Asparagine synthetase modulates glutaminase inhibitor sensitivity through metabolic reprogramming and serves as a prognostic biomarker in hepatocellular carcinoma.

Glutamine addiction represents a metabolic vulnerability in hepatocellular carcinoma (HCC), making glutaminase inhibitor CB-839 therapy a promising approach. However, effective therapeutic strategies are not yet available. In this study, we aim to investigate the potential role of asparagine synthetase (ASNS) as a target for HCC therapy during CB-839 treatment. CB-839 suppressed HCC cell growth, triggered apoptosis, and induced oxidative stress along with the disruption of amino acid metabolism. Moreover, ASNS was induced by CB-839 treatment through the activation of the amino acid response pathway. ASNS was significantly upregulated in HCC tumor tissues and was positively associated with poor prognosis; indeed our results revealed that its overexpression facilitated the proliferation, migration, and invasion of HCC cells. Furthermore, ASNS increased glutaminolysis and glutathione synthesis through reprogramming glutamine metabolism to maintain intracellular redox homeostasis, thereby activating the mTOR pathway that contributed to HCC progression. ASNS knockdown sensitized HCC cells to CB-839 both in vitro and in vivo. Overall, ASNS modulated the sensitivity to CB-839 in HCC through metabolic reprogramming, potentially serving as a biomarker for CB-839 response and a promising therapeutic target for HCC.