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类胰腺癌中致癌 KRAS 下游信号通路的依赖性。虽然 KRAS 本身迄今为止已被证明难以直接药理学靶向，但一系列潜在的可药物化信号传导效应器将突变 KRAS 的信号传导至下游。这些效应器包括： 1) RAF/MEK/ERK 激酶级联； 2）PLC/PKC和PI3K/AKT介导的磷脂信号传导； 3) 由 RALA/B 和 RAC1 介导的 GTPase 信号转导。这些下游效应子的一小部分已被询问在胰腺癌前体的引发中是否发挥必要的作用。然而，尚未对维持已建立的胰腺癌的必要作用进行基因评估，这使得我们合理规划这些途径的治疗靶向的能力存在很大差距。因此，我们建议在小鼠和人类模型系统中对这些潜在的可成药下游介质进行基因剖析。该项目的中心假设是：首先，突变体 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传导介质；其次，下游介质的遗传和药理学联合靶向将揭示新的治疗弱点；第三，不同的 Kras 突变等位基因可能对个别信号通路的治疗破坏具有不同的敏感性。 为了检验这些假设，我们提出以下具体目标：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) 利用人类胰腺癌 3D 类器官培养系统，结合不同下游 KRAS 介质的遗传和药理学抑制，以确定新的治疗协同作用和脆弱性； 3) 创建并比较新的基于 ESC 的基因工程小鼠模型，涉及 Kras G12R 和 Q61H。该项目利用高度创新的“Speedy”GEMM ESC 小鼠技术，包括新的专用囊胚注射设施，以及患者来源的肿瘤类器官 3D 类器官培养的新技术。总之，这些研究将首次对已确定的 Kras 驱动的胰腺癌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，其中利用这些依赖性来获得治疗效果。

 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.