Lenvatinib (LEN) is a key first-line therapy for advanced hepatocellular carcinoma (HCC), but intrinsic and acquired resistance limits its long-term efficacy. We aimed to identify novel mechanisms underlying LEN resistance and to explore clinically relevant biomarkers and therapeutic targets that may improve patient stratification and treatment outcomes.

LEN-resistant (LenR) HCC cell lines were established by chronic drug exposure and analyzed by RNA sequencing. The functional role of tryptophan 2,3-dioxygenase (TDO2) was evaluated by overexpression, knockdown, and pharmacological inhibition with 680C91 in vitro and in vivo. Clinical relevance was assessed by measuring baseline serum kynurenine levels in 58 HCC patients treated with LEN.

LenR cells showed greater tumor growth in vivo, epithelial-mesenchymal transition-like features, and increased kynurenine secretion. RNA sequencing identified TDO2 upregulation as a prominent molecular alteration in LenR cells. TDO2 overexpression reduced LEN sensitivity, whereas TDO2 knockdown or pharmacological inhibition restored sensitivity in vitro without apparent cytotoxicity of 680C91 alone. In xenograft models, combined treatment with LEN and 680C91 significantly suppressed tumor growth compared with LEN alone. In the clinical cohort, elevated baseline serum kynurenine levels were significantly associated with progressive disease (AUC=0.8459) and poorer overall survival (p = 0.0291), suggesting its utility as a predictive biomarker.

TDO2 contributes to LEN resistance in HCC, and baseline serum kynurenine may serve as a non-invasive biomarker of TDO2 associated resistance. TDO2 inhibition represents a potential therapeutic strategy for overcoming LEN resistance.