Targeting BRAF and MEK in tumors with BRAF and RAS mutations.

描述（由申请方提供）：MAP激酶通路的组成性激活是人类肿瘤中的常见事件，该通路中的激活突变以互斥方式发生在RAS、BRAF和上游受体酪氨酸激酶（RTK）中。我们发现具有激活BRAF（V600 E）突变的肿瘤细胞对MEK 1/2的选择性抑制剂PD 0325901选择性敏感。其中MAPK被其他上游事件（突变RAS、RTK激活）激活的肿瘤通常对MEK抑制不太敏感或具有抗性。该提案的主要目的是确定RAF/MEK通路抑制剂敏感性和耐药性的遗传预测因子。提出了三个目标。在目标1中，我们将比较RAF、MEK和siRNA敲低这些靶标的药理学抑制。这些研究将用于验证RAF选择性抑制剂PLX 4032的体内选择性，并确定致癌BRAF是否通过MEK激酶以外的效应子促进细胞存活。BRAF突变通过MEK非依赖性效应子促进肿瘤发生的可能性具有重要意义。如果突变型BRAF的所有致癌作用都是通过MEK/MAPK介导的，那么MEK和pan-RAF抑制剂应该同样有效。然而，如果BRAF的重要作用是由其他效应物介导的，则预测RAF抑制剂将是上级的。我们的初步数据还表明，一些BRAF突变型肿瘤对MEK抑制的纯细胞生长抑制反应和许多RAS突变型肿瘤对MEK抑制的抗性可能是平行信号传导途径（如PI 3 K/AKT途径）中其他遗传改变的结果。BRAF突变通常与黑色素瘤中的PTEN缺失共存。因此，在目的2中，我们将确定PTEN功能的丧失是否足以将V600 E BRAF突变肿瘤对MEK（和RAF）抑制的反应从细胞毒性反应转化为细胞抑制反应。最后，在目标3中，我们将确定在具有RAS突变的肿瘤中，同时发生的p1101 PI 3激酶（PIK 3CA）突变是否赋予对RAS敲低（通过shRNA）或MEK抑制的抗性。如果PTEN缺失或PIK 3CA突变足以使BRAF和RAS突变细胞对RAF/MEK途径抑制产生耐药性，我们将在此类肿瘤中测试RAF/MEK和PI 3 K/AKT途径抑制剂的组合。这些努力的长期目标将是通过确定最有可能产生反应的肿瘤的遗传特征来加速MEK和RAF抑制剂的开发。此外，通过确定RAF和MEK抑制剂耐药的遗传预测因子，概述的研究将为研究信号传导抑制剂的合理组合提供分子基础，这些信号传导抑制剂适用于肿瘤在RAS/RAF和PI 3 k/Akt途径中均存在突变的患者。 公共卫生相关性：RAS和BRAF突变是人类肿瘤中最常见的遗传改变之一，表达这些突变的癌症对标准细胞毒性化疗的反应很差。该提案的主要目标是确定RAS/RAF/MEK通路抑制剂敏感性和耐药性的遗传预测因子，并利用这些信息开发癌症治疗的新策略。

DESCRIPTION (provided by applicant): Constitutive activation of the MAP kinase pathway is a common event in human tumors and activating mutations in this pathway occur in RAS, BRAF and upstream receptor tyrosine kinases (RTK) in a mutually exclusive fashion. We find that tumor cells with activating BRAF(V600E) mutations are selectively sensitive to PD0325901, a selective inhibitor of MEK1/2. Tumors in which MAPK is activated by other upstream events (mutant RAS, RTK activation) are typically less sensitive or resistant to MEK inhibition. The primary objective of this proposal is to identify genetic predictors of sensitivity and resistance to inhibitors of the RAF/MEK pathway. Three aims are proposed. In Aim 1, we will compare pharmacologic inhibition of RAF, MEK and siRNA knockdown of these targets. These studies will serve to validate the in vivo selectivity of the RAF selective inhibitor PLX4032 and determine whether oncogenic BRAF promotes cell survival via effectors other than MEK kinase. The possibility that BRAF mutations promote tumorigenesis through MEK-independent effectors has important implications. If all of the oncogenic effects of mutant BRAF are mediated through MEK/MAPK, MEK and pan-RAF inhibitors should be equally effective. However, if important effects of BRAF are mediated by other effectors, RAF inhibitors would be predicted to be superior. Our preliminary data also suggests that the purely cytostatic response of some BRAF mutant tumors to MEK inhibition and the resistance of many RAS mutant tumor to MEK inhibition may be the result of additional genetic alterations in parallel signaling pathway such as the PI3K/AKT pathway. BRAF mutations commonly co-exist with PTEN loss in melanoma. Therefore, in Aim 2, we will determine whether loss of PTEN function is sufficient to convert the response of V600E BRAF mutant tumors to MEK (and RAF) inhibition from a cytotoxic to a cytostatic response. Finally, in Aim 3, we will determine in tumors with RAS mutations whether concurrent p1101 PI3 kinase (PIK3CA) mutations confer resistance to RAS knockdown (by shRNA) or MEK inhibition. If PTEN loss or PIK3CA mutations are sufficient to confer resistance of BRAF and RAS mutant cell to RAF/MEK pathway inhibition, we will test combinations of RAF/MEK and PI3K/AKT pathway inhibitors in such tumors. The long-term goal of these efforts will be to accelerate the development of MEK and RAF inhibitors by identifying the genetic profile of those tumors most likely to respond. Further, by identifying genetic predictors of resistance to RAF and MEK inhibitors, the studies outlined would provide a molecular basis for studies of rational combinations of signaling inhibitors tailored to patients whose tumors have mutations in both the RAS/RAF and PI3k/Akt pathways. PUBLIC HEALTH RELEVANCE: RAS and BRAF mutations are among the most common genetic alterations in human tumors and cancers expressing these mutations respond poorly to standard cytotoxic chemotherapies. The primary goals of this proposal are to identify genetic predictors of sensitivity and resistance to inhibitors of the RAS/RAF/MEK pathway and use this information to develop novel strategies for cancer therapy.