Weroha, S. John

Abstract About 50% of high-grade serous ovarian cancers (HGSOC) have defects in homologous recombination (HR) DNA repair. Intriguingly, HR-defective tumors are highly susceptible to poly(ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy. Mutations in BRCA1/2 or epigenetic silencing of BRCA1 via promoter hypermethylation contributes to ~30% of the HR defects, but other genetic alterations that cause HR deficiency in HGSOC remain unclear. To identify novel genes that may regulate HR, we studied Zinc Finger CCCH-Type Containing 18 (ZC3H18), encoded by ZC3H18. Notably, ZC3H18 lies within 16q24.2, a region with frequent copy number loss in HGSOC. Here we demonstrate that BRCA1 is transcriptionally activated by a previously unknown function of ZC3H18. We show that ZC3H18 interacts with an E2F site in the BRCA1 promoter where it facilitates recruitment of E2F4 to an adjacent E2F site to promote BRCA1 transcription. Consistent with the role of ZC3H18 in promoting BRCA1 expression, ZC3H18 depletion induces BRCA1 promoter methylation, reduces BRCA1 expression, disrupts HR, and sensitizes ovarian cancer cells to PARP inhibitors and platinum agents. Moreover, in patient-derived xenografts and primary HGSOC tumors, ZC3H18 mRNA levels were positively correlated with BRCA1 mRNA levels, further supporting the role of ZC3H18 in regulating BRCA1. Together, these findings suggest that loss and/or reduced ZC3H18 expression may help identify HGSOC patients most likely to benefit from PARP inhibitor and platinum-based therapies. Summary of the Unpublished Data/Significance/Innovation: These studies are innovative on several fronts. First, although BRCA1 methylation is a frequent event that mediates tumorigenesis and therapeutic responses in HGSOC, how BRCA1 methylation is regulated remains unknown. These studies will be the first to provide a mechanistic basis for how BRCA1 is methylated in HGSOC. Second, our studies are the first to show a role for ZC3H18 in any cancer. Third, we show that ZC3H18 regulates HR and that its depletion sensitizes ovarian cancer cells to PARP inhibitors and platinum therapy. Thus, these studies will provide important basis for developing ZC3H18 loss/decreased abundance as a novel biomarker for ovarian cancers that may respond to PARP inhibitors and platinum agents. Citation Format: Arun Kanakkanthara, Catherine J. Huntoon, Xiaonan Hou, Minzhi Zhang, Ethan P Heinzen, Ann L. Oberg, John S. Weroha, Scott H. Kaufmann, Larry M. Karnitz. Loss of ZC3H18 disrupts homologous recombination repair and sensitizes ovarian cancer cells to PARP inhibitors and DNA cross-linking agents [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 A33.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses—of more than 10-fold lower than previously reported for other cancers—significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Abstract Purpose: 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. Experimental Designs: 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. Results: 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. Conclusions: 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.

Background Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors. Methods Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient‐derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line. Results Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ‐H2AX. Inhibition of autophagy also increased ROS and γ‐H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy. Conclusions PARP inhibitor–induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild‐type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer.

Abstract BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. Significance: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

