Identification and validation of synthetic lethality in KRAS mutant lung adenocarcinoma patient derived xenografts

KRAS 突变肺腺癌患者异种移植物合成致死率的鉴定和验证

• 批准号: 9115563
• 负责人: Charles M. Rudin
• 金额: $ 57.08万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115563
• 关键词: Accounting; Address; Affinity; Alleles; Animals; Applications Grants; BRAF gene; Blood; Blood Volume; CRISPR screen; CRISPR/Cas technology; Cancer cell line; Candidate Disease Gene; Cell Culture Techniques; Cell Line; Cells; Characteristics; Clinical; Collection; Complex; Coupled; Dependency; Development; Doxycycline; Drops; Engineering; Enzymes; Epidermal Growth Factor Receptor; Feedback; Frequencies; Gene Targeting; Genes; Genomic approach; Growth; Guanosine Triphosphate; Housing; Human; Institution; KRAS2 gene; Lentivirus Vector; Libraries; Luciferases; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of lung; Measurement; Measures; Modeling; Monitor; Mus; Mutate; Mutation; Oncogenes; Oncogenic; Pathway interactions; Patients; Phosphotransferases; Population; Population Dynamics; Proteins; Proto-Oncogenes; Recurrence; Reporter; Reporter Genes; Reporting; Reproducibility; Research; Research Personnel; Sampling; Signal Transduction; Subfamily lentivirinae; Synthetic Genes; System; Technology; United States; Validation; Xenograft Model; Xenograft procedure; base; cancer genomics; cancer therapy; cancer type; carcinogenesis; cellular transduction; genetic manipulation; genome-wide; in vivo; knock-down; loss of function; member; mutant; mutational status; novel; novel strategies; novel therapeutics; nuclease; peripheral blood; personalized medicine; public health relevance; research study; screening; small hairpin RNA; targeted sequencing; targeted treatment; tumor; tumor growth; tumor microenvironment

 DESCRIPTION (provided by applicant): KRAS is the single most frequently mutated oncogene in human cancer, yet there are no targeted therapies available for patients whose cancers harbor these mutations. Mutant KRas is intrinsically difficult to target pharmacologically due to a number of factors and is considered by many to be an "undruggable" protein. An alternative approach to direct targeting is to target proteins to which KRAS mutant tumors have developed a artificial dependency, a condition termed synthetic lethality. Approaches to identifying synthetic lethal relationships in the context of mutant KRas have not been entirely successful; perhaps in part due to their reliance on 2D cell culture, insufficient sensitivity, anda lack of appropriate validation performed in multiple wild-type and mutant KRas systems. This project will take seek to address these weaknesses using KRas mutant lung adenocarcinoma PDXs harboring the most common mutant alleles (G12C (3), G12D (1), G12V (1), and G13D (1)) as well as wild-type KRAS. The first Aim is based on development of doxycycline inducible Cas9 nuclease PDX lines and in vivo CRISPR/Cas9 screening. Lung adenocarcinoma PDXs with varying KRAS mutation status will be transduced with an inducible Cas9 lentivirus and selected for expression of a constitutive reporter gene to obtain a pure population of transduced cells. These lines will be transduced with a pool of lentiviral sgRNAs targeting 19,050 human genes and xenografted into naïve mice. After tumors are established, a portion of each tumor will be excised, followed by induction of Cas9 expression by ad libitum doxycycline medicated chow. sgRNA frequencies will be compared across multiple tumors at baseline and after several weeks of in vivo selection. The second Aim uses an orthogonal approach to target validation in lung adenocarcinoma PDXs based on doxycycline inducible shRNAs and serial measurement of secreted luciferase in blood as a marker of clonal dynamics in vivo. In an orthogonal approach, lung adenocarcinoma PDXs will be independently transduced with lentiviral constructs that inducible express one of two secreted luciferases and either a control or gene targeting shRNA. After tumors are establish, luciferase and shRNA co-expression will be induced by doxycycline and subclonal tumor population dynamics measured by frequent collection of small volumes of peripheral blood. Deliverables from these studies are expected to include 1) doxycycline inducible Cas9 lentiviral vectors suitable for PDX transduction and positive selection; 2) lung adenocarcinoma PDX lines representing the most common KRas mutant alleles bearing these constructs; 3) candidates for synthetic lethal interactions in the context of KRAS mutations; 4) A novel platform for cross-validating our own synthetic lethal candidates and those of the KRAS Synthetic Lethality Network in lung adenocarcinoma PDXs harboring the most common KRAS mutant alleles.

 描述（由申请人提供）：KRAS 是人类癌症中最常见突变的癌基因，但由于多种因素，突变型 KRas 本质上难以在药理学上靶向，并且对于其靶向癌症含有这些突变的患者尚无可用的治疗方法。许多人认为这是一种“不可成药”的蛋白质，另一种直接靶向的方法是靶向 KRAS 突变肿瘤已产生人工依赖性的蛋白质，这种情况称为合成致死性鉴定方法。突变 KRas 背景下的致死关系尚未完全成功；可能部分原因是它们依赖于 2D 细胞培养、灵敏度不足以及缺乏在多个野生型和突变型 KRas 系统中进行的适当验证。寻求使用含有最常见突变等位基因（G12C (3)、G12D (1)、G12V (1) 和 G13D (1)）的 KRas 突变肺腺癌 PDX 来解决这些弱点第一个目标是基于强力霉素诱导型 Cas9 核酸酶 PDX 系的开发，以及体内 CRISPR/Cas9 筛选，将用诱导型 Cas9 慢病毒转导并选择表达不同 KRAS 突变状态的肺腺癌 PDX。组成型报告基因以获得纯的转导细胞群。这些细胞系将用慢病毒池进行转导。靶向 19,050 个人类基因的 sgRNA 异种移植到初始小鼠体内后，每个肿瘤的一部分将被切除，然后通过随意使用多西环素药物诱导 Cas9 表达，并在基线和之后比较多个肿瘤的 sgRNA 频率。第二个目标使用正交方法对基于肺腺癌 PDX 的靶点进行验证。多西环素诱导型 shRNA 和血液中分泌型荧光素酶的连续测量作为体内克隆动态的标记在正交方法中，将用可诱导表达两种分泌型荧光素酶之一以及对照或基因靶向的慢病毒构建体独立转导肺腺癌 PDX。 shRNA。肿瘤形成后，强力霉素和 shRNA 会诱导荧光素酶和 shRNA 共表达。通过频繁采集少量外周血来测量亚克隆肿瘤群体动态，预计这些研究的成果包括 1) 适合 PDX 转导和正选择的强力霉素诱导型 Cas9 慢病毒载体；2) 代表最常见 KRas 突变体的肺腺癌 PDX 系。带有这些构建体的等位基因；3）KRAS突变背景下合成致死相互作用的候选者；4）用于交叉验证我们的新平台自己的合成致死候选者以及 KRAS 合成致死网络中含有最常见 KRAS 突变等位基因的肺腺癌 PDX 的候选者。