Abstract B04: The bromodomain inhibitor INCB054329 enhances olaparib response in ovarian cancer cells by reducing homologous recombination efficiency
Authors: Wilson Andrew J. , Stubbs Matthew , Liu Phillip , Ruggeri Bruce , Khabele Dineo 
Published: August 2018

Abstract
Background: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair is a significant clinical problem. HR-proficient HGSOC tumors have poorer clinical outcomes and show relative resistance to DNA-damaging poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA-damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional downregulation of HR pathway components. An emerging class of epigenetic mediators of protumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical antitumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA-damaging agents.
Objective: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi.
Methods: Cultured HR-proficient ovarian cancer cell lines, OVCAR-3, OVCAR-4, and SKOV-3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi INCB054329 (Incyte Corporation), or the INCB054329/olaparib combination for 24-72 hours. Drug effects on HR repair efficiency were quantified by IF assays measuring formation of RAD51 foci after cisplatin-induced DNA damage, and GFP expression in cells co-transfected with I-Sce1 endonuclease and the DRGFP reporter plasmid. Sulforhodamine B (SRB) assays assessed cell growth. High-content immunofluorescence (IF) assays measured drug effects on adherent cell number, apoptosis (cleaved caspase-3), DNA damage (pH2AX), and cell cycle indices. Protein levels of the key HR component BRCA1, pH2AX and cleaved PARP were assessed by Western blot. Nude mice injected subcutaneously with SKOV-3 cells were treated by oral gavage with vehicle, INCB054329, olaparib, and combined INCB054329/olaparib for 19 days. Tumor volume was measured by serial caliper measurements. Harvested tumors were assayed for expression levels of HR components, and markers of proliferation, apoptosis, and DNA damage by Western blot and immunohistochemistry. Statistical significance (p&lt;0.05) was determined by Student’s t test.
Results: INCB054329 significantly reduced HR efficiency alone and in combination with olaparib in cultured cells. Consistent with reduced HR repair, the INCB054329/olaparib combination reduced levels of BRCA1 and increased levels of pH2AX, cleaved caspase-3, and cleaved PARP, compared to either drug alone. Moreover, the combination of INCB054329 and olaparib synergistically reduced cell growth following isobologram analyses of SRB experiments. Consistent with these results, cell cycle analyses demonstrated that the drug combination cooperatively reduced the number of adherent cells and cells in S phase, and induced a G0/G1 arrest. Similar drug effects were observed in vivo. Combined INCB054329/olaparib treatment inhibited tumor volume at an earlier time point than either drug alone (day 14 of treatment), and significantly reduced both tumor volume and weight at sacrifice compared to single-drug treatment. In tumors treated with the INCB054329/olaparib combination, we confirmed cooperative downregulation of BRCA1 and the proliferation marker PCNA, and cooperative upregulation of the apoptosis markers cleaved PARP and Bax, the cell cycle inhibitor p21, and pH2AX.
Conclusions: Our results suggest that BRDi reduce efficiency of HR repair in HR-proficient ovarian cancer cells, sensitizing them to PARPi therapy. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. This has the potential to benefit a substantial number of women with this devastating disease.
Citation Format: Andrew J. Wilson, Matthew Stubbs, Phillip Liu, Bruce Ruggeri, Dineo Khabele. The bromodomain inhibitor INCB054329 enhances olaparib response in ovarian cancer cells by reducing homologous recombination efficiency. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B04.