Epigenome reprogramming through H3K27 and H3K4 trimethylation as a resistance mechanism to DNA methylation inhibition in BRAFV600E-mutated CRC. [RNA-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: To investigate the gene expression changes by 5-azacitidine treatment in BRAFV600E mutant CRC PDX model, mice were treated with either vehicle or 5-azacitidine.

BRAFV600E突变型结直肠癌（colorectal cancer, CRC）与DNA高甲基化存在显著相关性，提示该肿瘤亚群具有独特的表观基因组表型。尽管如此，传统表观基因组治疗药物5-氮杂胞苷（5-azacitidine）可抑制DNA甲基转移酶活性，但在体内对BRAFV600E突变型结直肠癌的疗效提升并不充分。本研究采用患者来源的异种移植（patient-derived xenograft, PDX）模型，证实5-氮杂胞苷处理可有效降低DNA甲基化水平，但无法恢复基因表达模式。本研究通过无偏分析，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情况。研究人员在低甲基化区域附近观察到组蛋白乙酰化水平降低，而组蛋白甲基化水平升高，其中包括H3K27三甲基化与H3K4三甲基化。本研究结果显示，在药物诱导的低甲基化区域附近，抑制性组蛋白标记H3K27三甲基化（H3K27me3）出现代偿性升高，这提示DNA甲基化缺失的基因组区域存在多梳抑制复合体（polycomb repressive complex, PRC）的活性调控，从而维持了关键基因的沉默状态。通过EZH2抑制剂（EZH2 inhibitor）联合阿扎胞苷处理抑制PRC活性，可协同提升BRAFV600E突变型结直肠癌细胞的治疗效果。综上，在BRAFV600E突变型结直肠癌中，DNA高甲基化与H3K27三甲基化（H3K27me3）及PRC活性密切相关；同时阻断DNA甲基转移酶（DNA methyltransferase, DNMT）与EZH2的活性，有望成为BRAFV600E突变型结直肠癌患者的潜在治疗策略。实验设计：为探究5-氮杂胞苷处理对BRAFV600E突变型结直肠癌PDX模型的基因表达变化的影响，实验小鼠分别接受溶剂对照或5-氮杂胞苷处理。