Lysine 2-hydroxyisobutyrylation of HSP90AA1 mediates metformin-driven antitumour immune responses in lung cancer

Immune checkpoint inhibitors (ICIs) are the standard of care for advanced non-small-cell lung cancer (NSCLC) without targetable drivers, but resistance remains a challenge. Although the antidiabetic drug metformin has shown immunomodulatory potential, clinical evidence regarding its survival benefit in patients treated with ICIs is conflicting, and the molecular mechanisms driving potential synergy remain undefined. We conducted a multicentre, retrospective cohort study of patients with advanced NSCLC and type 2 diabetes treated with PD-1 or PD-L1 inhibitors at six tertiary hospitals in China. The primary outcome was durable clinical benefit (progression-free survival of 6 months or longer). Mechanistic studies used two in vivo models, a PBMC-humanized H1299 xenograft and an LLC syngeneic immunocompetent model, with the LLC tumours analysed by single-cell RNA sequencing (scRNA-seq). Lysine 2-hydroxyisobutyrylation (Khib) was quantified, HDAC3 and HSP90AA1 were modulated and quantitative proteomics mapped Khib sites. TBK1-IRF3 signalling and T-cell-mediated cytotoxicity were assessed. In total, 89 eligible patients were analysed. Concomitant metformin use ( n = 54) was independently associated with improved durable clinical benefit [43 (80%) of 54 versus 16 (46%) of 35; p = 0.001], prolonged progression-free survival (PFS; median 41.2 months versus 10.0 months; p = 0.005), and prolonged overall survival (OS; median not reached versus 16.4 months; p < 0.001) compared with non-use ( n = 35). In vivo, metformin augmented PD-1 blockade and reshaped intratumoural T-cell states, as revealed by scRNA-seq. Mechanistically, metformin downregulated tumour-intrinsic histone deacetylase 3 (HDAC3), preventing the enzymatic erasure of Khib. Proteomics identified heat shock protein 90 alpha (HSP90AA1) as the metformin-responsive hub. Metformin-induced Khib at conserved residues K435 and K559 strengthened the HSP90α–TBK1 interaction. K435R and K559R mutation of HSP90AA1 abolished metformin-induced HSP90α upregulation, disrupted HSP90α-TBK1 interaction, impaired TBK1 and IRF3 phosphorylation, and eliminated metformin-enhanced T-cell killing. Concomitant metformin use was associated with longer PFS and OS in patients with NSCLC receiving ICIs. Mechanistically, metformin potentiates anti-PD-1 efficacy via a tumour–intrinsic post-translational axis involving HDAC3 inhibition and the accumulation of Khib on HSP90AA1. These findings provide a rationale for combining metformin with immune checkpoint blockade in NSCLC.