Abstract 6051: Opposing function of USP5 in MRE11 endonuclease activity to evade anti-tumor immunity

Abstract Proliferating cancer cells frequently encounter replication stress, which is known to generate abnormal cytosolic DNA and trigger anti-tumor immune responses. Thus, the ability to evade immune surveillance is critical for the development and progression of malignant tumors for which the mechanisms are not fully understood. The MRE11 nuclease, essential for maintaining DNA integrity during cell proliferation, plays important roles in processing stalled replication forks during replication stress. This process produces single-stranded DNA (ssDNA) intermediates which activate the cGAS-STING cascade when released into the cytoplasm to trigger anti-tumor immunity. In this study, we demonstrate that in cancer cells experiencing endogenous replication stress induced by the loss of phosphorylation at tyrosine 211 of the proliferative protein PCNA (pY211-PCNA), MRE11 is modified by site-specific polyubiquitinations. The modifications activate the endonuclease activity of MRE11, favoring cytosolic ssDNA production and subsequent immune response, which sensitizes cells to killing by natural killer (NK) cells. In contrast, in cells expressing pY211-PCNA, the ubiquitin-specific protease USP5 is recruited to replication forks, where it deubiquitinates MRE11, selectively reducing its endonuclease activity, suppressing cytosolic ssDNA generation, and blunting the immune response. Elevated USP5 levels correlate with tumor metastasis and poor prognosis in breast cancer patients. Downregulation of USP5 results in enhanced NK cell infiltration and tumor suppression in immune-competent mouse xenograft models, but not in immune-deficient models. Inhibition of USP5 renders patient-derived tumor organoids susceptible to NK cell-mediated killing. Unbiased screening and biochemical verification identified two FDA-approved drugs as potent USP5 inhibitors. Treatment with these agents phenocopies USP5 downregulation by enhancing cytosolic ssDNA production, increasing sensitivity to NK-mediated killing, and tumor suppression in syngeneic mouse models. Furthermore, these treatments sensitize patient-derived tumor organoids (PDTOs) to cytotoxic killing by NK cells and patient-autologous primary peripheral blood mononuclear cells (PBMCs). This mechanistic insight advances our understanding of tumor progression and the programming of immune responses, potentially leading to novel therapeutic strategies that harness anti-tumor immunity. Citation Format: Chuan-Chun Lee, Wan-Rong Wu, Liang-Chih Liu, Ting-Yi Liao, You-Zhe Lin, Fang-Ying Lin, Yi-Chun Shen, Feng-Chi Chung, Yuan-Liang Wang, Chih-Hao Lu, Wei-Chung Cheng, Wei-Chao Chang, Yi-Chuan Li, Chih-Tung Lin, Chung-Yu Chen, Sheng-Wen Chen, Hsin-An Shih, Steven Lin, I-Wen Chou, Chen-Yuan Lin, Chang-Feng Chiu, Shao-Chun Wang. Opposing function of USP5 in MRE11 endonuclease activity to evade anti-tumor immunity [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 6051.