Background: Cholangiocarcinoma (CCA) is a highly lethal malignant tumour with increasing incidence. Current therapies exhibit limited benefits, which urgently demand the identification of novel therapeutic targets.

Objective: We aimed to identify potential therapeutic targets for CCA and broaden current therapies.

Design: Potential therapeutic targets for CCA were identified by sgRNA library screening and validated in preclinical models. Multi-omics sequencing and various experimental approaches were performed to validate the mechanism by which Aurora kinase B (AURKB) regulates CCA progression and the immune microenvironment, supported by clinical samples from public data sets and Tongji Hospital cohorts. The translational therapy was comprehensively validated in CCA organoid, patient-derived xenograft and preclinical murine models.

Results: AURKB was identified as a highly expressed and targetable kinase in CCA. Knockout of AURKB significantly inhibited CCA progression, reduced CD8 T cell exhaustion and enhanced antitumour response. Mechanistically, AURKB promoted the generation of histone H3 lysine 9 tri-methylation (H3K9me3)/serine 10 phosphorylation, leading to a decrease in the enrichment of H3K9me3 at the neutral cholesterol ester hydrolase 1 (NCEH1) promoter, thereby increasing NCEH1 expression and cholesterol levels in tumours. High AURKB expression in clinical samples predicted poorer outcomes in patients with CCA undergoing neoadjuvant chemoimmunotherapy and was associated with cholesterol accumulation within tumours. AURKB inhibitor or simvastatin can suppress CCA progression and significantly enhance sensitivity to chemoimmunotherapy. ⁺

Conclusions: AURKB regulates cholesterol levels and immune microenvironment in tumours, highlighting that targeting AURKB or adopting cholesterol-reducing strategy holds promise for CCA treatment, especially in conjunction with first-line chemoimmunotherapy.