USP5 suppresses MRE11 endonuclease function to facilitate tumor immune escape

Replication stress is a hallmark of cancer that generates abnormal cytosolic DNA, thereby activating intrinsic immune pathways that constrain tumor growth. To survive, malignant cells must counteract these immune surveillance mechanisms, yet the underlying regulatory processes remain incompletely understood. The MRE11 nuclease plays a dual role in maintaining genome stability and processing stalled replication forks, where its endonuclease activity produces single-stranded DNA (ssDNA) capable of activating the cGAS-STING axis to induce anti-tumor immunity. In this study, we uncover a ubiquitin-dependent mechanism that connects DNA replication stress to immune evasion. Loss of tyrosine 211 phosphorylation on PCNA (pY211-PCNA) induces endogenous replication stress and promotes site-specific polyubiquitination of MRE11. This modification enhances MRE11 endonuclease activity, resulting in cytosolic ssDNA accumulation, activation of innate immune signaling, and increased susceptibility to natural killer (NK) cell-mediated cytotoxicity. Conversely, in cells expressing pY211-PCNA, the deubiquitinase USP5 is recruited to replication forks, where it removes ubiquitin from MRE11, thereby attenuating its endonuclease activity and suppressing cytosolic ssDNA formation and immune activation. Clinically, elevated USP5 expression correlates with tumor metastasis and poor prognosis in breast cancer. Genetic ablation or pharmacologic inhibition of USP5 restores MRE11-dependent ssDNA production, promotes NK cell infiltration, and suppresses tumor growth in immune-competent but not immune-deficient mouse models. An unbiased drug screen identified two FDA-approved compounds as potent USP5 inhibitors, which mimic USP5 depletion by enhancing cytosolic ssDNA accumulation, triggering NK- and PBMC-mediated killing of patient-derived tumor organoids (PDTOs), and suppressing tumor growth in syngeneic mouse models. These results identify USP5 as a critical negative regulator of MRE11-driven immunogenic DNA processing and reveal a therapeutic strategy to potentiate anti-tumor immunity through targeted USP5 inhibition.