Abstract 4098: PARP1 and EZH2 independently limit 5'-3' end resection to promote rapid DNA repair after radiation

Abstract Cellular metabolism and epigenetic regulation have overlapping roles in cancer cell responses to radiation, with dynamic, reversible post-translational modifications including poly-ADP-ribosylation, O-GlcNAcylation and methylation emerging as key targets. This study explored crosstalk between poly-ADP-ribose polymerase 1 (PARP1), O-GlcNAc transferase (OGT), and the PRC2 subunit and histone methyltransferase EZH2 in the context of DNA double-strand break (DSB) repair. We focused on 5'-3' end resection, which determines repair pathway choice during S and G2 via guiding DSB repair away from non-homologous end joining (NHEJ) and toward homologous recombination (HR). DSB repair was examined in the human breast cancer cell line MCF7 using neutral comet assay and immunofluorescent detection of γH2AX, 53BP1, and BRCA1 foci. 5'-3' end resection was followed by tracking BrdU, RPA and RAD51 foci. We used CRISPR RNP to form pools of PARP1 and EZH2 knockout (KO) cells. After irradiation, both PARP1 KO and PARP inhibitor veliparib-treated cells displayed persistent DSBs and increased ssDNA, each restricted to S/G2 cells. While increased 5'-3' end resection may result from NHEJ defects, deregulation of resection may divert DSBs away from NHEJ to saturate cellular capacity for HR. Indeed, PARP inhibition enhanced HR repair based on a repair pathway reporter. Increasing protein O-GlcNAcylation in PARP1 KO or veliparib-inhibited cells restored DSB repair and suppressed hyper-resection, suggesting an independent, OGT-regulated determinant of pathway choice. Prior studies had implicated EZH2 and resulting histone H3 K27 trimethylation in promoting NHEJ and as a mediator of O-GlcNAcylation effects. Much like PARP1 KO, irradiated EZH2 KO cells displayed an S/G2 DSB repair defect associated with increased ssDNA. Hyper-resection in EZH2 KO was enhanced by PARP inhibition but modulating O-GlcNAcylation had no effect. Interestingly, along with increased nuclear BrdU foci, irradiated PARP1 KO and EZH2 KO each displayed cytosolic ssDNA foci. We infer that limiting NHEJ repair and/or promoting 5'-3' end resection in S/G2 can overwhelm HR, leading to hyper-resection, cytosolic DNA, and inflammation. Toward overcoming resistance to PARP inhibitors, O-GlcNac metabolism, OGT activity and its downstream mediator EZH2 offer attractive targets for therapeutic intervention. Overall, strategies aimed at deregulating 5'-3' end resection may help focus effects of genotoxic stress on proliferating cells and potentiate combinations with immunotherapies. Citation Format: Elena Efimova, Yue Liu, Sera Averbek, Isabelle Lomeli, SeokGyeong Choi, Sojung Ha, Woo-Young Kim, Stephen J. Kron. PARP1 and EZH2 independently limit 5'-3' end resection to promote rapid DNA repair after radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4098.