Synergistic oncogenesis in human cholangiocytes: IDH1 mutation and the biliary microenvironment to drive early transformation and a targetable metabolic dependency in patient-derived organoids.

Abstract

308 Background: Cholangiocarcinoma (CCA) is a lethal malignancy with limited understanding of its early developmental mechanisms. A significant subset of CCA harbors gain-of-function mutations in IDH1 (e.g., R132C), which promotes accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). A major barrier in dissecting early events is the lack of models that faithfully recapitulate human physiology. Patient-derived organoids (PDOs) retain the genetic and phenotypic diversity of the original tissue. Base editing now enables the introduction of specific nucleotide mutations into physiologically relevant models to study causal driver events. However, the oncogenic impact of IDH1 mutations on normal and pre-diseased cholangiocytes, and their synergy with factors like bile acids, are poorly defined. Methods: Normal and dilated cholangiocyte organoids were derived from clinical specimens. Cytosine base editing installed the IDH1 R132C mutation. Phenotypes (proliferation, invasion, CEA/CA19-9 secretion) and 2-HG levels were assessed, with/without bile acid challenge. Integrated transcriptomics and metabolomics delineated underlying reprogramming. Results: Introduction of the IDH1 R132C mutation into both normal and dilated cholangiocyte organoids induced a pro-oncogenic phenotype, including enhanced proliferation, invasion, and secretion of CEA/CA19-9, concomitant with 2-HG accumulation. Mechanistically, multi-omics analysis revealed that IDH1 R132C, particularly in dilated cholangiocytes, drives a transcriptional program characteristic of early biliary transformation, involving dysregulation of pathways governing cell fate (e.g., Hippo/YAP suppression) and metabolism (e.g., upregulated glutaminolysis). Crucially, bile acids acted as a potent synergistic cofactor, amplifying phenotypes and rewiring the metabolic network to create a dependency on specific biosynthetic pathways. This combination effectively locked organoids into a proliferative, precursor-like state. Conclusions: We have established a novel, physiologically relevant in vitro model that recapitulates the synergistic oncogenesis driven by IDH1 mutation and microenvironmental stress in early CCA. Our study moves beyond phenomenology to identify the critical early molecular and metabolic nodes activated during this transformation. Importantly, by targeting a key identified metabolic vulnerability (e.g., with a glutaminase inhibitor or a mutant IDH1 inhibitor), we were able to significantly attenuate the observed hyperproliferative and invasive phenotypes, and reduce tumor marker secretion. This work provides a preclinical platform for identifying and testing interceptive strategies aimed at reversing or halting early cholangiocarcinogenesis in high-risk settings.