Abstract 2222: Trimethylamine-N-oxide (TMAO) mediates immunotherapy resistance in advanced HCC

Abstract Recent approvals of immune checkpoint inhibitor (ICI) based regimens have transformed the clinical landscape of advanced hepatocellular carcinoma (HCC), however, clinical responses are only observed in a minority of patients. Trimethylamine-N-oxide (TMAO) is an amine oxide biosynthesized from trimethylamine (TMA), generated when gut bacteria metabolize dietary choline via the enzyme CutC. While prior studies have shown that TMAO concentrations positively correlate with the risk of several gastrointestinal malignancies, newer studies have demonstrated that TMAO may enhance antitumor immunity in some tumor types. TMAO may therefore play an important role in modulating the tumor immune response, but its significance in HCC is unknown. We prospectively collected whole blood from patients with HCC prior to initiating standard-of-care ICI-based therapy at Johns Hopkins (IRB #00267960). Clinical records were reviewed for demographic characteristics and clinical outcomes. Additionally, we studied the impact of acute and chronic choline supplementation on anti-PDL1 efficacy in a syngeneic murine model of HCC (Hep53.4 in C57BL/6 mice). Baseline choline, TMA, and TMAO plasma concentrations in clinical and preclinical specimens were quantified via liquid chromatography-mass spectrometry (LC-MS) utilizing the SCIEX Triple Quad 6500+ system. The clinical cohort included 42 patients with advanced HCC. Patients were predominantly male (81.0%) with Barcelona Clinic Liver Cancer (BCLC) stage C disease (73.8%) who underwent treatment with atezolizumab + bevacizumab (52.4%) or durvalumab + tremelimumab (31.0%). In a multivariable Cox model, higher plasma TMAO concentration at baseline was associated with inferior progression-free survival (PFS; HR 1.36 per interquartile range [IQR] increase, p=0.032) and overall survival (OS; HR 1.63 per IQR increase, p<0.001) after controlling for BCLC stage, albumin-bilirubin index, alpha fetoprotein (AFP) concentration, and baseline choline and TMA concentrations. In our preclinical model, mice given chronic choline supplementation (1% choline diet for 14 days prior to treatment with anti-PDL1 therapy) had increased plasma TMAO concentrations (p<0.001) and exhibited no response to anti-PDL1 therapy, compared to mice given standard and acute choline (1% choline diet starting concurrently with anti-PDL1 treatment) diets. Our clinical and preclinical results suggest that high plasma TMAO concentration is associated with inferior outcomes on ICI therapies in HCC. These findings contrast with existing preclinical studies that suggest TMAO may potentiate responses to ICI therapy. We hypothesize that chronic TMAO exposure may lead to immune cell exhaustion via promotion of an inflammatory microenvironment and oxidative stress. Studies examining the effect of CutC inhibition are ongoing in our laboratory and may provide further rationale for targeting TMAO in HCC. Citation Format: Mari Nakazawa, Vivian L. Raj, NV Rajeshkumar, Noushin Rastkari, Michael Davis, Elizabeth De Oliveria, Madelena Brancati, Ervin Griffin, Kabeer Munjal, James Leatherman, Sarah Mitchell, Marina Baretti, Won Jin Ho, Mark Yarchoan, Chi V. Dang. Trimethylamine-N-oxide (TMAO) mediates immunotherapy resistance in advanced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2222.