Epigenome reprogramming through H3K27 and H3K4 trimethylation as a resistance mechanism to DNA methylation inhibition in BRAFV600E-mutated CRC. [ChIP-Seq]. Epigenome reprogramming through H3K27 and H3K4 trimethylation as a resistance mechanism to DNA methylation inhibition in BRAFV600E-mutated CRC. [ChIP-Seq]

BRAFV600E-mutated colorectal cancer (CRC) exhibits a strong correlation with DNA hypermethylation suggesting this subgroup of tumors is uniquely presenting epigenomic phenotypes. Nonetheless, the traditional epigenomic therapeutic agent, 5-azacitidine, which inhibits DNA methyltransferase activity, did not yield sufficient improvements in the efficacies of BRAFV600E CRC in vivo. We utilized a patient-derived xenograft model and confirmed an effective reduction of DNA methylation levels upon 5-azacitidine treatment yet failed to restore gene expression patterns. This study unbiasedly explored the adaptive engagement of other epigenomic modifications upon the profound decrease in DNA methylation by azacitidine treatment. A loss of histone acetylation and a gain of histone methylations, including H3K27 and H3K4 trimethylation, were observed around hypomethylated regions. Our findings suggested a compensatory increase in repressive histone mark, H3K27 trimethylation, around treatment-induced hypomethylated regions, which suggests the involvement of polycomb repressive complex (PRC) activity around the genome with lost DNA methylation, therefore maintaining the suppression of key genes. Combined inhibition of PRC activity through EZH2 inhibitor with azacitidine treatment additively improved efficacies in BRAFV600E CRC cells. In conclusion, DNA hypermethylation exhibits a close association with H3K27me3 and PRC activity in BRAFV600E CRC, and simultaneous blockade of DNMT and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated CRC. Overall design: Chromatin Immunoprecipitation DNA sequencing (ChIP-seq) on histone marks heH3K4me3, H3K27ac, H3K27me3, and H3K9me3 in BRAFV600E-mutated CRC patient-derived xenograft (PDX).

BRAFV600E突变型结直肠癌（BRAFV600E-mutated colorectal cancer, CRC）与DNA高甲基化（DNA hypermethylation）密切相关，提示该肿瘤亚群具有独特的表观基因组表型。然而传统表观基因组治疗药物5-氮杂胞苷（5-azacitidine，一种DNA甲基转移酶（DNA methyltransferase, DNMT）活性抑制剂），在BRAFV600E突变CRC的体内实验中并未取得足够的疗效提升。本研究利用患者来源的异种移植模型（patient-derived xenograft, PDX），证实5-氮杂胞苷处理可有效降低DNA甲基化水平，但未能恢复基因表达模式。本研究无偏倚地探索了阿扎胞苷（azacitidine，即5-氮杂胞苷）处理导致DNA甲基化大幅降低后，其他表观基因组修饰的适应性调控。研究观察到低甲基化区域周围出现组蛋白乙酰化丢失以及H3K27三甲基化、H3K4三甲基化等组蛋白甲基化水平升高的现象。本研究结果提示，在治疗诱导的低甲基化区域周围，抑制性组蛋白标记H3K27三甲基化（H3K27me3）出现代偿性升高，这表明多梳抑制复合体（polycomb repressive complex, PRC）在DNA甲基化缺失的基因组区域发挥调控作用，从而维持关键基因的转录沉默。联合使用EZH2抑制剂阻断PRC活性与阿扎胞苷处理，可协同提升BRAFV600E CRC细胞的治疗效果。综上，BRAFV600E突变CRC中DNA高甲基化与H3K27me3及PRC活性密切相关，同时阻断DNMT与EZH2有望成为BRAFV600E突变CRC患者的潜在治疗策略。整体实验设计：对BRAFV600E突变CRC患者来源的异种移植模型的组蛋白标记H3K4me3、H3K27ac、H3K27me3及H3K9me3进行染色质免疫沉淀测序（Chromatin Immunoprecipitation DNA sequencing, ChIP-seq）。