Abstract 1375: Investigating synergy between CHK1 and PARP inhibitors in <i>BRCA2</i> mutant and restored cells
Authors: Hannah L. Smith, Lisa Prendergast, Nicola J. Curtin
Journal: Cancer Research (2020)

Abstract Introduction: PARP inhibitors (PARPi) effectively target BRCA mutated cancers by exploiting defects in homologous recombination repair (HRR) by synthetic lethality. PARPi cause replication stress, activating ATR, which signals to HRR and to S and G2/M cell cycle checkpoints via CHK1 and WEE1. Our aim was to determine if a CHK1 inhibitor (CHK1i) would be synthetically lethal with PARPi and to identify the underlying mechanism. Methods: Effects of the PARPi rucaparib and the CHK1i PF-477736 were investigated in V-C8 (BRCA2 mutant Chinese hamster fibroblasts) and V-C8.B2 (BRCA2 restored) cell lines. PARP activity was determined by immunoblot densitometry of the PAR product. CHK1, ATR and WEE1 activity was measured by CHK1S296, CHK1S345 and CDK1y15 phosphorylation, respectively, by Western blot. HRR activity was determined by γH2AX and RAD51 foci formation by immunofluorescence microscopy. Cytotoxicity was determined by colony formation assay and cell cycle analysis by flow cytometry using propidium iodide. Results: V-C8 cells had higher baseline PARP activity than V-C8.B2 cells (1.6 and 0.7 nmol/106 cells, respectively), and in both cell lines PARP activity was suppressed 99% by 1 µM rucaparib. ATR, CHK1 and WEE1 were activated by rucaparib (10 µM) in both V-C8 cells (2.1, 1.5 and 1.8-fold) and V-C8.B2 cells (1.7, 1.4 and 1.5-fold) respectively. Conversely, activation of CHK1 and WEE1 was completely inhibited by 50 nM PF-477736 in both cell lines, while upstream activation of ATR by PF-477736 was almost 2-fold in both cell lines. Rucaparib was 550x more cytotoxic to HRR-defective V-C8 cells compared to HRR-competent V-C8.B2 cells (LC50 &amp;lt;0.1 µM vs 8.8 µM; p&amp;lt;0.001 respectively). Combination treatment with PF-477736 (50 nM) sensitised V-C8.B2 cells to rucaparib cytotoxicity 4.9-fold but did not sensitise V-C8 cells. In V-C8.B2 cells, 10 µM rucaparib caused a 6-fold increase in γH2AX and a 4.8-fold increase in RAD51 foci in γH2AX positive cells, demonstrating both increased replication stress and HRR activity in response to DNA damage. Combination treatment with PF-477736 (50 nM) increased γH2AX foci 2-fold compared to rucaparib single agent and inhibited the rucaparib induced RAD51 foci formation entirely, showing complete inhibition of HRR in V-C8.B2 γH2AX positive cells. Conclusion: The CHK1i PF-477736 sensitised HRR competent, but not HRR defective V-C8 cells to rucaparib suggesting that the primary mechanism was through PF-477736 abrogation of HRR mediated by its direct inhibition of CDK1. Cell cycle analysis is ongoing to determine the impact of rucaparib plus PF-477736 modulation of CHK1 and WEE1 activities on S and G2/M checkpoint control. Citation Format: Hannah L. Smith, Lisa Prendergast, Nicola J. Curtin. Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells [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 1375.