LAUSR

DNA double strand breaks (DSB) results in the accumulation of abnormal nucleic fragments in the cytosol, which activates nucleic acid sensors such as cGAS, DHX9, and AIM2, leading to innate immune responses. Intriguingly, these sensors can influence DNA damage repair (DDR) within the nucleus conversely, which might indicate an unexpected feedback loop between these sensors and their functions in both the cytosol and nucleus. ZBP1, is also a classic nucleic acid sensor, which leads to programmed cell death and innate immune responses. Previous studies have demonstrated that ZBP1 plays a significant role in the downstream of DNA damage. However, it remains largely unclear whether ZBP1 exerts upstream feedback effects on DDR. ZBP1 was overexpressed in two human tumor cell lines, and its expression was induced in mouse L929 cell lines by IFN-γ. Subsequently, γ-H2AX foci were detected through immunofluorescence staining after DNA damage induced by etoposide, indicating that DNA damage repair was decelerated in cells expressing ZBP1. Immunohistochemical staining on ovarian cancer (OV) tissues demonstrated that the expression level of ZBP1 was positively correlated with that of γ-H2AX. Additionally, Co-IP assay revealed that ZBP1 bound with γ-H2AX, indicating ZBP1 localized to the DSB site. Then the DR-GFP and EJ5/GFP reporter system showed that ZBP1 inhibited Homologous Recombination (HR), but had no significant effect on Non-homologous End Joining (NHEJ). Subsequently, immunofluorescence and chromatin fractions extraction proved that ZBP1 affected DNA end resection. Co-IP assays verified that ZBP1 affected HR by binding to MRE11 and inhibiting MRE11 recruitment to chromatin. Furthermore, a kinase-specific phosphorylation site-prediction program, GPS 6.0, and western blot assay demonstrated that ZBP1 was phosphorylated at Ser106 by ATM, functioning as the initial signal for ZBP1 to inhibit downstream HR. Truncated mutants of ZBP1 for the binding site with MRE11, as well as a dephosphorylation mutation at Ser106, both attenuated the effect of ZBP1 in HR. In Addition, the results of flow cytometry and CCK8 assay on multiple ovarian cancer discovered ZBP1 overexpression cells displayed an enhanced sensitivity to PARP inhibitors. Moreover, the data from NANT study also indicated OV patients with high expression level of ZBP1 have better outcomes. This study reveals that ZBP1 is binding on the DSB site upon the exogenous DNA damage, where it undergoes phosphorylation at ser106 by ATM and subsequently binds to MRE11 to prevent downstream end resection and inhibit HR, ultimately enhancing the synthetic lethality effect of PARPi. These findings indicates a cellular protective mechanism that prevents excessive repair mediated by ZBP1 and also suggests that PARPi could be an effective treatment strategy for OV with elevated ZBP1 expression. Shi Shennan,Qiuyang Xu,Xuejiao Zhao,Gordon B. Mills,Qinglei Gao,Yong Fang. ZBP1 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in Ovarian cancer [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 2920.