Abstract 621: Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC)

Abstract

Abstract HGSOC is characterized by universal TP53 mutations and consequent G1 cell cycle dysfunction, thus relies on ATR/CHK1-mediated G2/M arrest for survival. CHK1 is also necessary for chromosome stability by mitigating replication stress (RS) and promoting homologous recombination (HR) DNA repair following DNA damage. However, clinical responses to CHK1 inhibitor (CHK1i) monotherapy have been variable in HGSOC, requiring new treatment strategies. We thus conducted a high throughput drug combination screening of a CHK1i, prexasertib (Prex) with 1,912 drugs in HGSOC cell lines (BRCA wild-type [BRCAwt: OVCAR5 and OVCAR8] and BRCA2 mutant [BRCA2m: PEO1]). In this screen, 24 drugs showed synergistic cytotoxicity with Prex, including 14 (58%) chemotherapies, 6 (25%) PI3K pathway inhibitors and 4 (17%) targeted drugs. We prioritized the PI3K pathway inhibitors given the PI3K pathway is activated in >45% of HGSOC and associated with increased cell survival, DNA repair and chemo-resistance. We confirmed the combination of Prex and a dual PI3K/mTOR inhibitor LY3023414 (LY302) yielded synergistic cytotoxicity (combination index<1) in a panel of HGSOC cell lines (OVCAR3, OVCAR5, OVCAR8, OV90 and PEO4 [all BRCA-proficient] and PEO1) by XTT and colony formation assays. OVCAR8 and PEO1 were used for further mechanistic studies. With clinically attainable concentrations of Prex (5 nM) and LY302 (200 nM), Prex+LY302 increased caspase 3/7 activation compared to Prex (increased 2,407% and 39%, respectively; P<0.01) or LY302 (increased 2,457% and 57%, respectively; P<0.001) in both cell lines. Prex+LY302 also enhanced the percentage of cells with ≥5 γH2AX foci relative to Prex (increased 39% and 22%, respectively; P< 0.001) or LY302 (increased 48% and 32%, respectively; P<0.001) in both cells, indicating greater DNA damage. We found Prex+LY302 induced a significant HR deficiency as manifested by decreased DR-GFP reporter activity compared to Prex (decreased 37% and 28%, respectively; P<0.01) or LY302 (decreased 44% and 63%, respectively; P<0.01), suggesting Prex+LY302 causes DNA damage likely via reducing HR functionality. Cell cycle analysis showed an imposed S phase arrest in cells treated with Prex+LY302 compared to those treated with Prex (increased 30% and 35%, respectively; P<0.05) or LY302 (increased 38% and 51%, respectively; P<0.01), implying Prex+LY302 increases RS or DNA damage during S phase. Supporting this notion, Prex+LY302 augmented RS as evidenced by increased phospho-RPA+/γH2AX+ populations compared with Prex (increased 37% and 38%, respectively; P<0.05) or LY302 (increased 80% and 79%, respectively; P<0.001). Overall, our results suggest that dual inhibition of CHK1 and PI3K pathways results in greater RS, DNA damage and subsequent cell death in HGSOC cells independent of BRCA mutation status. Citation Format: Tzu-Ting Huang, Jayakumar Nair, Ethan Brill, Xiaohu Zhang, Kelli Wilson, Lu Chen, Craig J. Thomas, Jung-Min Lee. Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC) [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 621.