Objectives Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. Design Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. Results We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH -mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. Conclusion Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits. Data are available in a public, open access repository. Sequencing datasets can be found at the European Genome-phenome Archive (EGA, Accession number: EGAS00001007309). Data used in this manuscript also includes previously published studies from public repositories: EGA (EGA00001000950) and Gene Expression Omnibus (GEO, GSE89749 and GSE89803). Codes used are available on GitHub ([https://github.com/chernycherny/CCA\_enhancers\_2023][1]). [1]: https://github.com/chernycherny/CCA_enhancers_2023

中文翻译：

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综合多组学增强子活性分析可识别不同病因胆管癌的治疗脆弱性

目的 胆管癌（CCA）是一种异质性恶性肿瘤，死亡率高，预后差，临床迫切需要新的治疗方法。对 CCA 表观基因组的了解很大程度上局限于异常 DNA 甲基化。增强子活性的失调已被确定会影响致癌作用并用于新疗法，但在 CCA 中尚未得到研究。我们的目标是通过增强子分析来确定 CCA 不同亚型的潜在治疗靶点。设计对不同的 CCA 进行了综合多组学增强子活性分析。使用一组不同的 CCA 细胞系、患者来源和细胞系来源的异种移植物来研究已确定的丰富途径和脆弱性。 NanoString、多重免疫组织化学染色和单细胞空间转录组学用于探索不同 CCA 的免疫原性。结果我们确定了三个不同的群体，与不同的病因和独特的途径相关。已确定途径的药物抑制剂可在体外和体内模型中减少肿瘤生长。第一组 (ESTRO) 大部分为吸虫阳性 CCA，显示雌激素信号传导激活并对 MTOR 抑制剂敏感。另一组 (OXPHO)，主要具有 BAP1 和 IDH 突变体 CCA，显示出激活的氧化磷酸化途径，并且对氧化磷酸化抑制剂敏感。最后一组 (IMMUN) 的免疫相关通路被激活，该组由免疫原性 CCA 亚型和具有马兜铃酸 (AA) 突变特征的 CCA 组成。瘤内 AA 突变负荷的差异与不同免疫细胞群的瘤内变异相关。结论 我们的研究阐明了增强子失调的机制，加深了对不同 CCA 亚型不同肿瘤发生过程的理解，具有潜在的显着治疗和临床益处。数据可在公共、开放访问存储库中获取。测序数据集可以在欧洲基因组-现象档案馆（EGA，登录号：EGAS00001007309）中找到。本手稿中使用的数据还包括先前发表的来自公共存储库的研究：EGA (EGA00001000950) 和基因表达综合 (GEO、GSE89749 和 GSE89803)。使用的代码可在 GitHub 上找到（[https://github.com/chernycherny/CCA\_enhancers\_2023][1]）。 [1]：https://github.com/chernycherny/CCA_enhancers_2023