Inhibition of DNA double strand break repair in cancer cells has been proposed as a new therapeutic approach for potentiation of radiotherapy. M3814 is a potent and selective inhibitor of DNA-PK, a key component of non-homologous end joining repair pathway and is currently under clinical investigation in radiation combination trials. We studied the mechanism of cancer cell response to combined treatment with ionizing radiation and M3814 using cancer cell lines with different p53 status. M3814 blocked the repair of radiation-induced DSBs and suppressed a negative regulatory loop between DNA-PK and ATM causing enhanced phosphorylation and activation of ATM. In the p53 wild-type cells, ATM and its targets, CHK2 and p53, were activated more strongly by the combination treatment than by radiation alone, leading to a complete p53/CHK2-dependent cell cycle block and premature cell senescence. The p53-null or mutant cancer cells were unable to fully arrest the cell cycle and a substantial fraction entered S phase with unrepaired DNA, leading to mutations, chromosomal aberrations, aneuploidy, micronucleation and ultimately cell death, primarily due to mitotic catastrophe. Two isogenic pairs of p53 null/wt cells (A549 and HT-1080) were generated and used to demonstrate the critical role of p53 in the choice of M3814-modified radiation response. Time-lapse imaging of cell death kinetics using a small panel of p53 wild-type and p53 null/mutant cancer lines revealed clear differences in outcome dependent on p53 status. However, standard growth/viability assays failed to show a role of p53 in the response type, due to inability to discriminate between cell cycle arrest and cell killing. Similarly, screening results obtained from large cancer cell panels (>100 lines) did not identify p53 as a marker of response. Our data revealed an important role of p53 in determining the mode of cancer cells' response to radiation in the presence of M3814 and suggest that p53 mutation status should be considered in the design of clinical trials. The failure of standard growth/viability screens with large cancer cell line panels to identify p53 status as a response indicator raises an alert for the utility of these screening platforms currently widely used in cancer drug discovery and development.

Citation Format: Qing Sun, Yige Guo, Xiaohong Liu, Frank Czauderna, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. TP53 status determines the fate of cancer cells exposed to ionizing radiation and DNA-PK inhibitor, M3814 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1845.