结直肠癌（CRC）的恶性转化中经常观察到肿瘤抑制基因的启动子高甲基化。然而，这种表观遗传机制在癌症中是否具有功能性，或仅是致癌过程的副作用，尚待阐明。在这项研究中，我们采用整合性多组学方法，在人类CRC样本和一组8种结肠癌细胞系中，识别到DNA甲基化和基因表达之间存在强相关性的候选基因。作为概念验证，我们结合了最近的CRISPR-Cas9表观遗传编辑工具（dCas9-TET1、dCas9-TET-IM）和一个自定义的阵列化gRNA库，调控了56个先前与CRC中强表观遗传抑制相关的启动子的DNA甲基化状态，并通过高容量细胞增殖筛选，监测了该DNA甲基化丧失的潜在功能后果。总的来说，这些DNA甲基化区域的表观调节对基因表达的重激活和癌细胞的存活率影响较小。有趣的是，我们发现在肿瘤环境中，RSPO2的表观重激活与p53-/-癌细胞系中的细胞增殖明显受损相关，并通过人类样本的进一步验证表明，RSPO2的表观沉默是腺瘤向癌变序列的中后期事件。这些结果强调了DNA甲基化作为CRC驱动机制的潜在作用，并为基于表观遗传重激活某些肿瘤抑制基因的新型治疗窗口的鉴定铺平了道路。© 2023年 BioMed Central有限公司，Springer Nature的一部分。

Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.© 2023. BioMed Central Ltd., part of Springer Nature.