NOTCH-YAP1/TEAD-DNMT1 Axis Drives Hepatocyte Reprogramming Into Intrahepatic Cholangiocarcinoma

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a devastating liver cancer with extremely high intra-and inter-tumoral molecular heterogeneity, partly due to its diverse cellular origins. We investigated clinical relevance and the molecular mechanisms underlying hepa-tocyte (HC)-driven ICC development. METHODS: Expression of ICC driver genes in human diseased livers at risk for ICC development were examined. The sleeping beauty and hy-drodynamic tail vein injection based Akt-NICD/YAP1 ICC model was used to investigate pathogenetic roles of SRY-box transcription factor 9 (SOX9) and yes-associated protein 1 (YAP1) in HC-driven ICC. We identified DNA methyltransferase 1 (DNMT1) as a YAP1 target, which was validated by loss-and gain-of-function studies, and its mechanism addressed by chromatin immunoprecipitation sequencing. RESULTS: Co-expression of AKT and Notch intracellular domain (NICD)/YAP1 in HC yielded ICC that represents 13% to 29% of clinical ICC. NICD independently regulates SOX9 and YAP1 and deletion of either, signifi- cantly delays ICC development. Yap1 or TEAD inhibition, but not Sox9 deletion, impairs HC-to-biliary epithelial cell (BEC) reprogramming. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directs HC-to-BEC/ICC fate switch through the repression of HC -specific genes regulated by master regulators for HC dif-ferentiation, including hepatocyte nuclear factor 4 alpha, hepatocyte nuclear factor 1 alpha, and CCAAT/enhancer-binding protein alpha/beta. DNMT1 loss prevented NOTCH/YAP1-dependent HC-driven cholangiocarcino-genesis, and DNMT1 re-expression restored ICC develop-ment following TEAD repression. Co-expression of DNMT1 with AKT was sufficient to induce tumor development including ICC. DNMT1 was detected in a subset of HCs and dysplastic BECs in cholestatic human livers prone to ICC development. CONCLUSION: We identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-to-BEC/ICC con-version, which may be relevant in cholestasis-to-ICC patho-genesis in the clinic.