Characterization of Differential GPX4 Essentiality Between Intrahepatic and Extrahepatic Cholangiocarcinoma via Leveraging of a Large-Scale Functional Genomic Screen.

Ferroptosis has emerged as a promising therapeutic vulnerability of diverse malignancies, yet the regulatory circuits adopted by each in cholangiocarcinoma (CCA) subtypes remain incompletely understood. We integrated the genome-wide CRISPR-Cas9 loss-of-function screens and transcriptomic profiles of the Cancer Dependency Map and then systematically assessed the essentiality of ferroptosis suppressor genes (FSGs) in the intrahepatic (iCCA) and extrahepatic (eCCA) subtypes. Nineteen and 16 essential FSGs were identified in iCCA and eCCA, respectively, among which GPX4 exhibited a significantly higher dependency in iCCA. Pharmacological inhibition of GPX4 with RSL3 markedly reduced cell viability and induced lipid peroxidation in iCCA cell lines, whereas eCCA cell lines displayed pronounced resistance associated with elevated GPX4 expression. A transcriptomic comparison revealed enrichment of WNT signaling in eCCA. Co-treatment with the tankyrase inhibitor XAV-939 and RSL3 enhanced growth inhibition of eCCA cells, indicating that WNT signaling contributed to ferroptosis resistance. These findings indicate that iCCA exhibits a preferential dependency on GPX4, whereas WNT-β-catenin signaling mediates resistance in eCCA. Collectively, the results clarify the molecular basis of subtype-specific ferroptosis vulnerability and offer a rationale for combinatorial therapeutic strategies that integrate GPX4 and WNT pathway inhibition when treating refractory eCCA.