Abstract

Introduction The clinical benefit of combining lenvatinib with PD-1 blockade in HCC is frequently constrained by adaptive resistance and the development of an immune-cold tumor microenvironment. Objectives This study aimed to elucidate the molecular mechanisms underlying adaptive resistance and immune exclusion during lenvatinib–PD-1 therapy in HCC, with a particular focus on a UBTF/HSP90A/MIF regulatory circuit. We examined whether genetic or pharmacologic targeting of macrophage migration inhibitory factor (MIF) could restore lenvatinib sensitivity, remodel the tumor immune microenvironment, and serve as a predictive biomarker in clinical cohorts. Methods Paired lenvatinib-sensitive and −resistant HCC models were interrogated using integrated multi-omic and functional approaches, including RNA sequencing, promoter pull-down assays, ChIP, luciferase reporter assays, PLA, and flow cytometry. Key findings were validated in patient-derived organoids and xenografts, as well as in an immunocompetent hydrodynamic HCC mouse model. Clinical relevance was evaluated in independent cohorts treated with lenvatinib plus anti–PD-1 therapy. Results UBTF directly bound to and transcriptionally activated the HSP90A promoter, resulting in increased HSP90A expression and stabilization of MIF. MIF signaling through CD74 co-activated the PI3K–AKT and MAPK pathways, sustaining tumor cell proliferation under lenvatinib pressure. Single-cell RNA sequencing and multiplex immunohistochemistry revealed macrophage enrichment and CD8 + T-cell exclusion in resistant tumors. Genetic ablation of Mif (Alb-Cre; Mif flox/flox ) or pharmacologic inhibition with 4-IPP (4-Iodo-6-phenylpyrimidine) restored lenvatinib sensitivity, reprogrammed the tumor immune microenvironment, and, when combined with PD-1 blockade, achieved superior tumor control and prolonged survival. In clinical datasets, low pretre