Transcriptomic profiling of epigenetic regulators and metabolic reprogramming in human cholangiocarcinoma

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Abstract
Background: Epigenetic alterations play an increasingly recognized role in carcinogenesis and in the development of resistance to anticancer therapies. Epigenetic enzymes (writers and erasers) and effectors (readers) are largely influenced by the availability of metabolites generated through one-carbon metabolism (OCM), the tricarboxylic acid (TCA) cycle, and acetyl-CoA synthesis (ACS). In this study we examined the expression of epigenetic and metabolic genes to investigate their interplay in cholangiocarcinoma (CCA). Method: We examined 257 epigenetic genes (EpiGs), 96 metabolic genes (MGs), and 189 rate-limiting enzymes (RLEs) in transcriptomic data from iCCA, eCCA, and normal bile ducts, healthy liver-derived organoids, and CCA tumoroids, alongside prognostic signatures. CRISPR-Cas9 DepMap data evaluated the impact of EpiGs and MGs on cell viability. HuCCT-1 iCCA cells were exposed to hypoxia (1% O 2 , 24 h) to assess EpiG responses. Transcriptomic deconvolution characterized EpiGs, MGs, and RLEs expression across four tumor microenvironment (TME) subtypes. Two mouse CCA models (TAZ/Akt and NICD1/Akt) underwent RNA-seq, complemented by multi-omic profiling (transcriptomic, proteomic, metabolomic) in TAZ/Akt livers. Results: Several EpiGs were upregulated in iCCA and eCCA, including writers ( DNMT1 , EZH2 , SUZ12 ), readers ( CBX3 , PHF20L1 , SMARCA4 ), and erasers ( HDAC1 , HDAC3 , KDM5C ). MGs in OCM, TCA, and ACS pathways were dysregulated (up: GART , IDH2 , TYMS ; down: ALDH1L1 , MAT1A , SHMT1 ). Integrated analyses identified 27 EpiGs and 8 MGs whose overexpression predicted poor survival. Subsets of EpiGs, MGs, and RLEs were linked to proliferative, high-recurrence iCCA subclasses. CRISPR screens highlighted 50 EpiGs and 23 MGs essential for CCA viability. Tumor microenvironment (TME) analyses revealed distinct immune-stromal subclasses with coherent epigenetic-metabolic signatures. In CCA cells, hypoxia induced epigenetic programs that mirrored those in CCA patients. Transcriptomic analyses in human and multi-omic analyses (transcriptomic, metabolomic, and proteomic studies) in mouse CCA livers highlighted rewiring of nucleotide, one-carbon, lipid, and mitochondrial pathways, with evidence of metabolic-epigenetic crosstalk. Conclusion: EpiGs and MGs are markedly altered in both human and experimental CCA, with several changes particularly enriched in aggressive molecular subclasses associated with poor prognosis. We observed substantial rewiring of epigenetic cofactor-related MG expression in CCAs. Functional assays validated new targets among EpiGs (e.g., CBX3 , CHD4 , DEK , SMARCA4 , and TRIM28 ) and MGs ( TYMS and IDH2 ) in CCA.

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