DataSheet1_Metformin Combining PD-1 Inhibitor Enhanced Anti-Tumor Efficacy in STK11 Mutant Lung Cancer Through AXIN-1-Dependent Inhibition of STING Ubiquitination.PDF

Background: Non-small-cell lung cancer (NSCLC) with STK11 mutation showed primary resistance to immune checkpoint inhibitors (ICIs). The glucose-lowering drug metformin exerted anti-cancer effect and enhanced efficacy of chemotherapy in NSCLC with KRAS/STK11 co-mutation, yet it is unknown whether metformin may enhance ICI efficacy in STK11 mutant NSCLC.
Methods: We studied the impact of metformin on ICI efficacy in STK11 mutant NSCLC in vitro and in vivo using colony formation assay, cell viability assay, Ki67 staining, ELISA, CRISPR/Cas9-mediated knockout, and animal experiments.
Results: Through colony formation assay, Ki67 incorporation assay, and CCK-8 assay, we found that metformin significantly enhanced the killing of H460 cells and A549 cells by T cells. In NOD-SCID xenografts, metformin in combination with PD-1 inhibitor pembrolizumab effectively decreased tumor growth and increased infiltration of CD8+ T cells. Metformin enhanced stabilization of STING and activation of its downstream signaling pathway. siRNA-mediated knockdown of STING abolished the effect of metformin on T cell-mediated killing of tumor cells. Next, we found that CRISPR/Cas9-mediated knockout of the scaffold protein AXIN-1 abolished the effect of metformin on T cell-mediated killing and STING stabilization. Immunoprecipitation and confocal macroscopy revealed that metformin enhanced the interaction and colocalization between AXIN-1 and STING. Protein-protein interaction modeling indicated that AXIN-1 may directly bind to STING at its K150 site. Next, we found that metformin decreased K48-linked ubiquitination of STING and inhibited the interaction of E3-ligand RNF5 and STING. Moreover, in AXIN-1−/− H460 cells, metformin failed to alter the interaction of RNF5 and STING.
Conclusion: Metformin combining PD-1 inhibitor enhanced anti-tumor efficacy in STK11 mutant lung cancer through inhibition of RNF5-mediated K48-linked ubiquitination of STING, which was dependent on AXIN-1.

背景：携带STK11突变的非小细胞肺癌（Non-small-cell lung cancer, NSCLC）对免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）存在原发性耐药。降糖药物二甲双胍（metformin）可发挥抗癌作用，并增强KRAS/STK11共突变型非小细胞肺癌的化疗疗效，但目前尚不明确二甲双胍是否可增强STK11突变型非小细胞肺癌对免疫检查点抑制剂的疗效。
方法：我们通过集落形成实验、细胞活力检测、Ki67染色、酶联免疫吸附试验（ELISA）、CRISPR/Cas9介导的基因敲除及动物实验，在体外和体内研究了二甲双胍对STK11突变型非小细胞肺癌中免疫检查点抑制剂疗效的影响。
结果：通过集落形成实验、Ki67掺入实验及CCK-8实验，我们发现二甲双胍可显著增强T细胞对H460细胞与A549细胞的杀伤作用。在NOD-SCID异种移植瘤模型中，二甲双胍联合PD-1抑制剂帕博利珠单抗（pembrolizumab）可有效抑制肿瘤生长，并增加CD8+ T细胞的浸润。二甲双胍可增强STING（干扰素基因刺激蛋白）的稳定性并激活其下游信号通路。小干扰RNA（siRNA）介导的STING敲除可抵消二甲双胍对T细胞介导的肿瘤细胞杀伤的增强作用。进一步研究发现，CRISPR/Cas9介导的支架蛋白AXIN-1（AXIN-1）基因敲除可抵消二甲双胍对T细胞介导的肿瘤杀伤及STING稳定性的增强作用。免疫共沉淀与共聚焦显微镜成像结果显示，二甲双胍可增强AXIN-1与STING之间的相互作用及共定位。蛋白质相互作用建模分析表明，AXIN-1可直接结合STING的K150位点。此外，我们发现二甲双胍可降低STING的K48连接泛素化水平，并抑制E3连接酶RNF5与STING的相互作用。在AXIN-1基因敲除的H460细胞中，二甲双胍无法改变RNF5与STING的相互作用。
结论：二甲双胍联合PD-1抑制剂可通过抑制RNF5介导的STING的K48连接泛素化，增强STK11突变型肺癌的抗肿瘤疗效，且该作用依赖于AXIN-1。