Functional characterization of BAP1 mutations in genome edited cholangiocarcinoma organoids: role in cell death and drug responses

Cholangiocarcinoma (CCA) is a genetically heterogeneous malignancy of the bile ducts with limited effective treatments and variable chemotherapeutic responses. BRCA1-associated protein 1 (BAP1), a tumor suppressor gene frequently mutated in CCA, encodes a nuclear deubiquitinating enzyme involved in chromatin remodeling and cell death regulation. In this study, we investigated the role of BAP1 mutations in programmed cell death and drug response using patient-derived and prime-edited CCA organoids (CCAOs). BAP1-mutant organoids exhibited impaired activation of apoptosis and necroptosis, as evidenced by reduced cleaved caspase-3 and pMLKL expression. Transcriptomic analysis revealed BAP1-dependent gene expression changes including enrichment of pathways related to stress response, ion transport, and metabolic detoxification. Interesting, BAP1 mutant CCAOs showed enhanced sensitivity to sorafenib, a multikinase inhibitor commonly used in biliary tract cancer. These findings highlight BAP1 as a modulator of cell death and a potential predictive biomarker for sorafenib response in CCA, with implications for personalized therapy design. Overall design: investigate how BAP1 influences cell death pathways in prime-edited CCAOs. CCAOBAP1-WT was prime edited, FACSorted and clonally expended. Clones were screened by Western blot for BAP1 expression and one clone was selected based on substantial reduction of BAP1 protein level. Three technical replicates of targeted and control were sequenced