COLO829 treatment with PLX4032 and/or MITF knockdown. Homo sapiens

Thousands of enhancers are characterized in the human genome, yet few have been shown important in cancer. Inhibiting oncokinases, such as EGFR, ALK, HER2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by upregulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific MET enhancers for multiple common tumor types, including a melanoma lineage-specific MET enhancer that displays inducible chromatin looping and MET gene induction upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of tumor lineage-enriched transcription factors mediating innate resistance to oncokinase therapy. Overall design: COLO829 human melanoma cell line harboring the BRAFV600E mutation was treated with BRAF inhibtior PLX4032 (Vemurafenib) and/or a hairpin against MITF

人类基因组中已有数千个增强子（enhancer）得到功能注释，但其中极少被证实与癌症发生发展相关。靶向抑制致癌激酶（oncokinase），如EGFR、ALK、HER2与BRAF，是当前癌症治疗的核心手段，但该类疗法常因HGF受体（HGF receptor, MET）上调介导的先天性耐药而受阻。介导此类靶向治疗应答的基因组调控机制目前仍未阐明。
本研究针对多种常见肿瘤类型鉴定出了谱系特异性的MET增强子，其中包括黑色素瘤谱系特异性MET增强子——该增强子在BRAF抑制剂处理后可诱导染色质环（chromatin looping）形成并介导MET基因的表达上调。表观基因组学（epigenomic）分析显示，黑色素细胞特异性转录因子MITF介导了该增强子的调控功能。对MET增强子内的MITF结合基序实施小于7bp的靶向基因组缺失，可抑制可诱导的染色质环形成与先天性耐药，同时保留MITF依赖型、抑制剂诱导的黑色素瘤细胞分化功能。由此可见，表观基因组学分析可指导调控DNA的功能性干预，从而解除介导致癌激酶治疗先天性耐药的肿瘤谱系富集转录因子的促癌与抑癌功能之间的偶联。
实验整体设计：携带BRAFV600E突变的人黑色素瘤细胞系COLO829分别经BRAF抑制剂PLX4032（维莫非尼，Vemurafenib）、靶向MITF的短发夹RNA（hairpin against MITF）单药或联合处理。