Abstract 264: The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations

Abstract

Abstract Background: Chk1/2 are major cell cycle regulators in p53-deficient tumors, such as HGSOC. Chk1 plays a critical role in DNA repair, facilitating the BRCA2-RAD51 interaction by phosphorylating the BRCA2 C-terminal domain and Thr309-RAD51. Clinical activity with the Chk1/2 inhibitor, LY2606368 mesylate monohydrate (LY), has been observed in solid tumors. We hypothesize that Chk1 inhibition would sensitize HGSOC to PARP inhibition (PARPi) by preventing nuclear RAD51 foci formation, thus impairing DNA repair. We investigated potential synergy of the combination of LY and the PARPi, olaparib (O), in HGSOC cell lines, testing lower concentrations than clinically attained. Materials and Methods: We examined cytotoxicity, DNA damage, and nuclear RAD51 foci formation by LY and/or O using XTT assay, comet assay and immunofluorescence (IF), in 3 HGSOC cell lines: two BRCA1/2 wild type (CAOV3, OV90), and one with BRCA2 mutation (PEO1). We examined a dose range based on clinically achievable concentrations of LY (0.53μM–1.34μM) and O (7.8μM–11.0μM) and calculated IC50 concentrations for cytotoxicity. The combination index (CI) was calculated to evaluate synergism. DNA damage and nuclear RAD51 foci formation were examined. DMSO was used as vehicle control. All experiments were performed in at least three replicates in all cell lines. Results are presented as mean ± SEM. Results: LY alone yielded cytotoxicity in CAOV3, OV90, and PEO1 with IC50 6.34nM, 35.2nM, 12.6nM, respectively. O 5μM monotherapy resulted in 47%, 13%, and 69% cytotoxicity in CAOV3, OV90, and PEO1. LY/O combination showed 51%, 58% and 82% cell injury in CAOV3, OV90 and PEO1. CI values indicated cytotoxicity synergism with the combination. LY/O (5nM/5μM) treatment showed greater DNA damage than O alone in OV90 and PEO1 (p<0.05), and a similar trend was observed in CAOV3 (20.2% ±3.10 vs. 6.71% ±1.08, p = 0.05). The mean percentage of cells with ≥ 5 nuclear RAD51 foci decreased with LY/O (5nM/5μM) combination therapy compared to O alone in CAOV3 (9.03% ±1.85 vs. 36.3% ±7.45, p<0.01) and OV90 (3.66% ±1.84 vs. 22.9% ±3.08, p<0.01), but not in PEO1 (1.26% ± 1.26 vs. 3.73% ±3.73). Ongoing work includes cell cycle analysis and immunoblotting to examine biochemical changes in DNA repair and cell cycle checkpoint pathways. Conclusions: Our preliminary results suggest synergistic activity of the combination of LY and PARPi in HGSOC cell lines using concentrations that are lower than clinically attainable in patients. Citation Format: Yeong-ran Ahn, Takuhei Yokoyama, Minshu Yu, Nicolas Gordon, Elise C. Kohn, Jung-min Lee. The cell cycle checkpoint kinase 1/2 inhibitor, LY2606368 with PARP inhibition results in synergistic cytotoxicity in high-grade serous ovarian cancer (HGSOC) at lower than physiologically administered concentrations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 264.