The use of Poly ADP ribose polymerase inhibitors (PARPi) has revolutionized the treatment of homologous recombination (HR) deficient ovarian cancer tumors. A subset of these tumors exhibit genetic and acquired resistance to PARPi treatment. We have previously shown histone deacetylase inhibitors (HDACi) resensitizes ovarian cancer cells to PARPi. The objective of this study is to examine the effectiveness of combination HDACi and PARPi treatment in two in vitro models of mouse ovarian cancer cells. To investigate these effects, we used the following three ID8 murine ovarian cancer epithelial cell lines: TP53− / − (HR deficient), TP53− / −/BRCA2− / − (HR proficient), and an olaparib resistant line TP53− / −/BRCA2− / −-OR (ID8_OR). We also created ovarian cancer organoids from these three lines. In short, 8-week old female C57BL/6 mice were injected with 7 million untreated cells in PBS. Following the formation of ascites, mice were sacrificed and organoids were derived from both ascites and tumors. For viability assays, cells were pretreated with entinostat (Ent, 0.25uM) for 24h, followed by 72h (2D cells) or 7 days (organoids) of Ent (0-2uM), olaparib (Ola, 0-40uM), or in combination. Cell proliferation and viability was assessed using MTS and ATP-based assays. For immunofluorescence and western blotting analysis, cells were treated with 0.5uM Ent, 10uM Ola or the combination.TP53 adherent cells and organoids treated with 0.5uM Ent + 10uM of Ola significantly reduced cell proliferation when compared to Ola alone (p= 0.0028, 0.0021 Two-Way ANOVA). When compared to Ola and Ent alone, TP53/BRCA2 adherent cells treated with 0.125uM of Ent + 2.5uM of Ola also reduced cell proliferation (p=0.0011 and p=0.0072, 0.0845). Lastly, ID8_OR adherent cells and organoids treated with 0.125uM of Ent + 2.5μM of Ola significantly reduced cell proliferation when compared to Ola alone (p=0.0018, 0.0264).Immunofluorescences analysis revealed, when treated with Ola and Ent combination, adherent TP53 (p= 0.002, n.s), TP53/BRCA2 (p=0.0033, 0.0315), and ID8_OR (n.s) cells displayed increased expression of DNA damage marker gH2AX when compared to Control and Ola alone. We also observed decreased expression of RAD51 in cells treated with combination when compared to control and Ola alone, TP53 (p= <0.0001, 0.0023) and ID8_OR (n.s). In our organoids, we found an increase in gH2AX across all three cell lines when treated with Ola+Ent compared to control and Ola alone: TP53 (p= <0.0001, 0.0057), TP53/BRCA2 (n.s), and ID8_OR (p= 0.0116, 0.0151). Similarly, we found a decrease in RAD51 when cells were treated with Ola+Ent compared to control and Ola alone: TP53 (p= n.s, 0.0418) and ID8_OR (n.s). Lastly, preliminary western blot analysis of the organoids, revealed a decreases in proliferation marker PCNA in both adherent cells and organoids when comparing the control to combination treatment. In conclusion, the combination of Ola and Ent treatment restores responsiveness of two in vitro models of genetic and acquired PARPi resistance through increasing DNA damage and reducing DNA repair and proliferation. Citation Format: Bisiayo E. Fashemi, Lilian N. van Biljon1, Tyler Woodard, Vijayalaxmi Gupta, Mary M. Mullen, Benjamin Bitler, Dineo Khabele. Entinostat restores sensitivity to olaparib in two in vitro models of PARPi resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB164.

Highlights Vaginal masses after hysterectomy for benign indications should be investigated. Opportunistic salpingectomies may decrease incidence of epithelial ovarian cancers. Consider regular pelvic exams for women with asymptomatic prolapsed fallopian tubes.

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Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p< 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 795.