Abstract LB-398: DNA-PKcs protects irradiated cells from persistent DNA damage signaling and accelerated senescence

Abstract

Abstract The DNA-dependent protein kinase catalytic subunit DNA-PKcs has long been considered as a promising target in cancer, based on its roles in DNA damage response and the potential for inhibitors to enhance sensitivity to genotoxic therapy. Multiple investigational DNA-PK inhibitors remain under evaluation, though several have failed in development and none have reached the clinic. DNA-PKcs has been assigned diverse roles in DNA damage response that overlap with ATM including direct phosphorylation of H2AX to form γH2AX and promoting double-strand break (DSB) repair by conventional non-homologous end-joining (NHEJ). Nonetheless, a recent report implicated DNA-PKcs as a negative regulator of ATM. This may resolve the paradox that while ATM inhibitors block H2AX phosphorylation, prevent ionizing radiation induced foci (IRIF) formation and suppress terminal arrest and onset of cellular senescence, DNA-PK inhibitors induce persistent γH2AX, delay IRIF resolution and promote senescence. Toward better defining DNA-PKcs roles in radiation response, we examined MCF7 mammary carcinoma cells for effects of DNA-PKcs shRNA knockdown or treatment with specific inhibitors on DNA damage response and senescence after irradiation. We confirmed a requirement for DNA-PK in resolution of γH2AX and IRIF by 24 h after irradiation. Rather than return to proliferation, surviving cells with DNA-PK defects developed characteristic features of senescence within five days in culture. To examine if this effect of DNA-PKcs inhibition was mediated by blocking DSB repair, we irradiated MCF7 cell lines expressing shRNAs targeting NHEJ factors Ku70, Ku80, XLF, XRCC4, PAXX, and LIG4 as well as scrambled control in the presence or absence of DNA-PKcs inhibitor NU7441. Strikingly, IRIF resolution at 24 h was similar to control in cell lines lacking NHEJ factors while NU7441 increased IRIF persistence and enhanced senescence in all cell lines. These results suggest that a critical role for DNA-PKcs in radiation response is to attenuate DNA damage signaling and prevent cellular senescence. Treating irradiated DNA-PKcs knockdown cells with the ATM inhibitor KU55933 at 24 h resolved persistent γH2AX and IRIF and blocked senescence. Taken together, our data demonstrate that DNA-PKcs regulates IRIF resolution and cell senescence by targeting ATM functions rather than directly affecting DSB repair. Citation Format: Yue Liu, Elena V. Efimova, Aishwarya Ramamurthy, Stephen J. Kron. DNA-PKcs protects irradiated cells from persistent DNA damage signaling and accelerated senescence [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 LB-398.