The deficiency of ALKBH5 promotes lenvatinib resistance and CD8+ T cell exhaustion via accumulation of the cholesterol metabolite 27HC in hepatocellular carcinoma

Lenvatinib is a first-line treatment option for advanced hepatocellular carcinoma (HCC), but resistance development is a challenge. A comprehensive and in-depth exploration of the molecular mechanisms underlying Lenvatinib resistance may provide novel insights and strategies for enhancing its therapeutic effectiveness. We established Lenvatinib-sensitive (LS) and Lenvatinib-resistant (LR) HCC models both in vitro and in vivo. Dot blot and Western blot analyses confirmed that the m6A methylation levels were elevated in the LR models, while the expression of ALKBH5 was significantly reduced. Lentivirus-mediated transfection of HCC cells was employed to validate the inhibitory role of ALKBH5 in Lenvatinib resistance in HCC. Mechanistically, we integrated sequencing results from mRNA and methylated RNA immunoprecipitation (MeRIP), followed by validation through RNA immunoprecipitation (RIP), MeRIP-qPCR, dual luciferase reporter assays, Liquid chromatography and tandem mass spectrometry (LC-MS/MS), Enzyme-linked immunosorbent assay (ELISA), and animal experiments (including subcutaneous tumor models and HCC orthotopic mouse models). Results confirmed that ALKBH5 deficiency in LR cells elevated m6A methylation of TBX3 mRNA, thereby enhancing its stability. TBX3 bound to the promoter region of CYP27A1, stimulating its transcription and promoting the synthesis of the cholesterol metabolite 27-hydroxycholesterol (27HC). 27HC could inhibit ferroptosis, thereby protecting LR cells from the targeting effects of Lenvatinib. Additionally, T cell-mediated tumor cell killing and flow cytometry assays evaluated that HCC-derived 27HC promoted CD8⁺ T cell exhaustion. Our findings indicated that the deficiency of ALKBH5 mediated the enhanced synthesis of the cholesterol metabolite 27HC, which in turn inhibited ferroptosis in HCC cells and the cytotoxicity of CD8⁺ T cells, leading to Lenvatinib resistance in HCC cells. While the specific role of 27HC was strongly supported, the potential contributions of other CYP27A1-derived metabolites to this phenotype remain a possibility.