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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：该FOA支持的研究目标是“确定其抑制作用将诱导癌症中的合成致死性，依赖于突变的Kras等位基因表达的癌症”，“重点是“四个或多个经常观察到的一个或多个”中的一个或多个等位基因中的一个或多个较高的kras kras kras kras cras cras cras cras cras cars cancer cancers cancreser cancr and cancr cancr cancr cancr cancr cancr cancr cancr cancr cancr cancr cancr cancr c。在2D组织培养中的筛选”。为了实现这一目标，我们在三个机构组成了一支由五名调查人员组成的全面团队。我们的团队将应用三个完整且高度创新的高级屏幕，以识别和验证抑制作用将引起KRAS突变胰腺导管腺癌（PDAC）的抑制作用。我们的每个筛选都与先前发表的基于RNAi的合成致死筛查有很大不同。我们将不仅专注于K-RAS G12D和G12V，还专注于G12R，G12R是PDAC中第三次最常见的KRAS突变，并且我们认为其性质与其他G12突变体的特性不同。我们提出了三个特定的目的：（1）一个可靠的化学库筛选，将K-RAS效应子信号传导的药物抑制剂从细胞抑制剂转化为细胞毒性活性；（2）识别癌症信号通路成分的聚焦遗传筛选，其激活使成瘾成瘾对突变体K-ras；（3）一个无偏的，全基因组的功能获得的插入诱变筛选，以鉴定其过表达的基因克服了成瘾对突变体KRAS的基因。 AIM 1将使用专门选择的化合物使用强大的化学库筛选（药物敏感性和耐药性测试，DSRT），以允许快速临床过渡的阳性结果。 AIM 2和3将采用完整的创新功能遗传筛选。 AIM 2将采用初步数据中显示的以信号为中心的方法（癌症工具包），以识别抑制剂耐药性的已知和未知机制，而AIM 3将采用全基因组无偏见的方法（CDT/MS），是质谱基于质谱的方法，并在蛋白质级别上读取蛋白质级别，从而启用了构造的范围，从而启动了一个依据，并提供了更详细的依据。目标1和2共享信号焦点，而目标2和3共享一个概念主题。在整个研究中，我们将利用低通道KRAS突变胰腺癌患者衍生的异种移植（PDX）衍生的细胞系。虽然最初的目标1屏幕将在常规的高吞吐量2D分析中完成，但命中率将在包括胰腺器官在内的3D培养模型中进行。 AIM 2和3屏幕将在2D和3D培养物以及肿瘤小鼠的体内进行，并将在2D和3D培养中进行验证。然后，AIMS 1-3的最高点击将在PDX原位胰腺癌模型中进一步验证。我们将采用途径和网络分析，并希望在三种筛选方法中发现重要的命中率显着重叠。这些策略的信息将在所有平台上集成，以确定用于药物口语抑制和诱导KRAS突变胰腺癌细胞中细胞毒性的最佳合成杀伤力靶标。