Comprehensive genetic dissection of druggable KRAS targets

 描述（由申请人提供）：本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KRAS本身迄今已被证明对直接药理学靶向是难治的，但一系列潜在的可药物化信号传导效应物从突变KRAS向下游传递信号传导。这些效应器包括：1）RAF/MEK/ERK激酶级联; 2）由PLC/PKC和PI 3 K/AKT介导的磷脂信号传导;和3）由RALA/B和RAC 1介导的GTT信号传导。这些下游效应子的一小部分已经被询问在胰腺癌前体的起始中的必要作用。然而，没有一个基因在维持已建立的胰腺癌中的必要作用方面进行了遗传评估，这使得我们在合理规划这些途径的治疗靶向方面存在很大的差距。因此，我们建议在小鼠和人类模型系统中对这些潜在的可药用下游介质进行遗传解剖。该项目的中心假设是：第一，由突变Kras诱导的复合表型的单个组分将依赖于不同的下游信号传导介质;第二，下游介质的组合遗传和药理学靶向将揭示新的治疗弱点;第三，不同的突变Kras等位基因可能对单个信号传导通路的治疗中断具有不同的敏感性。 为了验证这些假设，我们提出了以下具体目的：1）建立新的基于GEMM ESC的小鼠模型，允许在已建立的胰腺肿瘤中定时失活Raf 1、Pik 3ca、PLCε、RalA/B和Rac 1; 2）使用人胰腺癌3D类器官培养系统将不同下游KRAS介质的遗传和药理学抑制结合联合收割机以鉴定新的治疗协同作用和弱点; 3）建立并比较Kras G12 R和Q61 H基因工程小鼠模型。该项目利用高度创新的“快速”GEMM ESC小鼠技术，包括新的专用囊胚注射设施，以及用于患者源性肿瘤类器官的3D类器官培养的新技术。总之，这些研究将首次提供一个全面的分析下游信号依赖性在既定的Kras驱动的胰腺癌。我们预计这些研究将为未来临床试验的设计提供信息，在这些临床试验中，这些依赖性被用于治疗增益。

 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.