Comprehensive genetic dissection of druggable KRAS targets

 描述(由申请人提供)：该研究项目旨在全面确定小鼠和人胰腺癌中致癌KRAS下游的信号通路依赖性。虽然到目前为止，KRAS本身已被证明对直接药物靶向无效，但一系列潜在的可药物信号效应器从突变的KRAS向下游传递信号。这些效应包括：1)RAF/MEK/ERK激酶级联；2)PLC/PKC和PI3K/AKT介导的磷脂信号转导；3)Rala/B和RAC1介导的GTP酶信号转导。这些下游效应器中的一小部分已经被询问在胰腺癌前体启动中的必要作用。然而，没有一个是关于维持已建立的胰腺癌所必需的作用的遗传学评估，这在我们合理规划这些通路的治疗靶向的能力方面留下了很大的空白。因此，我们建议在小鼠和人类模型系统中从基因上解剖这些潜在的可用药的下游介体。该项目的中心假设是：第一，突变的Kras诱导的复合表型的各个组成部分将依赖于不同的下游信号调节因子；第二，下游调节因子的遗传和药物靶向的结合将揭示新的治疗易感性；第三，不同的突变Kras等位基因可能对个别信号通路的治疗干扰具有不同的敏感性。 为了验证这些假设，我们提出了以下具体目标：1)创建新的基于GEMM ESC的小鼠模型，允许在已建立的胰腺肿瘤中定时灭活RAF1、PIK3CA、PLCε、RALA/B和RAC1；2)结合不同KRAS下游介体的遗传和药物抑制作用，以确定新的治疗协同效应和脆弱性；以及3)创建并比较包括Kras G12R和Q61H在内的新的基于胚胎干细胞的基因工程小鼠模型。该项目利用了高度创新的“快速”GEMM ESC小鼠技术，包括一种新的专用胚泡注射设施，以及用于患者来源的肿瘤有机化合物的3D有机培养的新技术。总而言之，这些研究将首次对已建立的Kras驱动的胰腺癌的下游信号依赖性进行全面分析。我们预计，这些研究将为未来临床试验的设计提供信息，在这些试验中，这些依赖性被利用来获得治疗收益。

 DESCRIPTION (provided by applicant): This research project seeks to comprehensively determine signaling pathway dependencies downstream from oncogenic KRAS in mouse and human pancreatic cancer. While KRAS itself has so far proven refractory to direct pharmacologic targeting, an array of potentially druggable signaling effectors convey signaling downstream from mutant KRAS. These effectors include: 1) the RAF/MEK/ERK kinase cascade; 2) Phospholipid signaling mediated by PLC/PKC and PI3K/AKT; and 3) GTPase signaling mediating by RALA/B and RAC1. A small subset of these downstream effectors have been interrogated for requisite roles in the initiation of pancreatic cancer precursors. However, none have been genetically evaluated with respect to a requisite role in the maintenance of established pancreatic cancer, leaving substantial gaps in our ability to rationally plan therapeutic targeting of these pathways. We therefore propose to genetically dissect these potentially druggable downstream mediators in both mouse and human model systems. The central hypotheses of this project are: First, that the individual components of the composite phenotype induced by mutant Kras will be dependent upon different downstream signaling mediators; second, that combined genetic and pharmacologic targeting of downstream mediators will reveal novel therapeutic vulnerabilities; and third, that different mutant Kras alleles may be differentially sensitive to therapeutic disruption of individual signaling pathways. To test these hypothesis, we propose the following Specific Aims: 1) To create new GEMM ESC-based murine models allowing for the timed inactivation of Raf1, Pik3ca, PLCε, RalA/B and Rac1 in established pancreatic tumors; 2) To combine genetic and pharmacologic inhibition of different downstream KRAS mediators to identify new therapeutic synergies and vulnerabilities using a human pancreatic cancer 3D organoid culture system; and 3) To create and compare new ESC-based genetically engineered mouse models involving Kras G12R and Q61H. The project leverages highly innovative "Speedy" GEMM ESC mouse technology, including a new dedicated blastocyst injection facility, as well as novel techniques for 3D organoid culture of patient-derived tumor organoids. Together, these studies will for the first tim provide a comprehensive analysis of downstream signaling dependencies in established Kras-driven pancreatic cancer. We anticipate that these studies will inform the design of future clinica trials in which these dependencies are exploited for therapeutic gain.