USP10 inhibition with GL-320 enhances chemo-immunotherapy efficacy and overcomes resistance in NSCLC

Platinum-based chemotherapy combined with immunotherapy remains the frontline regimen for advanced non-small cell lung cancer (NSCLC), yet most patients ultimately relapse due to acquired resistance. A major contributor to this resistance is hyperactivation of the DNA damage response (DDR), which enables tumor cells to repair chemotherapy-induced lesions, evade cytotoxicity, and suppress immunogenic signaling. The deubiquitinating enzyme USP10 stabilizes multiple DDR and pro-survival proteins, making it an attractive target for re-sensitizing resistant tumors. Inhibiting USP10 may attenuate DNA repair, disrupt oncogenic pathways, and enhance anti-tumor immune activation. GL-320, a potent and selective small-molecule USP10 inhibitor developed in our laboratory, was evaluated across NSCLC models using integrated biochemical, cellular, and molecular assays. MTT and colony formation studies demonstrated robust, dose-dependent suppression of proliferation and clonogenic survival, while CellTiter-Glo and combination colony assays showed that GL-320 effectively re-sensitized cisplatin-resistant cells. CETSA confirmed direct intracellular USP10 engagement. Comet assays and immunofluorescence imaging revealed increased DNA fragmentation and elevated gammaH2AX, PARP1, and 53BP1 foci, indicating impaired DNA repair capacity. Flow cytometry demonstrated early G₀/G₁ arrest accompanied by ATM/ATR activation and increased pChk1 and gammaH2AX. Co-treatment with ATM and pan-caspase inhibitors confirmed the involvement of DDR signaling and caspase-dependent apoptosis. Broader mechanistic profiling showed modulation of DDR and checkpoint proteins (MSH2, p21, MDM2, p53), destabilization of survival factors (HDAC6, CD36), and induction of autophagy (p62, Beclin-1, LC3-II). Importantly, GL-320 induced strong immunogenic stress, marked by increased GRP78/BiP, caspase-8 activation, calreticulin exposure, and reduced CD47 expression, hallmarks of endoplasmic reticulum stress and immunogenic cell death. Collectively, GL-320 mediated USP10 inhibition disrupts DDR signaling, activates autophagy and caspase-dependent apoptosis, and triggers immunogenic cell death, thereby restoring cisplatin sensitivity and potentially enhancing response to immunotherapy. These multifaceted effects position USP10 as a promising therapeutic target for overcoming treatment resistance and stimulating anti-tumor immunity in NSCLC.