COP1 Deficiency in BRAFV600E Melanomas Confers Resistance to Inhibitors of the MAPK Pathway [CHIP-seq]

Aberrant activation of the mitogen-activated protein kinase (MAPK) cascade promotes oncogenic transcriptomes. Despite efforts to inhibit oncogenic kinases, such as BRAFV600E, tumor responses in patients can be heterogeneous and limited by drug resistance mechanisms. Here, we describe patient tumors that acquired COP1 or DET1 mutations after treatment with the BRAFV600E inhibi-tor vemurafenib. COP1 and DET1 constitute the substrate adaptor of the E3 ubiquitin ligase CRL4COP1/DET1, which targets transcription factors, including ETV1, ETV4, and ETV5, for pro-teasomal degradation. MAPK-MEK-ERK signaling prevents CRL4COP1/DET1 from ubiquitinating ETV1, ETV4, and ETV5, but the mechanistic details are still being elucidated. We found that pa-tient mutations in COP1 or DET1 inactivated CRL4COP1/DET1 in melanoma cells, stabilized ETV1, ETV4, and ETV5, and conferred resistance to inhibitors of the MAPK pathway. ETV5, in partic-ular, enhanced cell survival and was found to promote the expression of the pro-survival gene BCL2A1. Indeed, the deletion of pro-survival BCL2A1 re-sensitized COP1 mutant cells to vemu-rafenib treatment. These observations indicate that the post-translational regulation of ETV5 by CRL4COP1/DET1 modulates transcriptional outputs in ERK-dependent cancers, and its inactivation contributes to therapeutic resistance. ChIP-seq of ETV5 and 2 histone modifications (H3K4me3, H3K27ac ) in A375 cells was performed on the following genotypes in two replicates: Cop1 wildtype and Cop1 SNP as well as treatment with ERK inhibitor (Vertex11e for 1 h). We had two pooled input sample as control, one per genotype.

丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）级联反应的异常激活可促进致癌转录组的形成。尽管学界已针对BRAFV600E等致癌激酶开发了多种抑制策略，但患者的肿瘤应答仍存在显著异质性，且受限于各类耐药机制。本研究报道了经BRAFV600E抑制剂维莫非尼（vemurafenib）治疗后获得COP1或DET1突变的患者肿瘤样本。COP1与DET1共同构成E3泛素连接酶（E3 ubiquitin ligase）CRL4COP1/DET1的底物适配子，该复合物可靶向包括ETV1、ETV4及ETV5在内的转录因子进行蛋白酶体降解。MAPK-MEK-ERK信号通路可阻止CRL4COP1/DET1对ETV1、ETV4及ETV5进行泛素化修饰，但其具体分子机制仍有待阐明。我们的研究发现，黑色素瘤细胞中COP1或DET1的患者源性突变可使CRL4COP1/DET1复合物失活，进而稳定ETV1、ETV4及ETV5的蛋白水平，并使肿瘤细胞对MAPK通路抑制剂产生耐药性。其中ETV5尤为关键，它可增强细胞存活能力，并被发现能够促进促存活基因BCL2A1的表达。事实上，敲除促存活基因BCL2A1可使COP1突变细胞重新对维莫非尼治疗产生敏感性。上述研究结果表明，CRL4COP1/DET1对ETV5的翻译后调控可调节ERK依赖型癌症中的转录输出，而该通路的失活会促成治疗耐药性的产生。我们针对A375细胞开展了ETV5及两种组蛋白修饰（H3K4me3、H3K27ac）的染色质免疫共沉淀测序（ChIP-seq）实验，实验针对以下分组设置两次生物学重复：Cop1野生型细胞、Cop1 SNP细胞，以及使用ERK抑制剂Vertex11e处理1小时的细胞组。我们设置了两份混合输入样本作为对照，每种基因型对应一份对照样本。