Metformin promotes anti-tumor immunity in STK11 mutant NSCLC through AXIN1-dependent upregulation of multiple nucleotide metabolites

Background: Metformin has pleiotropic effects beyond glucose reduction, including tumor inhibition and immune regulation. It enhanced the anti-tumor effects of programmed cell death protein 1 (PD-1) inhibitors in serine/threonine kinase 11 ( STK11) mutant non-small cell lung cancer (NSCLC) through an axis inhibition protein 1 (AXIN1)-dependent manner. However, the alterations of tumor metabolism and metabolites upon metformin administration remain unclear. Methods: We performed untargeted metabolomics using liquid chromatography (LC)- mass spectrometry (MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis. Results: According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, most metabolites were annotated into metabolism, including nucleotide metabolism. Next, the differentially expressed metabolites in H460 (refers to H460 cells), H460_met (refers to metformin-treated H460 cells), and H460_KO_met (refers to metformin-treated Axin1(-/- )H460 cells) were distributed into six clusters based on expression patterns. The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis. We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated. Furthermore, these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes (STING) pathway independently of AXIN1. Conclusion: Relying on AXIN1, metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC, indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.