Acetylation-stabilized METTL3 drives anti-PD-1 resistance in lung cancer by promoting CD8+ T cell exhaustion via SPP1 translation

Anti-programmed cell death protein 1 (anti-PD-1) therapy is a standard treatment for advanced lung cancer, but its efficacy is often limited by resistance, driven largely by an immunosuppressive tumor microenvironment. Methyltransferase-like 3 (METTL3), a core N6-methyladenosine (m 6 A) writer, plays a key role in immune regulation and has emerged as a critical modulator of the immunosuppressive tumor niche. Beyond its canonical m 6 A-catalytic function, METTL3 also exhibits non-catalytic activities and is itself regulated by acetylation. However, its non-catalytic and acetylation-dependent roles in anti-PD-1 resistance remain to be elucidated. METTL3 expression in anti-PD-1-resistant lung cancer cells was assessed by immunohistochemistry and Western blotting. Its in vivo function was evaluated using subcutaneous and orthotopic tumor models under anti-PD-1 treatment. CD8 + T cell exhaustion was examined by single-cell RNA sequencing and flow cytometry. Mechanistic studies included genetic manipulation, polysome profiling, co-immunoprecipitation, and RNA immunoprecipitation. Clinical relevance was validated in human lung cancer samples via immunohistochemistry and multiplex immunofluorescence. Statistical analyses involved unpaired two-tailed Student’s t -test, one-way/two-way ANOVA, and Pearson’s correlation. METTL3 was upregulated in anti-PD-1-resistant lung cancer cells and it promoted anti-PD-1 resistance in both subcutaneous and orthotopic mouse models. Mechanistically, METTL3 induced CD8 + T cell exhaustion via upregulation of secreted phosphoprotein 1 (SPP1). This occurred independently of its m 6 A catalytic activity, potentially through direct interaction with eukaryotic translation initiation factor 3 subunit H (EIF3H) to facilitate SPP1 translation. Furthermore, METTL3 protein stability was enhanced by lysine acetyltransferase 5 (KAT5)-mediated acetylation, which attenuated ubiquitin-dependent degradation. Finally, critical nodes of the KAT5-METTL3-SPP1 axis were validated in human lung cancer cells, and METTL3 expression positively correlated with KAT5, SPP1, and CD8 + T cell exhaustion markers in clinical samples. We identified a novel KAT5-METTL3-SPP1 axis that drives anti-PD-1 resistance by inducing CD8 + T cell exhaustion. Notably, METTL3 functions through a non-m 6 A catalytic mechanism involving translation and acetylation. These findings provide new mechanistic insights and potential therapeutic targets for overcoming anti-PD-1 resistance in lung cancer.