PO.ET09.07 · 实验与分子治疗
用GL-320抑制USP10增强NSCLC中化疗-免疫治疗疗效并克服耐药
USP10 inhibition with GL-320 enhances chemo-immunotherapy efficacy and overcomes resistance in NSCLC
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
铂类化疗联合免疫治疗仍是晚期非小细胞肺癌（NSCLC）的一线方案，但大多数患者最终因获得性耐药而复发。这种耐药的一个主要原因是DNA损伤反应（DDR）的过度激活，它使肿瘤细胞能够修复化疗诱导的损伤、逃避细胞毒性并抑制免疫原性信号。去泛素化酶USP10稳定多种DDR和促存活蛋白，使其成为重新致敏耐药肿瘤的有吸引力的靶点。抑制USP10可能减弱DNA修复、破坏致癌通路并增强抗肿瘤免疫激活。GL-320是我们实验室开发的一种强效且选择性的小分子USP10抑制剂，在NSCLC模型中通过整合的生化、细胞和分子检测进行了评估。MTT和集落形成研究显示了强健的、剂量依赖性的增殖和克隆存活抑制，而CellTiter-Glo和联合集落检测显示GL-320有效地使顺铂耐药细胞重新致敏。CETSA证实了细胞内直接的USP10结合。彗星实验和免疫荧光成像揭示了DNA片段化增加和gammaH2AX、PARP1及53BP1焦点升高，表明DNA修复能力受损。流式细胞术显示早期G₀/G₁期阻滞，伴随ATM/ATR激活以及pChk1和gammaH2AX增加。与ATM和泛caspase抑制剂共处理证实了DDR信号和caspase依赖性凋亡的参与。更广泛的机制分析显示DDR和检查点蛋白（MSH2、p21、MDM2、p53）的调节、存活因子（HDAC6、CD36）的失稳以及自噬的诱导（p62、Beclin-1、LC3-II）。重要的是，GL-320诱导强烈的免疫原性应激，表现为GRP78/BiP增加、caspase-8激活、钙网蛋白暴露和CD47表达降低，这些是内质网应激和免疫原性细胞死亡的标志。总之，GL-320介导的USP10抑制破坏DDR信号、激活自噬和caspase依赖性凋亡并触发免疫原性细胞死亡，从而恢复顺铂敏感性并可能增强对免疫治疗的反应。这些多方面的作用使USP10成为克服NSCLC治疗耐药并刺激抗肿瘤免疫的有前景的治疗靶点。
查看英文原文 English abstract
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.
利益披露 Disclosure
S. Dorandish, None..
K. Bhayekar, None..
P. Parajuli, None..
A. Samarbakhsh, None..
B. Kushwaha, None..
Y. Ge, None..
N. Gavande, None.