e17039 Background: Treatment strategies are needed for platinum-resistant recurrent ovarian cancer. Glucocorticoid receptor (GR) is associated with poor prognosis in ovarian cancer. GR signaling inhibits chemotherapy-induced cell death via transcriptional regulation of anti-apoptotic proteins. Non-selective (mifepristone) and selective GR antagonists (Relacorilant, Corcept Therapeutics) are under clinical development (eg. NCT02046421, NCT03776812). We hypothesized that GR antagonism would improve chemotherapy sensitivity in ovarian cancer patient-derived xenograft (PDX) models. Methods: Five PDX models were selected based on GR expression above median RNA seq levels. Tissues were transplanted into 300 female SCID-Bg mice at the Mayo Clinic. Once tumor diameter reached 0.2 cm, mice were randomized into treatment arms (2-25 mice per arm) and treated intraperitoneal (i.p): control (9% saline, CTL) on day 0 (D0), 1, 7, 8, 14, 15, 21, 22; mifepristone (MIF), 15 mg/kg D0, 1, 7, 8, 14, 15, 21, 22; relacorilant (134), 20 mg/kg D0, 1, 7, 8, 14, 15, 21, 22; carboplatin (CBDCA) at the MTD, 51 mg/kg D1, 8, 15, 22; CBDCA + MIF; CBDCA + 134. Tumor area was measured by ultrasound weekly (D0-D28), and the ratio calculated relative to D0. Tumor growth curves were compared between arms for each PDX via a linear mixed effects regression test of coincident curves. Time to progression was also evaluated and will be presented. Results: In 1/5 models (PH160), CBDCA + MIF significantly suppressed tumor growth compared to CBDCA alone (p < 0.05, Table 1). Addition of 134 to CBDCA did not suppress tumor growth compared to CBDCA alone in any model. In the 2 models with no difference of CBDCA versus CTL (PH160, PH454), addition of a GR antagonist to CBDCA suppressed tumor growth versus CTL. Conclusions: Addition of GR antagonists can improve response to chemotherapy. Work is ongoing to identify the GR transcriptional network, tumor, and host characteristics which predict responses to GR antagonists. [Table: see text]

e17038 Background: Carcinosarcomas (CS) are aggressive gynecologic malignancies defined by a biphasic tumor histology of carcinomatous and sarcomatous components. High expression of glucocorticoid receptor (GR) is associated with aggressive tumor biology in triple-negative breast, ovarian, and endometrial cancers. GR activation results in transcriptional regulation of epithelial-mesenchymal transition (EMT) genes. Therefore, we hypothesized that the sarcomatous component of Müllerian CS may be associated with relatively higher GR expression compared to the carcinomatous portion. Methods: Müllerian CSs from the University of Chicago tissue bank diagnosed from 5/19/2009 – 8/25/2014 were identified and pathology confirmed (RL). In addition, we evaluated GR expression in a mouse PDX CS model. Immunohistochemical expression of nuclear estrogen (ER), progesterone (PR), androgen (AR), and GR was quantified using the H-score. Scores between the sarcomatous and carcinomatous components were compared using a paired t-test. Results: Mean GR H-score was relatively higher in the sarcomatous vs carcinomatous component, overall (144.44 vs 38.89, P = 0.0014). Mean H-score also differed between sarcomatous and carcinomatous components for ER (3.06 vs 63.06, P = 0.016), PR (1.67 vs 47.22, P = 0.004), and AR (0 vs 2.78, P = 0.056). The significantly higher sarcomatous GR expression was replicated in IHC evaluation of the mouse PDX model. Conclusions: GR expression is significantly higher in the sarcomatous than the carcinomatous component of Müllerian CS, while ER, PR, and AR expression is significantly lower. Recent data suggest that an EMT gene signature is more prominently expressed in the sarcomatous versus carcinomatous component. We hypothesize that GR expression and activity may mediate EMT gene expression and represent a therapeutic target for CS. [Table: see text]

