Lactylation Converts ABHD6 into a Mitochondrial Regulator That Drives Lenvatinib Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) frequently develops resistance to lenvatinib, a first-line tyrosine kinase inhibitor. Resistance arises from heterogeneous mechanisms involving metabolic reprogramming and mitochondrial adaptation, implicating regulators of these processes as potential therapeutic targets. In this study, we identified α/β hydrolase domain containing 6 (ABHD6) as a critical driver of lenvatinib resistance by perturbing mitochondrial dynamics. Ligand binding at the S148 catalytic site allosterically controlled a molecular switch between canonical enzymatic and noncanonical scaffolding functions of ABHD6, and the proresistance function was independent of catalysis but required an unoccupied catalytic site. In resistant HCC, the Warburg effect elevated lactate, leading to K245 lactylation of ABHD6. This modification triggered the mitochondrial translocation of ABHD6, in which it functioned as a scaffold that competitively bound the fission regulator mitochondrial fission 1 (FIS1) and displaced dynamin-related protein 1 (DRP1). Disruption of the fission machinery stabilized hyperfused mitochondria, thereby conferring lenvatinib resistance by suppressing drug-induced apoptosis and ROS generation. Both inhibiting lactate production and enforcing occupancy of the S148 site with substrates or a specific inhibitor blocked formation of the ABHD6-FIS1 complex, reactivated mitochondrial fission, and restored lenvatinib sensitivity. This study identified a lactate-driven functional switch in ABHD6 and established that targeting this allosteric mechanism is an effective therapeutic strategy to overcome lenvatinib resistance. SIGNIFICANCE: Lactylated ABHD6 moonlights as a regulator of mitochondrial dynamics by sequestering FIS1 to drive lenvatinib resistance in hepatocellular carcinoma, which can be overcome by targeting ABHD6 to disrupt the non-canonical scaffolding function.