Overcoming Resistance to RAF Inhibition in BRAF-Mutant Colorectal Cancer

BRAF癌基因突变是结直肠癌（CRC）的常见事件。这些突变与不良结局和对基于表皮生长因子受体（EGFR）的治疗不敏感相关。尽管RAF抑制剂已成功治疗BRAF突变型恶性黑色素瘤，但BRAF突变型CRC的缓解率却令人惊讶地低。这些不同的治疗结果的基础仍然不完全清楚。我们小组的初步研究发现，PLX 4720对CRC细胞系中MAPK途径的抑制不完全。在某些情况下，这可能是由于增强的EGFR依赖性信号传导，但这并不能解释这种情况下的所有耐药。我们的目标是确定BRAF突变型结直肠癌中赋予RAF抑制剂从头耐药性的机制，希望能够实现更有效的治疗。首先，将通过TCGA数据集的分析来鉴定与BRAF突变型CRC相关的差异表达基因;这些基因将与基于正在进行的系统功能筛选来改变对MAPK通路抑制剂的应答的基因整合。将对排名靠前的基因进行机制研究，以阐明它们赋予耐药性的分子基础，同时，将扩大正在进行的功能筛选，以鉴定在BRAF突变结直肠细胞中具有RAF抑制作用的合成致死基因。在这里，主要候选基因的验证将优先考虑潜在的药物基因-几个候选基因已经被提名。将在细胞培养和异种移植模型中探索靶向药物与RAF抑制剂的合理组合。最后，联合RAF和MEK抑制剂的临床试验将在DF/HCC进行，试图通过增强MAPK通路的抑制来提高疗效，并可能防止耐药性的出现。将在治疗前、治疗中和进展后收集肿瘤活检，并将使用全外显子组和转录组测序来鉴定可能导致耐药的基因组改变。总之，这项工作应该提供一个严格的分析耐药性MAPK抑制剂和新的治疗方法，以克服他们。

Mutation of the BRAF oncogene is a common event in colorectal cancer (CRC). These mutations are associated with adverse outcome and insensitivity to epidermal growth factor receptor (EGFR) based therapy. Whereas RAF inhibitors have been successful in the treatment of BRAF-mutant malignant melanoma, response rates in BRAF-mutant CRC are surprisingly low. The basis for these disparate treatment outcomes remains incompletely understood. Preliminary studies from our groups found that suppression of the MAPK pathway by PLX4720 is incomplete in CRC lines. In some cases, this may be due to augmented EGFR-dependent signaling, but this does not explain all resistance in this setting. Our objective is to identify mechanisms operant in BRAF-mutant colorectal cancer that confer de novo resistance to RAF inhibitors, in hopes of enabling more efficacious therapeutics. First, differentially expressed genes linked to BRAF-mutant CRC will be identified by analysis of the TCGA dataset; these genes will be integrated with those that modify response to MAPK pathway inhibitors based on ongoing systematic functional screens. Top-ranking genes will be subjected to mechanistic studies to elucidate the molecular basis by which they confer resistance, in parallel, ongoing functional screens will be expanded to identify genes that are synthetic lethal with RAF inhibition in BRAF-mutant colorectal cells. Here, validation of leading candidates will be prioritized for genes that are potentially druggable-several candidates have already been nominated. Rational combinations of targeted agents with RAF inhibitors will be explored in cell culture and in xenograft models. Finally, clinical trials of combined RAF and MEK inhibitors will be performed at DF/HCC in an attempt to improve efficacy by enhancing suppression of the MAPK pathway and possibly prevent the emergence of drug-resistance. Tumor biopsies will be collected pre-treatment, on-treatment and post-progression, and whole exome and transcriptome sequencing will be used to identify genomic alterations that may drive resistance. Altogether, this work should provide a rigorous analysis of resistance to MAPK inhibitors and new therapeutic approaches to overcome them.