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

项目摘要/摘要 精确肿瘤学的目标是为特定的患者量身定做目前和未来的癌症治疗。 对他们的肿瘤进行系统的基因组评估。因此，大规模的癌症基因组测序 这些努力是实现这一目标的重要第一步。这样的研究已经发现了类似凯尔奇的 ECH相关蛋白1(Keap1)，核因子红系样蛋白2(NFE2L2；以下简称NFE2L2)的负调控因子 NRF2基因在大约30%的肺癌中发生突变。NRF2是一种转录因子，调节一种 协调细胞对氧化应激反应的基因网络。Keap1基因的失活突变 导致肿瘤细胞中固有的NRF2活性和氧化应激反应通路的激活。的 注意，30%的肺腺癌患者携带致癌的KRAS突变。在这些KRAS突变的肿瘤中， 20%的人表现出Keap1功能突变的丧失，这意味着氧化应激反应的重要性 在这种肿瘤类型中，启动和/或维持途径。其中基因突变的频率很高 PATH使其成为治疗肺癌和其他癌症类型的有吸引力的靶点。 目的：本研究将(1)明确Keap1功能丧失在正常肺和正常肺中的作用。 癌症的发展；(2)高通量鉴定Keap1突变细胞系中的药物靶点 已知化学抑制剂靶向~5000个基因的定制sgRNA文库(3)测试TOP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和 与Kras和Keap1融合的基因的功能可以作为治疗癌症的药理学靶点。三 为实现这些目标，提出了具体目标： 目的1：利用体内实验确定Keap1功能缺失突变的作用 已发表的连接Cre和Cas9表达的慢病毒载体将用于同时启动 含致癌Kras基因工程小鼠肺上皮中肿瘤和Keap1的编辑 和P53功能缺失突变。将分析肿瘤负担和肿瘤分级的变化。 目的2：鉴定针对Keap1突变肿瘤细胞的新药物靶点 高通量筛选将确定Keap1突变细胞中的合成遗传相互作用 利用sgRNA文库靶向大约5000个推定或真正的药物靶点的努力。这些基因 将使用各种含有Lost的小鼠等基因肺腺癌细胞株来验证相互作用 Keap1的功能突变。 目的3：假想的Keap1突变型合成致死基因的体内功能验证 肺腺癌的条件模型将被用来探索和验证功能丧失。 利用体内CRISPR基因编辑以Keap1突变体特有的方式合成致死基因候选。

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.