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）：本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点”，“重点关注四种最常见的等位基因中的一种或多种......在一种或多种主要的突变型KRAS依赖性癌症中，胰腺...“，并利用先进的屏幕“，超越了目前的屏幕在二维组织培养”。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其抑制将诱导KRAS突变胰腺导管腺癌（PDAC）合成致死性的靶点。我们的每一个屏幕都与以前发表的基于RNAi的合成致死屏幕有很大不同。我们将不仅关注K-Ras G12 D和G12 V，还关注G12 R，这是PDAC中第三常见的KRAS突变，我们认为其性质与其他G12突变体不同。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效应器信号传导的药理学抑制剂从细胞抑制活性转化为细胞毒性活性;（2）一个集中的遗传筛选，以鉴定其活化克服对突变K-Ras的成瘾的癌症信号传导通路组分;和（3）无偏倚的全基因组功能获得性插入诱变筛选，以鉴定其过表达克服对突变型KRAS的成瘾的基因。目标1将使用一个强大的化学库筛选（药物敏感性和耐药性测试，DSRT）的化合物，专门选择，以允许快速临床过渡的积极结果。目标2和3将采用互补的创新功能获得性基因筛选。目标2将采用以信号为中心的方法（癌症工具包），在初步数据中显示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方法（CDt/MS），该方法基于质谱，在蛋白质水平上独特地读出，从而实现比传统功能基因组筛选更便宜，更快和更信息化的过程。目标1和目标2有一个共同的信号重点，而目标2和目标3有一个共同的概念主题。我们将在整个研究中使用低传代KRAS突变胰腺癌患者来源的异种移植物（PDX）来源的细胞系。虽然最初的Aim 1筛选将在传统的高通量2D测定中完成，但命中的验证将在包括胰腺类器官的3D培养模型中完成。目标2和3筛选将在2D和3D培养以及荷瘤小鼠体内进行，并将在2D和3D培养中验证命中。然后将在PDX原位胰腺癌模型中进一步验证来自目标1-3的最高命中。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命中的显著重叠。来自这些策略中的每一种的信息将在所有平台中整合，以确定用于药理学抑制和诱导KRAS突变胰腺癌细胞中细胞毒性的最佳合成致死靶标。