion: Our studies identify a novel drug combination targeting a genetic alteration (e.g., ARID1A) common in CCOC that is highly effective and tolerable. BRD4i in combination with ATRi or WEE1i was synergistic, decreasing survival, colony formation with an increase in DNA damage and apoptosis in ARID1AMUT models. Using our novel CCOC drug development PDX platform, we demonstrated that BRD4i-ATRi combination therapy is more effective than standard chemotherapy or monotherapy alone with acceptable toxicity in ARID1AMUTPDXs. This abstract is also being presented as Poster B24. Citation Format: Yasuto Kinose, Hyoung Kim, Haineng Xu, Sergey Medvedev, Erin George, Sarah Gitto, Margaret Whicker, Dorothy Hallberg, Lauren Schwartz, Gordon Mills, Victor E. Velculescu, Tian-Li Wang, Ronny Drapkin, Fiona Simpkins. Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear-cell ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR11.

Abstract A67: Understanding HIF1A-mediated therapy in clear cell ovarian cancer

Abstract Previously, we have used an integrated approach to use genome-scale screening in RNAi and CRISPR-Cas9 to identify several putative cancer dependencies. From these putative dependencies, we discovered EGLN1 was a significant and selective dependency in ovarian cancer and EGLN1 dependency was further enriched in clear-cell ovarian cancers. Genetic knockout and small-molecule inhibition of EGLN1 reduced cell proliferation and increased apoptosis in vitro and in vivo. We found the function of EGLN1 as a cancer dependency is through its canonical role as a negative HIF1A regulator and knockout of HIF1A, but not HIF2A, rescues genetic knockout and small-molecule inhibition of EGLN1. Genetic and small-molecule suppression of VHL, downstream of EGLN1 in the degradation of HIF1A, similarly reduces proliferation and increases apoptosis, and is rescued by HIF1A knockout. Further, we found that forced expression of a mutated HIF1A that blocks EGLN1-dependent hydroxylation reduces proliferation and promotes apoptosis. Using RNA-sequencing, we identified several putative downstream targets of HIF1A and confirmed that HIF1A directly binds and drives transcription of negative MYC regulator MXI1. Further, we found that knockout of MXI1 reduced the effects of EGLN1-mediated suppression, indicating HIF1A-driven MYC suppression as one mechanism underlying EGLN1 dependency. Further understanding of HIF1A-mediated suppression of ovarian cancer progression will be critical to potential small-molecule therapy. Citation Format: Colles Price, Stanley Gill, Zandra V. Ho, Shawn M. Davidson, Erin Merkel, James M. McFarland, Lisa Leung, Andrew Tang, Maria Kost-Alimova, Aviad Tsherniak, Oliver Jonas, Francisca Vazquez, William C. Hahn. Understanding HIF1A-mediated therapy in clear cell ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A67.

The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.

Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention. APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages. One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues. These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

Abstract A73: BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines

Abstract Clear-cell ovarian carcinoma is a rare histologic subtype of epithelial ovarian cancers. Clear-cell ovarian cancers tend to be less responsive to platinum-based chemotherapy compared to high-grade serous ovarian cancers. In a dataset of large-scale CRISPR knockout and RNAi screens of cancer cell lines (depmap.org), a subset of ovarian clear-cell carcinoma cell lines were sensitive to knockout or knockdown of BCL2L1, encoding antiapoptotic protein BCL-XL. Most clear-cell ovarian cancer cell lines also express BCL2L1 mRNA and protein. We therefore tested a panel of ovarian cancer cell lines for response to BH3 mimetic drugs, which inhibit different members of the BCL-2 family of antiapoptotic proteins. Initial data showed that a subset of ovarian clear-cell carcinoma cell lines was sensitive to single-agent BCL-XL inhibition with A1331852 and dual BCL-2/BCL-XL inhibition with ABT-263. In contrast, these cell lines were relatively insensitive to single-agent BCL-2 inhibition or MCL1 inhibition. We are currently applying BH3 profiling, a functional assay for apoptotic priming, to test the response of ovarian clear-cell carcinoma cell lines to BCL-XL inhibition using a BCL-XL specific peptide, HRK. Collectively, these studies suggest that ovarian clear-cell carcinomas may be sensitive to BCL-XL inhibition, either alone or in combination with chemotherapy. Citation Format: Elizabeth H. Stover, Maya K. Gelbard, Ursula A. Matulonis, Matthew Meyerson. BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A73.

