Identification of synthetic lethal interactors in pancreatic cancer

胰腺癌中合成致死相互作用因子的鉴定

• 批准号: 8967017
• 负责人: ADRIENNE D COX
• 金额: $ 74.47万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8967017
• 关键词: Adenocarcinoma Cell; Alleles; Biological Assay; CMV promoter; Cancer Etiology; Cancer Model; Cancer Patient; Cell Culture Techniques; Cell Line; Cessation of life; Clinical; Collaborations; Colon; Cytostatics; Data; Drug resistance; Engineering; Epitopes; Frequencies; Funding Opportunities; Genes; Genetic Screening; Genome; Goals; Growth; In Vitro; Insertional Mutagenesis; Institution; KRAS2 gene; Lung; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Modeling; Mus; Mutation; Organoids; Outcome; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway Analysis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Process; Property; Proteins; Publishing; RNA Interference; RNA Splicing; Reading; Relative (related person); Research Personnel; Research Support; Resistance; Role; Scheme; Serial Passage; Signal Pathway; Signal Transduction; Subfamily lentivirinae; System; Testing; Therapeutic; Validation; Viral; Wood material; Xenograft procedure; addiction; base; cancer cell; cancer therapy; cytotoxic; cytotoxicity; drug sensitivity; functional genomics; gain of function; genetic selection; genome-wide; in vivo; inhibitor/antagonist; innovation; mouse model; mutant; novel; novel strategies; overexpression; pancreatic cancer cells; programs; public health relevance; screening; small molecule libraries; targeted treatment; tissue culture; tumor; tumor xenograft

 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.

 描述（由申请人提供）：该 FOA 支持的研究目标是“确定其抑制将诱导依赖于突变 KRas 等位基因表达的癌症合成致死性的靶标”，“重点关注四种最重要的基因中的一种或多种”。经常观察到的等位基因...在一种或多种主要突变型 KRas 依赖性癌症中，例如胰腺...”，以及利用先进筛选的组织“超出了 2D 培养中当前的筛选”。为了实现这一目标为了实现这一目标，我们在三个机构组建了一个由五名研究人员组成的综合团队，我们的团队将应用三种互补且高度创新的先进筛选来识别和验证抑制 KRAS 突变胰腺导管腺癌 (PDAC) 的靶标。我们的每个筛选都与之前发表的基于 RNAi 的合成致死筛选有很大不同，我们不仅关注 K-Ras G12D 和 G12V，而且关注第三个最常见的 G12R。 PDAC 中的 KRAS 突变以及我们认为其特性与其他 G12 突变体不同的特性我们提出了三个具体目标：(1) 强大的化学库筛选，将 K-Ras 效应信号传导的药理学抑制剂从细胞抑制活性转变为细胞毒性活性；重点遗传筛选，以确定其激活可克服突变 K-Ras 成瘾的癌症信号通路成分；以及（3）无偏倚的全基因组功能获得性插入诱变筛选，以识别其过度表达可克服成瘾的基因；目标 1 将使用选定化合物的强大化学库筛选（药物敏感性和耐药性测试，DSRT），以实现阳性结果的快速临床转化；目标 2 和 3 将采用互补的创新功能获得性遗传筛选。目标 2 将采用初步数据所示的以信号传导为中心的方法（癌症工具包），以便能够识别已知和未知的抑制剂耐药机制，而目标 3 将应用全基因组无偏倚的方法方法 (CDt/MS) 基于质谱，在蛋白质水平上进行独特的读取，从而比传统的功能基因组筛选更便宜、更快且信息更丰富，目标 1 和 2 具有相同的信号焦点，而目标 2 和3 共享一个概念主题。我们将在整个研究中使用低传代的 KRAS 突变胰腺癌异种移植 (PDX) 细胞系，而最初的 Aim 1 筛选将以传统的高通量进行。 2D 测定、命中验证将在 3D 培养模型中进行，包括胰腺类器官 Aim 2 和 3 筛选将在 2D 和 3D 培养以及荷瘤小鼠体内进行，命中将在 2D 中进行验证。然后，我们将应用路径和网络分析来进一步验证目标 1-3 的热门结果。三种筛选方法中重要命中的显着重叠将在所有平台上整合来自每种策略的信息，以确定 KRAS 突变胰腺癌细胞中药理抑制和细胞毒性诱导的最佳合成致死靶点。