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

项目1：在过去的几年里，伴随着新药的临床测试而来的是相当大的兴奋 死于黑色素瘤。特别是，RAF抑制剂PLX4032在给药时显示出显著的效果 对携带BRAFV600E突变的黑色素瘤患者。不幸的是，这种药物的总体临床益处 针对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。 关于治疗的问题。 我们实验室的工作已经发现了与MEK突变体的出现有关的不同的抗性机制 MEK抑制剂CI-1040处理BRAFV600E细胞后的等位基因及CRAF的过表达 用BRAF抑制剂PLX-4720处理BRAFV600E细胞后，MAPK38/CoT激酶的表达也不受影响。多么 然而，总的来说，这些新近发现的抗性机制仍然没有明确的定义，而且， 毫无疑问，抵抗RAF抑制的其他机制仍有待发现。 我们建议的目的是系统地寻找存在于植物体内的新型抗性机制， 以及肿瘤来源的MAPK抑制耐药肿瘤的脆弱性。将通过1)解决此问题 询问患者来源的MAPK抑制抗性中存在的遗传性和非遗传性改变 肿瘤，通过整个外显子组和转录组测序，以及2)识别独有的新的依赖关系 通过联合RNAi筛选和合成致死性RNAi筛选MAPK抑制耐药肿瘤 MAPK抑制剂的存在。此外，我们将建立临床前的活体小鼠模型，以表征 最近发现的抗性机制。 在完成这项研究后，我们将对抗药性的机制有了更多的了解 在黑色素瘤中，并确定了可能的组合治疗的新药物靶点，可以压倒 抵抗。

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.