ChaC1-based drug screenings identify a synergistic lethal effect of auranofin and proteasome inhibitors in hepatocellular carcinoma cells

The γ-glutamylcyclotransferase, ChaC1, is an enzyme catalyzing glutathione (GSH) degradation. In this study, we performed a ChaC1-activity-directed pharmacological screening from FDA-approved drug library to identify GSH-sensitive agents, and found that ChaC1 overexpression mediated glutathione depletion largely enhanced auranofin (AUR)-induced cell death in hepatocellular carcinoma (HCC) cells. AUR elicited sustained activation of oxidative and endoplasmic reticulum (ER) stress pathways in ChaC1-overexpressed HCC cells, exemplified by Nrf2 and ATF4 upregulation. Proteomic analyses identified DNA Damage Inducible Transcript 4 (DDIT4) as one of the key pro-death effectors in this process. Mechanistic investigation revealed ChaC1/AUR-driven cytotoxicity was attenuated by disulfide reducing agents (e.g., NAC and TCEP), while maintaining resistance to canonical programmed death pathway inhibitors targeting Apoptosis, Necroptosis, and Ferroptosis. To mimic ChaC1 overexpression by inducing endogenous ChaC1 high expression, a complementary ChaC1-induction-based screening was performed and revealed Proteasome inhibitors (e.g., bortezomib, ixazomib, delanzomib) as potent inducers of endogenous ChaC1 via ATF4-dependent transcriptional activation. Combinational treatment of AUR and Proteasome Inhibitor (PI) synergistically led to catastrophic cell death, reversible by NAC or protein synthesis inhibitor CHX, yet refractory to blockade of Apoptosis, Necroptosis or Ferroptosis. And PI/AUR co-treatment recapitulated the phenotype of DDIT4 induction, while genetic disruption of the ATF4-ChaC1 axis abolished both cell death and DDIT4 upregulation. This functional convergence demonstrates that ChaC1 activation, either achieved through genetic manipulation or pharmacological induction, creates a synthetic lethal context for AUR in HCC cells. Together, our study establishes a ChaC1-based pharmacological discovery platform that identifies Proteasome Inhibitor and auranofin combination as a mechanistically rational anti-HCC strategy, providing both methodological innovation in drug repurposing screening and immediate translational potential for HCC treatment.