Development of a novel platform for the identification of synthetic lethal genes in a Kras and Keap1-mutant mouse model of lung adenocarcinoma.

开发一个新平台，用于鉴定 Kras 和 Keap1 突变小鼠肺腺癌模型中的合成致死基因。

• 批准号: 9469983
• 负责人: Rodrigo Romero
• 金额: $ 4.29万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9469983
• 关键词: Address; Affect; Alpha Cell; Antioxidants; Biology; Cancer cell line; Candidate Disease Gene; Cell Line; Cell Transplants; Cells; Chemicals; Clinical Management; Clustered Regularly Interspaced Short Palindromic Repeats; Coupling; Custom; Development; Disease; Drug Targeting; Enterobacteria phage P1 Cre recombinase; Epithelium; Event; Exhibits; Frequencies; Future; Gene Expression Profile; Gene Mutation; Generations; Genes; Genetic; Genetic Predisposition to Disease; Genetically Engineered Mouse; Genomics; Genotype; Glutamine; Glutathione; Goals; Human; In Vitro; KRAS2 gene; Knock-out; Knowledge; Large-Scale Sequencing; Lentivirus; Lentivirus Vector; Lethal Genes; Libraries; Lung; Lung Adenocarcinoma; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Mediator of activation protein; Metabolic Pathway; Modeling; Mus; Mutate; Mutation; Neoplasm Transplantation; Non-Small-Cell Lung Carcinoma; Oncogenic; Oxidative Stress; Pathway interactions; Patients; Pharmacology; Proteins; Publishing; Role; TP53 gene; Testing; Transplantation; Tumor Burden; Validation; base; biological adaptation to stress; cancer cell; cancer genome; cancer therapy; cancer type; cell growth; experimental study; feeding; fitness; functional loss; genome sequencing; high throughput screening; in vivo; inhibitor/antagonist; loss of function; loss of function mutation; mouse model; mutant; mutant mouse model; neoplastic cell; new therapeutic target; novel; nuclear factor-erythroid 2; oxidative damage; precision oncology; response; small molecule inhibitor; targeted treatment; therapy design; transcription factor; tumor; tumor initiators

PROJECT SUMMARY/ABSTRACT The goal of precision oncology is to tailor present and future cancer therapies to specific patients based on the systematic genomic assessment of their tumors. Therefore, large-scale cancer genome sequencing efforts represent an important first step towards achieving this goal. Such studies have identified Kelch-like ECH Associated Protein 1 (KEAP1), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2, to be mutated in approximately 30% of all lung cancers. NRF2 is a transcription factor that regulates a network of genes that coordinate the cellular response to oxidative stress. Inactivating mutations in KEAP1 result in constitutive NRF2 activity and activation of the oxidative stress response pathway in cancer cells. Of note, 30% of lung adenocarcinoma patients carry oncogenic KRAS mutations. Of these KRAS mutant tumors, 20% display loss of function mutations in KEAP1, implicating the importance of the oxidative stress response pathway in initiation and/or maintenance in this tumor type. The high frequency of mutation of genes in this pathway makes it an attractive target for therapy in lung cancer and other cancer types. Objectives: This study will (1) define the role of Keap1 loss of function in vivo in the normal lung and in cancer development; (2) high-throughput identification of drug targets in Keap1-mutant cell lines utilizing a custom sgRNA library targeting ~5000 genes with known chemical inhibitors (3) test the requirement of top candidate genes for tumor maintenance in murine lung adenocarcinoma that may reveal new pathways and genes that converge with Kras and Keap1 function that can be pharmacologically targeted to treat cancer. Three specific aims are proposed to address these objectives: Aim 1: Determine the role of Keap1 loss of function mutations using in vivo A published lentiviral vector coupling Cre and Cas9 expression will be used to simultaneously initiate tumors and edit Keap1 in the lung epithelium of genetically engineered mice containing oncogenic Kras and p53 loss of function mutations. Changes in tumor burden and tumor grade will be analyzed. Aim 2: Identification of novel drug targets specific to Keap1-mutant tumor cells Synthetic genetic interactions in Keap1-mutant cells will be determined by high-throughput screening efforts utilizing an sgRNA library targeting ~5000 putative or bona-fide drug targets. These genetic interactions will be validated using various murine isogenic lung adenocarcinoma cell lines containing loss of function mutations in Keap1. Aim 3: Functional validation of putative Keap1-mutant synthetic lethal genes in vivo A conditional model of lung adenocarcinoma will be used to explore and validate the functional loss of synthetic lethal gene candidates in a Keap1-mutant specific fashion utilizing in vivo CRISPR gene editing.

项目总结/摘要 精准肿瘤学的目标是根据特定患者的情况， 对他们的肿瘤进行系统的基因组评估因此，大规模癌症基因组测序 这些努力是实现这一目标的重要的第一步。这些研究已经确定了Kelch样 ECH相关蛋白1（KEAP 1），核因子红细胞2-样2（NFE 2L 2;下文中 NRF 2在大约30%的肺癌中发生突变。NRF 2是一种转录因子，它调节细胞内的 协调细胞对氧化应激反应的基因网络。KEAP 1失活突变 导致癌细胞中组成型NRF 2活性和氧化应激反应途径的活化。的 值得注意的是，30%的肺腺癌患者携带致癌KRAS突变。在这些KRAS突变肿瘤中， 20%显示KEAP 1功能缺失突变，暗示氧化应激反应的重要性 在这种肿瘤类型中启动和/或维持的途径。这种基因突变的高频率 这使得它成为肺癌和其他癌症类型治疗的一个有吸引力的靶标。 目的：本研究将（1）确定Keap 1功能丧失在正常肺和肺组织中的作用。 癌症的发展;（2）在Keap 1突变细胞系中高通量鉴定药物靶点， 定制的sgRNA文库靶向约5000个基因与已知的化学抑制剂（3）测试的要求， 小鼠肺腺癌中肿瘤维持的候选基因，可能揭示新的途径， 与Kras和Keap 1功能趋同的基因，可以被靶向治疗癌症。三 为实现这些目标提出了具体目标： 目的1：使用体内方法确定Keap 1功能缺失突变的作用 已发表的偶联Cre和Cas9表达的慢病毒载体将用于同时启动 在含有致癌Kras基因的基因工程小鼠的肺上皮中的肿瘤和编辑Keap 1 和p53功能缺失突变。将分析肿瘤负荷和肿瘤分级的变化。 目的2：鉴定对Keap 1突变肿瘤细胞具有特异性的新型药物靶标 将通过高通量筛选确定Keap 1突变细胞中的合成遗传相互作用 利用靶向约5000个推定或真正的药物靶标的sgRNA文库的努力。这些遗传 将使用各种鼠等基因肺腺癌细胞系验证相互作用， Keap 1的功能突变。 目的3：推定的Keap 1突变体合成致死基因的体内功能验证 肺腺癌的条件模型将用于探索和验证肺腺癌的功能丧失。 在一个实施方案中，本发明涉及利用体内CRISPR基因编辑以Keap 1突变体特异性方式合成致死基因候选物。