Abstract IA25: Personalizing treatment for women with rare epithelial ovarian cancers (EOC): Lessons learned

Abstract Prior to the mid-2000s, all epithelial ovarian cancer subtypes were treated identically worldwide. In 2005, the Gynecologic Oncology Group (GOG) established the Rare Tumor Committee. Over the past almost 15 years, GOG (subsequently merged into NRG Oncology) has been a leader in the development of clinical trials for rare gynecologic tumors, and a number of lessons have been learned. Initially the Rare Tumor Committee was faced with the challenge of determining on which rare gynecologic cancers it should focus. Lesson 1: There is no universal definition of a rare cancer. Ultimately, the group decided to concentrate on trials of rare EOC. Prior to this point, there were essentially no prospective data on these histotypes. Lesson 2: The study of rare EOC was energized by a number of converging factors. Refinement of histopathologic criteria, advances in the molecular biology, and publication of hypothesis-generating observational studies all played a major role in moving the field forward. Lesson 3: Significant challenges and barriers to conducting clinical trials of rare cancers exist and need to be fully understood. These include small numbers of cases, long accrual times, few interested investigators, less attention by the scientific community, low funding priority, low priority by pharma, fewer patient advocates, and lack of standard bioinformatics methods and trial designs. Lesson 4: In designing rare EOC clinical trials, one must balance scientific value with feasibility. Over time and with trial and error, it became obvious that accrual to clinical trials for recurrent clear-cell and low-grade serous ovarian cancers was completely feasible, but the unexpected extreme rarity of mucinous ovarian cancer led to a failed international trial. Lesson 5: Trials for rare EOC in the front-line or salvage setting may have different implications for feasibility. Front-line trials for clear-cell ovarian cancer, both randomized and nonrandomized, have been feasible. Front-line randomized trials for mucinous ovarian cancers do not appear to be feasible. A new clinical trial for front-line treatment of low-grade serous ovarian cancer has recently been activated; feasibility is as yet unknown. Lesson 6: Accurate pathologic diagnosis is essential to quality rare EOC clinical trials. In some instances, prospective digital pathology review was used as an eligibility criterion to optimize efficiency. Lesson 7: Characterization of the molecular signatures of rare EOC and availability of targeted agents significantly facilitated the development of innovative clinical trials. We now have excellent information regarding the common molecular aberrations in clear-cell carcinoma, low-grade serous carcinoma, and mucinous carcinoma. Lesson 8: Strategies for clinical trial design for rare EOC should include consideration of certain principles. These principles include determination of feasibility of phase II versus randomized phase II versus phase III designs; clinical trials that seek large benefits in selected populations with small sample size; tolerating a higher degree of uncertainty; relaxation of type I and type II errors; adaptive and Bayesian trial designs; rigorous use of early stopping rules; use of surrogate endpoints (response, PFS, or DFS, etc.); full exploitation of modern imaging techniques; strong translational research endpoints; and, in absence of known effective standard treatment, use of randomized phase II design to compare two or more experimental treatments. Finally, Lesson 9: Organizational aspects may be critical in the conduct of rare EOC trials. Key ingredients for successful conduct of rare tumor trials may include national or international networks involving centers of excellence; use of cancer registries to identify, screen, and recruit patients; utilization of retrospective or observational studies for hypothesis-generating data; quality tumor repositories; understanding differences in regulatory and funding issues across the world; and regular consultations between regulatory bodies, pharmaceutical companies, patient advocates, and investigators. All of these lessons will hopefully assist future investigators in the development of clinical trials for rare EOC. Citation Format: David M. Gershenson. Personalizing treatment for women with rare epithelial ovarian cancers (EOC): Lessons learned [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA25.

Abstract B67: Analysis of function and inhibition of PGE2 pathway members MRP4 and EP4 in treatment of ovarian cancer

Abstract Ovarian cancer has the highest mortality incidence of all gynecologic malignancies in the United States. The majority of ovarian cancer cases lead to recurrent disease that is often incurable and fatal due to innate or acquired chemoresistance; therefore, novel therapeutic interventions are desperately needed. Cyclooxygenases–COX-1 and COX-2–are enzymes that catalyze the production of prostaglandin E2 (PGE2), an important inflammatory lipid mediator that is functionally linked to progression of many cancers, including breast and ovarian cancer. PGE2 is exported from the cell via multidrug resistance-associated protein 4 (MRP4) where it acts in a paracrine and autocrine manner by activating a family of four G-protein coupled receptors (EP1-4) that are linked to different intracellular signaling pathways. EP2 and EP4 can activate PKA/cAMP, PI3K and ERK pathways. We hypothesize that the EP4 receptor has increased expression in ovarian cancer and that binding of its cognate ligand, PGE2, will drive ovarian cancer progression. We also hypothesize that alternation of the tumor microenvironment via MRP4 will also lead to inhibition of EP4-mediated signaling and affect phenotypes associated with ovarian cancer progression. In order to test this hypothesis, we analyzed the expression of the EP4 and MRP4 in a human ovarian cancer tissue microarray (TMA) as well as human ovarian cancer cell lines. Immunohistochemical analysis of EP4 on the TMA composed of varying histologies, including serous, endometrioid, and clear-cell, as well as normal ovarian tissue, revealed that EP4 was expressed in 38.7% of ovarian cancer tissues, whereas EP4 had no or low expression in 10 normal ovarian tissue samples. Immunohistochemistry of MRP4 also revealed increased expression in ovarian cancer histologies compared to normal ovarian tissue. Serous, endometrioid, and clear-cell subtypes presented with a majority of 4+ and 3+ staining intensities compared to normal ovarian tissue, which presented with mostly 2+ and 1+ staining, and none of the normal ovarian tissue presented with 4+ intensity. EP4 and MRP4 also has increased expression in multiple ovarian cancer cells lines including those representing low-grade serous, clear-cell, and high-grade serous ovarian cancer. Treatment of these cell lines with an EP4 antagonist resulted in decreased proliferation and migration compared to vehicle control. Consistent with the pharmacologic data, treatment of ovarian cancer cell lines with siRNA directed against the EP4 receptor led to decreased proliferation and migration. Inhibition of PGE2 export via MRP4 inhibitor Ceefourin and probenecid results in increased sensitization of ovarian cancer cell lines to treatment with paclitaxel. Based on these data, targeting of the PGE2 EP4 receptor and PGE2 export via MRP4 should be investigated further for the treatment of ovarian cancer. Citation Format: Jocelyn Reader, McMillan Ching, Cong (Ava) Fan, Sulan Wu, Paul Staats, Teklu Legesse, Olga Goloubeva, Ningbo Jian, Mark Carey, Amy Fulton, Dana Roque, Gautam Rao. Analysis of function and inhibition of PGE2 pathway members MRP4 and EP4 in treatment of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B67.

