Defining and Modeling Resistance to RAF/MEK Inhibition in Human Melanoma

项目1：在过去的几年里，伴随着新药的临床试验， 黑色素瘤特别地，RAF抑制剂PLX 4032在施用时显示出显著的功效。 BRAFV 600 E突变的黑色素瘤患者。不幸的是， 黑色素瘤的靶向药物仍然有限，主要是由于几个月后出现耐药性 的治疗。 我们实验室的工作发现了不同的抗性机制，包括MEK突变体的出现 用MEK抑制剂CI-1040处理BRAFV 600 E细胞后，CRAF等位基因的表达 或MAPK 38/COT激酶在用BRAF抑制剂PLX-4720处理BRAFV 600 E细胞后的变化。如何 然而，通常，这些最近发现的抗性机制仍然不明确，并且， 毫无疑问，对RAF抑制的抗性的其它机制仍有待发现。 我们的建议的目的是进行系统的搜索新的耐药机制， 和肿瘤衍生的MAPK抑制抗性肿瘤的脆弱性。这将通过以下方式解决：1） 询问患者来源的MAPK抑制抗性细胞中存在的遗传以及“非遗传”改变， 肿瘤，通过全外显子组和转录组测序，和2）确定独特的新的依赖性， MAPK抑制抗性肿瘤，通过合并RNAi筛选，和合成致死RNAi筛选， MAPK抑制剂的存在。此外，我们将建立临床前体内小鼠模型，以表征 最近发现的抵抗机制 完成这项研究后，我们将获得对抗性机制的理解， 在黑色素瘤，并确定了可能的新的药物靶点，为组合治疗，可以压倒 阻力

Project 1: Over the past years, considerable excitement has accompanied the clinical testing of new drugs in melanoma. In particular, the RAF-inhibitor, PLX4032, has showed remarkable efficacy when administered to melanoma patients harboring the BRAFV600E mutation. Unfortunately, the overall clinical benefit of targeted agents in melanoma remains limited, mainly due to the appearance of resistance after a few months of treatment. Work in our lab has discovered different resistance mechanisms involving the appearance of MEK mutant alleles upon treatment of BRAFV600E cells with the MEK inhibitor CI-1040, and the overexpression of CRAF or of MAPK38/COT kinase upon treatment of BRAFV600E cells with the BRAF inhibitor PLX-4720. How general, however, these recently-discovered resistance mechanisms are, remains poorly defined, and, undoubtedly, additional mechanisms of resistance to RAF inhibition remain to be discovered. The purpose of our proposal is to perform a systematic search for novel resistance mechanisms present in, and vulnerabilities of, tumor-derived MAPK-inhibition resistant tumors. This will be addressed by 1) interrogating genetic, as well as "non-genetic" alterations present in patient-derived MAPK-inhibition resistant tumors, by whole-exome and transcriptome sequencing, and 2) identifying novel dependencies unique to MAPK-inhibition resistant tumors, by pooled RNAi screening, and synthetic lethal RNAi screening in the presence of MAPK-inhibitors. In addition, we will establish pre-clinical in vivo mouse models to characterize the recently discovered resistance mechanisms. Upon completion of this research, we will have gained understanding of the resistance mechanisms operant in melanoma, and identified possible new drug targets for combinatorial treatments that could overpower the resistance.