Glutamine synthetase loss in β-catenin–mutant hepatocellular carcinoma promotes tumor burden through macrophage metabolic reprogramming

BACKGROUND AND AIMS: Activating β-catenin gene ( CTNNB1 ) mutations are seen in 30% of all hepatocellular cancer (HCC). These tumors are a molecularly distinct subclass characterized in the majority of cases by the presence of tumor-wide glutamine synthetase (GS), increased glutamine, mTOR activation, and susceptibility to mTOR inhibitors. Here, we investigate the impact of GS loss from β-catenin-mutated HCCs. APPROACH: TCGA was assessed for CTNNB1 -mutated HCCs with differential Glul (encoding GS) expression for survival. Glul was conditionally deleted from hepatocytes and/or macrophages in HCCs co-expressing mutant- CTNNB1 (T41A) and mutant Nrf2 in mice. Macrophage depletion was also performed by Clodranate treatment. Tumors were characterized by histology and single-cell spatial transcriptomics. RESULTS: CTNNB1 -mutated HCC patients with low Glul showed poor survival. β-Catenin-mutated HCCs lacking GS exhibited aggressive disease due to altered glutamate/glutamine availability, forcing metabolic adaptation through upregulation of macrophage Glul , permitting mTOR activation and susceptibility to mTOR inhibitors, but switching macrophage function from immunosurveillance to immunosuppression. Glul loss from tumors did not interfere with β-catenin-dependent tumor zonation and responsiveness to β-catenin inhibition. Depleting macrophages using clodronate or conditionally deleting Glul from macrophages in GS-deficient, β-catenin-mutant HCCs decreased tumor burden and improved survival. CONCLUSIONS: We demonstrate the unique metabolic dependency of β-catenin-mutated HCCs on GS in tumor cells, which is diverted to macrophages upon GS elimination in tumor cells. This adaptation alters macrophage metabolism and function, leading to compromised immunosurveillance and greater tumor burden. Our study reveals a metabolic dynamic between HCC cells and macrophages with an impact on tumor biology.