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（由适用提供）：KRAS是人类癌症中最常见的癌基因，但对于癌症含有这些突变的患者尚无靶向疗法。由于多种因素，突变的KRAS在本质上很难在药理学上靶向，并且许多因素被认为是“不可用”的蛋白质。直接靶向的另一种方法是靶向KRAS突变肿瘤发展到人工依赖性的靶蛋白，这种依赖性称为合成致死性。在突变的KRAS背景下鉴定合成致死关系的方法并不完全成功。也许部分原因是它们缓解了2D细胞培养，灵敏度不足以及在多种野生型和突变的KRAS系统中进行的缺乏适当的验证。该项目将寻求使用具有最常见的突变等位基因（G12C（3），G12D（1），G12V（1）和G12V（1）和G13D（1））以及野生型Kras的KRAS突变肺腺癌PDX（G12C（3），G12C（3），G12C（3），G12C（3），G12C（3），G12C（3），G12C（3），以及野生型Kras。第一个目的是基于强力霉素诱导的Cas9核酸酶PDX系和体内CRISPR/CAS9筛选的发展。具有不同KRAS突变状态的肺腺癌PDX将以诱导的CAS9慢病毒转移，并选择用于表达本构报告基因以获得纯粹的翻译细胞群体。这些线将用靶向19,050个人类基因的慢病毒SGRNA池翻译成幼稚的小鼠。建立肿瘤后，将超过每个肿瘤的一部分，然后通过多余的强力霉素药物诱导Cas9表达。在基线和体内选择数周后，将在多个肿瘤的多个肿瘤中进行SGRNA频率。第二个目标使用一种正交方法来靶向基于强力霉素可诱导的shRNA的肺腺癌PDX中的靶向验证，以及对血液中分泌的荧光素酶的序列测量作为体内克隆动力学的标志。在正交方法中，肺腺癌PDX将以慢病毒构建体独立翻译，这些溶液构建体能够诱导两个分泌的荧光素酶之一，并且是靶向shRNA的对照或基因。建立肿瘤后，荧光素酶和SHRNA共表达将由强力霉素和亚克隆肿瘤种群动力学诱导，这些动力学经常通过少量的外周血收集来测量。这些研究的可交付成果预计包括1）强力霉素诱导的Cas9慢病毒载体适合PDX转移和阳性选择； 2）肺腺癌PDX线，代表具有这些构建体的最常见的Kras突变等位基因； 3）在KRAS突变的背景下进行合成致命相互作用的候选者； 4）一个新颖的平台，用于交叉验证我们自己的合成致命候选者以及肺腺癌PDX中具有最常见的KRAS突变等位基因的KRAS合成致死性网络的平台。