Auto-S-fatty acylation of PKM2 regulates its tetramerization and glycolytic metabolism in cancer cells.

Cancer cells rely on aerobic glycolysis to fuel proliferation, yet how lipid metabolism dynamically regulates glycolysis remains incompletely understood. Here, we identify pyruvate kinase M2 (PKM2) is regulated by auto-S-fatty acylation. Using bioorthogonal chemical reporters, we demonstrate that PKM2 is S-fatty acylated at a conserved cysteine residue (Cys474), preferentially incorporating C18 stearate and C16 palmitate. This modification destabilizes PKM2's active tetramer, thereby suppressing pyruvate kinase activity and altering the abundance of glycolytic intermediates. Genetic ablation of PKM2 S-fatty acylation (C474S mutant) enhances mitochondrial respiration, alters glycolytic metabolite abundance, and impairs lung cancer cell growth in vitro and in vivo. Our findings establish PKM2 auto-S‑fatty acylation as a mechanism that links fatty acid availability to glycolysis and tumor cell growth, highlighting a potential vulnerability in cancers dependent on aerobic glycolysis.