Lactylation of Mutation-Prone RAS Residues Mimics Oncogenic Mutations and Drives Constitutive Activation.

Background: Elevated expression of wild-type RAS contributes to tumorigenesis, yet the mechanisms underlying its mutation-independent hyperactivity remain unclear. Lactylation, a lactate-derived post-translational modification, has emerged as a regulator in cancer, but its role in RAS activation is unknown. Here, we investigated whether lactylation of RAS at specific residues drives its constitutive activation and the underlying mechanisms. Methods: Lactylation sites on RAS were identified by mass spectrometry. A site-specific lactylation system was employed to introduce lactyl-lysine into RAS at K117 and K147. Structural effects were analyzed via molecular dynamics simulations. RAS activity was assessed through GTP-binding and GTPase assays; protein stability was evaluated using ubiquitination mutants and cycloheximide chase. The responsible enzymes were defined by in vitro lactylation and delactylation assays. Functional impact was tested via colony formation, mammosphere assays, and xenograft models. Clinical relevance was examined in breast cancer tissues and survival databases, and drug synergy was assessed by combining lactate-lowering agents with MEK inhibitors. Results: We reported that RAS was lactylated at K117 and K147, 2 residues mutated in cancers. Mechanistically, RAS lactylation was catalyzed by TIP60 and reversed by SIRT2. Lactylation at these residues recapitulated features of oncogenic mutations by disrupting GTP interaction, impairing intrinsic guanosine triphosphatase (GTPase) activity, and competitively antagonizing K48-linked ubiquitination at K147 to stabilize RAS. Functionally, RAS lactylation promoted malignant transformation and tumorigenesis, phenocopying oncogenic mutations at the same sites. Inhibition of lactate production sensitized lactylated RAS-driven cancer cells to drugs targeting RAS mutations, revealing a therapeutic vulnerability. Clinically, RAS lactylation was elevated in breast cancer tissues and correlated with poor prognosis. Employing a site-specific lactylation system, we further confirmed that lactylation alone phenocopied mutation-driven RAS activation. Conclusions: Our study redefines lactylation as a functional "mutation mimic" mechanism, which not only recapitulates the effects of oncogenic mutations but also bridges the gap between genetic and epigenetic drivers in cancer. This suggests that targeting the lactylation pathway alone or in combination with mutation-directed therapies represents a promising strategy for treating wild-type RAS-driven cancers.