Identification of synthetic lethal interactors in pancreatic cancer

 描述(申请人提供)：本FOA支持的研究目标是“确定其抑制作用将在依赖突变的KRAS等位基因表达的癌症中诱导合成致命性的靶点”，重点是“在一个或多个主要的突变KRAS依赖的癌症中，如胰腺……中最常观察到的四个等位基因中的一个或多个”，并利用先进的筛查“在2D组织培养中超越当前的筛查”。为了实现这一目标，我们在三个机构组建了一个由五名调查人员组成的整合良好的团队。我们的团队将应用三种互补和高度创新的先进筛选来识别和验证其抑制将在KRAS突变的胰腺导管腺癌(PDAC)中诱导合成致命性的靶点。我们的每一个屏幕都与之前发表的基于RNAi的合成致命屏幕有很大不同。我们将不仅关注K-RAS G12D和G12V，还将关注G12R，这是PDAC中第三个最常见的KRAS突变，我们认为其特性不同于其他G12突变体。我们提出了三个具体目标：(1)一个强大的化学文库筛选，以将K-RAS效应信号的药物抑制物从细胞抑制活性转化为细胞毒活性；(2)一个集中的遗传筛选，以识别其激活克服对突变K-RAS成瘾的癌症信号通路组件；以及(3)一个无偏见的，全基因组功能获得的插入突变筛选，以确定其表达克服对突变KRAS成瘾的基因。AIM 1将使用一个强大的化学资料库筛选(药物敏感性和耐药性测试，DSRT)，专门选择化合物，以允许阳性结果的快速临床过渡。AIMS 2和AIMS 3将采用互补的创新功能获得基因筛查。AIM 2将采用初步数据显示的以信号为中心的方法(癌症工具包)，能够识别已知和未知的抑制剂耐药机制，而AIM 3将应用基于质谱学的全基因组无偏方法(CDT/MS)，并在蛋白质水平上唯一地读出，从而实现比传统功能基因组筛选更便宜、更快和更具信息量的过程。目标1和目标2共享一个信号焦点，而目标2和目标3共享一个概念主题。在我们的研究中，我们将使用低传代KRAS突变的胰腺癌患者来源的异种移植(PDX)来源的细胞系。虽然最初的Aim 1筛查将在传统的高通量2D分析中进行，但HITS的验证将在包括胰腺器官在内的3D培养模型中进行。目的2和3将在2D和3D培养以及荷瘤小鼠体内进行筛选，并将在2D和3D培养中验证命中。然后，来自AIMS 1-3的最高命中率将在PDX原位胰腺癌模型中进一步验证。我们将应用路径和网络分析，并期望在三种筛选方法中找到重要的重叠。来自这些策略的信息将在所有平台上整合，以确定在KRAS突变的胰腺癌细胞中进行药理抑制和诱导细胞毒性的最佳合成致死靶点。

 DESCRIPTION (provided by applicant): The goal of research supported by this FOA is "to identify targets whose inhibition would induce synthetic lethality in cancers dependent on the expression of mutant KRas alleles", with a "focus on one or more of the four most frequently observed alleles...in one or more of the predominant mutant KRas-dependent cancers e.g., pancreas...", and utilizing advanced screens "that go beyond the current screens in 2D tissue culture". To accomplish this goal, we have assembled a well-integrated team of five investigators at three institutions. Our team will apply three complementary and highly innovative advanced screens to identify and validate targets whose inhibition would induce synthetic lethality in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). Each of our screens differs substantially from those in previously published RNAi-based synthetic lethal screens. We will focus not only on K-Ras G12D and G12V but also on G12R, the third most frequent KRAS mutation in PDAC and one whose properties we believe differ from those of other G12 mutants. We propose three specific aims: (1) a robust chemical library screen to convert pharmacologic inhibitors of K-Ras effector signaling from cytostatic to cytotoxic activities; (2) a focused genetic screen to identify cancer signaling pathway components whose activation overcomes addiction to mutant K-Ras; and (3) an unbiased, genome-wide gain-of-function insertional mutagenesis screen to identify genes whose overexpression overcomes addiction to mutant KRAS. Aim 1 will use a powerful chemical library screen (Drug Sensitivity and Resistance Testing, DSRT) of compounds selected specifically to allow rapid clinical transition of positive results. Aims 2 and 3 will employ complementary innovative gain-of-function genetic screens. Aim 2 will take a signaling-centric approach (Cancer Toolkit) shown in preliminary data to be able to identify both known and unknown mechanisms of inhibitor resistance, whereas Aim 3 will apply a genome-wide unbiased approach (CDt/MS) that is mass spectrometry-based and uniquely reads out at the protein level, thereby enabling a cheaper, faster and more informative process than conventional functional genomic screens. Aims 1 and 2 share a signaling focus, whereas Aims 2 and 3 share a conceptual theme. We will utilize low passage KRAS-mutant pancreatic cancer patient-derived xenograft (PDX)-derived cell lines throughout our studies. While the initial Aim 1 screens will be done in conventional high throughput 2D assays, validation of the hits will be done in 3D culture models including pancreatic organoids. Aim 2 and 3 screens will be done in both 2D and 3D culture as well as in vivo in tumor-bearing mice, and hits will be validated in 2D and 3D culture. The top hits from Aims 1-3 will then be further validated in PDX orthotopic pancreatic cancer models. We will apply pathway and network analysis, and expect to find significant overlap of important hits among the three screening approaches. Information from each of these strategies will be integrated across all platforms to identify the best synthetic lethal targets for pharmacologic inhibition and induction of cytotoxicity in KRAS-mutant pancreatic cancer cells.