HSPA6 Confers Lenvatinib Resistance in Hepatocellular Carcinoma by Promoting Phase Separation–Mediated TXNRD1 Stabilization to Suppress Ferroptosis

Abstract The development of therapy resistance compromises the long-term efficacy of lenvatinib in advanced hepatocellular carcinoma (HCC), highlighting the need to characterize the molecular drivers of resistance. Through integrated multiomics analysis combining genome-wide CRISPR screening, transcriptomics, and proteomics, we identified Heat shock protein family A member 6 (HSPA6) as a critical driver of lenvatinib resistance. HSPA6 was consistently upregulated in resistant cell lines and patient tumors, and high expression correlated with poor treatment response and survival. HSPA6 recruited the deubiquitinase USP9X to stabilize the antioxidant enzyme thioredoxin reductase 1 (TXNRD1), thereby suppressing lenvatinib-induced ferroptosis. Furthermore, HSPA6 underwent drug-enhanced liquid–liquid phase separation through its intrinsic disordered region 1 domain, facilitating the assembly of biomolecular condensates that reinforce TXNRD1 stability and ferroptosis resistance. Leveraging these insights, canagliflozin, an FDA-approved sodium-glucose cotransporter 2 inhibitor, was repurposed as a direct HSPA6-targeting compound that disrupted this resistance axis, restored ferroptosis sensitivity, and synergized with lenvatinib in patient-derived models. This work unveils a dynamic, condensate-driven mechanism of drug resistance and offers a readily translatable strategy to overcome lenvatinib resistance in HCC. Significance: HSPA6 drives lenvatinib resistance and can be targeted by repurposing canagliflozin to restore ferroptosis sensitivity and overcome therapy resistance in hepatocellular carcinoma.