Abstract A47: Preclinical efficacy of CPI-1688, a novel EZH2 inhibitor, in epithelial ovarian cancer with alterations in the SWI/SNF chromatin remodeling complex

Abstract Epithelial ovarian cancer is a paradigm for exploring precision medicine in the context of genetic alteration in the SWI/SNF chromatin remodeling complex. For example, the ARID1A subunit of the SWI/SNF complex is mutated in more than 50% of clear cell ovarian carcinomas. In addition, CARM1 is amplified/overexpressed in ~20% of high-grade serous ovarian cancer. CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 regulates the SWI/SNF genome-wide distribution by post-transcriptionally methylating SWI/SNF core subunit BAF155. EZH2 is the catalytic subunit of the polycomb repressive complex that typically suppresses its gene expression through generating lysine 27 trimethylation on histone H3 (H3K27me3). Our previous studies established that inhibition of the methyltransferase activity of EZH2 is synthetically lethal with ARID1A mutation or CARM1 overexpression due to the antagonistic roles played by the SWI/SNF and EZH2/PRC2 complexes. Here we report the preclinical efficacy of CPI-1688, a highly selective second-generation EZH2 methyltransferase inhibitor, in ARID1A mutated clear-cell ovarian cancer cells and CARM1 overexpressed high-grade serous ovarian cancer cells. Notably, the antitumor effects conferred by CPI-1688 correlate with ARID1A mutation or CARM1 expression status. In addition, at the pharmacodynamic levels, the observed efficacy correlates with a reduction in H3K27me3 both in vitro and in vivo. This suggests that the observed antitumor effects are due to on-target inhibition of EZH2 methyltransferase activity. Finally, this observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes. Together, these results indicate that CPI-1688 is a promising new therapeutic for ovarian cancer with ARID1A mutation or CARM1 overexpression. Citation Format: Elizabeth Magno, Sergey Karakashev, Jennifer Mertz, Patrick Trojer, Rugang Zhang. Preclinical efficacy of CPI-1688, a novel EZH2 inhibitor, in epithelial ovarian cancer with alterations in the SWI/SNF chromatin remodeling complex [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A47.

Pretreatment with methanolic extract of Pistacia lentiscus L. increases sensitivity to DNA damaging drugs in primary high-grade serous ovarian cancer cells

IntroductionOvarian cancer remains the most lethal gynecologic cancer in women. Despite achievements in surgical and systemic therapy, most patients develop platinum resistance. Thus, new strategies to increase or reverse the platinum sensitivity in ovarian cancer patients are urgently needed. In this study, we aimed to investigate the anti-cancer effect of the leaves of Pistacia lentiscus L medicinal plants used in Algerian traditional medicine on ovarian cancer cells in vitro. MethodsFour newly established primary cell lines derived from ascites of patients with high-grade serous and clear cell ovarian cancer were used to test the anti-cancer effect of the leaves of Pistacia lentiscus L. An experimental study was performed to study the therapeutic effects of the plant leaves substances in patient derived models. The anti- proliferative activity of ethanolic, acetonic and methanolic plant leaves extracts was measured by cell viability assays, and the apoptotic effect assessed using flow cytometry analysis. The impact on constitutive active oncogenic pathways and cytokine release in cell culture supernatant were monitored by Western blotting and ELISA, respectively. ResultsSequencing analysis confirmed the presence of mutations in several genes, such as TP53, RB1, PIK3C, which are commonly mutated in high-grade serous ovarian cancer. Obtained results indicated a cytotoxic effect of the methanolic extract of P. lentiscus L (MEPL) on primary cell line cultures, inhibiting PI3K/AKT and MAPK/ERK signaling pathways, and decreasing the release of IL6 and VEGF by the malignant cells. Moreover, treatment with MEPL enhanced the sensitivity to platinum-based chemotherapy in our primary cell lines of patients. ConclusionMethanolic extract of P. lentiscus L might be a promising candidate for novel therapeutic approaches in combination with classic chemotherapy for patients with high-grade serous ovarian cancer.