AUTHOR=Wang Zhiguo , Lu Conghua , Zhang Kejun , Lin Caiyu , Wu Fang , Tang Xiaolin , Wu Di , Dou Yuanyao , Han Rui , Wang Yubo , Hou Chao , Ouyang Qin , Feng Mingxia , He Yong , Li Li 

TITLE=Metformin Combining PD-1 Inhibitor Enhanced Anti-Tumor Efficacy in STK11 Mutant Lung Cancer Through AXIN-1-Dependent Inhibition of STING Ubiquitination

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2022.780200

DOI=10.3389/fmolb.2022.780200

ISSN=2296-889X

ABSTRACT=<p><bold>Background:</bold> Non-small-cell lung cancer (NSCLC) with <italic>STK11</italic> 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 <italic>KRAS/STK11</italic> co-mutation, yet it is unknown whether metformin may enhance ICI efficacy in <italic>STK11</italic> mutant NSCLC.</p><p><bold>Methods:</bold> We studied the impact of metformin on ICI efficacy in <italic>STK11</italic> mutant NSCLC <italic>in vitro</italic> and <italic>in vivo</italic> using colony formation assay, cell viability assay, Ki67 staining, ELISA, CRISPR/Cas9-mediated knockout, and animal experiments.</p><p><bold>Results:</bold> 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<sup>+</sup> T cells. Metformin enhanced stabilization of STING and activation of its downstream signaling pathway. siRNA-mediated knockdown of <italic>STING</italic> 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 <italic>AXIN-1</italic><sup><italic>−/−</italic></sup> H460 cells, metformin failed to alter the interaction of RNF5 and STING.</p><p><bold>Conclusion:</bold> Metformin combining PD-1 inhibitor enhanced anti-tumor efficacy in <italic>STK11</italic> mutant lung cancer through inhibition of RNF5-mediated K48-linked ubiquitination of STING, which was dependent on AXIN-1.</p>