CRISPR Screen Reveals Pathways and Factors Driving Tyrosine Kinase Inhibitor Resistance in Hepatocellular Carcinoma

ABSTRACT

Tyrosine kinase inhibitor (TKI) resistance severely limits clinical outcomes in hepatocellular carcinoma (HCC), highlighting the urgent need to elucidate its underlying molecular mechanisms. In this study, an unbiased genome‐wide CRISPR/Cas9 screening identified novel key factors related to the therapeutic responsiveness of TKI in HCC. By integrating data from 20 datasets encompassing 322 samples, a comprehensive TKI therapeutic response landscape for HCC was constructed. GO and Reactome enrichment analyses revealed that dysregulated RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways modulate TKI sensitivity, with close links to antitumor immunity. This study identified GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37 as key genes mediating TKI resistance in HCC. These six genes were found to be highly expressed in HCC and significantly associated with HCC patient prognosis. Drug sensitivity assays identified a significant association between their expression and responsiveness to TKI agents. In‐house quantitative real‐time PCR validated their differential expression levels in normal hepatocytes, parental HCC cells, and TKI‐resistant HCC sublines. ssGSEA, TIMER2, and ESTIMATE analysis revealed that their expression modulates HCC immune infiltration. Bibliometric analysis revealed a growing focus on immunotherapy‐based combination regimens to overcome TKI resistance. Ferroptosis, epithelial‐mesenchymal transition and hypoxia were new research directions, which were closely related to the pathways investigated in this study. In conclusion, this study identified RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways, as well as GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37, as novel directions and targets for TKI–immunotherapy combination strategies, providing new insights for overcoming TKI resistance in HCC.