Impact of HSPA6 on lenvatinib resistance in HCC via phase separation–mediated TXNRD1 stabilization and ferroptosis suppression.

4119 Background: Lenvatinib is a first-line treatment for advanced hepatocellular carcinoma (HCC), but its efficacy is frequently limited by intrinsic and acquired resistance. The underlying molecular mechanisms remain incompletely understood, and strategies to overcome resistance are urgently needed. Methods: We integrated genome-wide CRISPR/Cas9 screening, transcriptomic profiling of lenvatinib-resistant HCC cells, and proteomic analysis of patient tumors to identify key mediators of resistance. Results: Heat shock protein family A (Hsp70) member 6 (HSPA6) emerged as a central driver of both intrinsic and acquired resistance, was consistently upregulated in resistant models, and was associated with poor response and reduced survival in lenvatinib-treated patients. Functional studies demonstrated that HSPA6 knockdown sensitized HCC cells and xenograft tumors to lenvatinib, whereas HSPA6 overexpression conferred resistance both in vitro and in vivo . Mechanistically, HSPA6 recruited the deubiquitinase ubiquitin-specific protease 9X (USP9X) to stabilize thioredoxin reductase 1 (TXNRD1), thereby suppressing lenvatinib-induced ferroptosis. Moreover, lenvatinib enhanced HSPA6 liquid–liquid phase separation (LLPS) through its intrinsically disordered region 1 (IDR1), facilitating the formation of HSPA6–USP9X–TXNRD1 condensates that reinforced TXNRD1 stability. Through structure-based virtual screening, we identified canagliflozin, an FDA-approved sodium–glucose cotransporter 2 (SGLT2) inhibitor, as a direct HSPA6 binder that disrupted this complex, restored ferroptosis sensitivity, and synergized with lenvatinib in preclinical models. Conclusions: Our study defines a novel HSPA6-driven resistance axis that integrates chaperone function, phase separation, and redox homeostasis to suppress ferroptosis in HCC. Targeting this axis with canagliflozin represents a promising therapeutic strategy to overcome lenvatinib resistance.