5510 Background: Single agent aromatase inhibitor (AI) therapy is associated with limited clinical activity in ovarian cancer (OC) and endometrial cancers (EC). AI therapy was associated with a progression free survival (PFS) at 12 weeks of only 20% in relapsed OC (Bowman et al, 2002) and a median PFS of 1 month in relapsed EC (Rose et al, 2000). In Estrogen Receptor (ER) positive metastatic breast cancer, clinical studies had shown a significant prolongation of PFS with the addition of the cyclin kinase 4/6 inhibitor ribociclib to AI (Hortobagyi et al, 2016). Here, we report the results of a phase 2 clinical trial of the combination of ribociclib and letrozole in patients with relapsed ER positive OC or EC. Objectives: Primary endpoint was the proportion of patients with relapsed ER positive OC or EC alive and progression-free after 12 weeks of therapy (PFS12) with the combination of ribociclib given at a dose of 400 mg orally daily and letrozole 2.5 mg orally daily. A PFS of 45% was considered a favorable result based on the data referenced above from Bowman et al. Methods: Eligibility criteria included patients with relapsed ER positive OC or EC, with measurable disease, not previously treated with ribociclib or AIs. Xenografts were created from CT guided tumor biopsies at baseline to assess feasibility. Results: A total of 40 patients were enrolled (20 with OC and 20 with EC) ) with a median age of 61 years (range: 30-82) and 64.5 (range: 52-75) in the OC and EC groups respectively. Among the OC patients, 17 had high grade serous carcinomas and 3 had low grade serous carcinomas. 11 EC patients had endometrioid cancers (3 with grade 1 tumors) and 9 had high grade serous tumors. Ten out of 20 OC patients and 11/20 EC patients were alive and progression-free at 12 weeks (PFS12 of 50 and 55%, respectively). The most common grade 3 or higher adverse events (occurring in at least 5 pts) were leukopenia (18%), lymphopenia (18%), neutropenia (13%), and fatigue (13%). 34 tumor biopsies were suitable for injection into mice and 44% engrafted. ER expression persisted through multiple passages in mice. Two of three EC PDX models exhibited improved PFS with letrozole/ribociclib compared to letrozole alone. Conclusions: The combination of ribociclib and letrozole is associated with a promising 50% and 55% PFS12 in patients with ER positive relapsed OC or EC respectively. Creation of xenograft tumor models from CT guided biopsies of OC and EC tumors was feasible. Clinical trial information: NCT02657928.

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance. Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR. Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway. Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance. Citation Format: Rachel M. Hurley, Ksenija Nesic, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Paula A. Schneider, Xiaonan Hou, Cristina Correia, Karen S. Flatten, Giada V. Zapparoli, Alexander Dobrovic, Kevin K. Lin, Thomas C. Harding, Andrea E. Wahner Hendrickson, Elizabeth M. Swisher, Matthew Wakefield, S. John Weroha, Clare L. Scott, Scott H. Kaufmann. Loss of RAD51C promoter hypermethylation confers PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5885.

Abstract Background: Endometrioid carcinoma (EC) and clear cell carcinoma (CC) histotypes of epithelial ovarian cancer are understudied compared with the more common high-grade serous carcinomas (HGSC). We therefore sought to characterize EC and CC transcriptomes in relation to HGSC. Methods: Following bioinformatics processing and gene abundance normalization, differential expression analysis of RNA sequence data collected on fresh-frozen tumors was completed with nonparametric statistical analysis methods (55 ECs, 19 CCs, 112 HGSCs). Association of gene expression with progression-free survival (PFS) was completed with Cox proportional hazards models. Eight additional multi-histotype expression array datasets (N = 852 patients) were used for replication. Results: In the discovery set, tumors generally clustered together by histotype. Thirty-two protein-coding genes were differentially expressed across histotype (P &lt; 1 × 10−10) and showed similar associations in replication datasets, including MAP2K6, KIAA1324, CDH1, ENTPD5, LAMB1, and DRAM1. Nine genes associated with PFS (P &lt; 0.0001) showed similar associations in replication datasets. In particular, we observed shorter PFS time for CC and EC patients with high gene expression for CCNB2, CORO2A, CSNK1G1, FRMD8, LIN54, LINC00664, PDK1, and PEX6, whereas, the converse was observed for HGSC patients. Conclusions: The results suggest important histotype differences that may aid in the development of treatment options, particularly those for patients with EC or CC. Impact: We present replicated findings on transcriptomic differences and how they relate to clinical outcome for two of the rarer ovarian cancer histotypes of EC and CC, along with comparison with the common histotype of HGSC. Cancer Epidemiol Biomarkers Prev; 27(9); 1101–9. ©2018 AACR.