Abstract Metabolic dysfunction-associated steatohepatitis (MASH) is the fastest growing cause of hepatocellular carcinoma (HCC), yet the metabolic dependencies underlying MASH-HCC remain poorly defined. Here, we identify mitochondrial proton conductance as a central determinant of HCC progression and therapeutic vulnerability. Human HCC exhibited increased mitochondrial efficiency and glycolytic flux with reduced oxidative flux, consistent with large-scale HCC dependency datasets showing strong enrichment for downregulated mitochondrial pathways. To functionally interrogate this bioenergetic liability, we performed an unbiased PRISM barcoded-cell-line screen using the mitochondrial uncoupler BAM15. Lineage-level analysis revealed broad cytotoxicity with marked enrichment of sensitivity in HCC, which was validated in HepG2 cells, where BAM15 demonstrated greater potency than sorafenib, a frontline therapy for advanced HCC. We next evaluated mitochondrial uncoupling in vivo using two MASH-HCC models: (1) high-fat, fructose, and cholesterol diet with thermoneutral housing plus low-dose DEN, and (2) cocoa-butter diet plus DEN, each recapitulating metabolic injury and tumor development. In both models, BAM15 significantly reduced tumor burden and improved liver function. These effects persisted in PPARα-null mice, indicating that uncoupling exerts its efficacy through direct disruption of mitochondrial efficiency rather than secondary activation of fatty acid oxidation. To assess translational relevance, we tested the long-acting liver-targeted mitochondrial uncoupler TLC-1180, which similarly decreased tumor burden and improved hepatic function. Finally, liver-specific deletion of Ucp2 reduced hepatic proton conductance, increased tumorigenesis, and impaired liver function, establishing a mechanistic link between diminished uncoupling capacity and enhanced carcinogenic progression. Together, these data demonstrate that suppressed proton conductance is a fundamental metabolic adaptation that promotes HCC growth, and that enforced mitochondrial uncoupling directly counteracts this program. These findings position hepatic proton conductivity as a mechanistically grounded therapeutic target and support mitochondrial uncoupling, particularly liver-directed approaches, as a rational strategy for treating MASH-HCC. Citation Format: Elizabeth Rachel Marie Zunica, Kim Pedersen, Anan L. Cole, Analisa L. Taylor, Elizabeth C. Heintz, Megan D. Dousay, Bolormaa Vandanmagsar, Monika Sharma, Odinakhon Shamieva, Lucas Kniess Debarba, Marcus DaSilva Goncalves, Tomislav Jelesijevic, Rees G. Matthew, Jennifer A. Roth, Martin J. Ronis, Christopher L. Axelrod, John P. Kirwan. Hepatic proton conductance as a bioenergetic vulnerability and therapeutic target in MASH-associated HCC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7331.
Zunica et al. (Fri,) studied this question.