Abstract 2817: ATR-mediated phosphorylation of RIPK1 inhibits DNA damage-induced necoptosis and chemosensitivity in ovarian cancer

Abstract Background: Cell death under DNA damage induced by chemotherapy is considered a highly significant and effective treatment for epithelial ovarian cancer. Nevertheless, ovarian cancer to date persists as the leading cause of death among all gynecological malignancies, as the majority of patients eventually obtain chemotherapy resistance. chemotherapy-resistant patients, while the underlying mechanism remains unclarified. When tumor cells are undergoing persistent DNA damage, ATR is activated, yet the cell fate mediated by ATR remains unknown. RIPK1 is the crucial regulatory protein for cell death, which is activated by upstream signals and mediates RIPK1-dependent cell apoptosis and necroptosis. However, the mechanism through which RIPK1 regulates cell death in DNA damage still requires further exploration. Methods and Results: Western blot and flow cytometry were employed to detect the cell death induced by etoposide-induced DNA damage, which revealed this mode of cell death was dependent on RIPK1 activation-induced necroptosis. Through Co-immunoprecipitation, it was determined that RIPK1 and ATR had an obvious interaction. Subsequently, by using ATR inhibitors or si-RNA to knockdown ATR, the activation of RIPK1 increased, the necroptosis induced by DNA damage was enhanced, and the downstream Necrosome formed within a shorter period. Furthermore, a kinase-specific phosphorylation site-prediction program, GPS 6.0, and a mass spectrometry analysis were employed to determine that RIPK1 is phosphorylated at Ser335 by ATR. Additionally, the WT and S335A mutant plasmids were complemented in RIPK1 knockout tumor cells, which revealed that in the case of the S335A mutation, the repression of RIPK1 was released, and downstream necroptosis was also augmented. Finally, RIPK1 was knocked out with the complementation of WT and S335A in ovarian cancer cell lines. The results demonstrated that ATR phosphorylation of RIPK1 at S335 led to chemoresistance, while the acquisition of the S335A mutation significantly enhanced chemosensitivity, as indicated by the intense activation of the necroptosis pathway, decreased cell viability, and increased apoptotic cells. Conclusions: Our study reveals a pathway that governs ATR-mediated RIPK1 regulation and RIPK1-dependent cell death under DNA damage, suggesting for the first time that the ATR-RIPK1 axis plays an unrecognized role in integrating DNA damage checkpoints and cell death. Parallel to its other substrates, ATR directly inhibits RIPK1 and downstream necrosome formation by phosphorylation at Ser335, thereby suppressing RIPK1-dependent cell death triggered by DNA damage. This work broadens our comprehension of the interplay between DNA damage and cell death regulation, which could facilitate the development of therapeutic drugs aimed at enhancing DNA damage-induced tumor cell death and promoting chemosensitivity. Citation Format: Qiuyang Xu, Shen-nan Shi, Xiaofei Jiao, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. ATR-mediated phosphorylation of RIPK1 inhibits DNA damage-induced necoptosis and chemosensitivity 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 2817.