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

 描述（由申请人提供）：KRAS是人类癌症中最常见的单一突变癌基因，但对于携带这些突变的癌症患者尚无靶向治疗。由于许多因素，突变型KRas本质上难以靶向靶蛋白，并且被许多人认为是“不可用药的”蛋白质。直接靶向的另一种方法是靶向KRAS突变型肿瘤已经产生人工依赖性的蛋白质，这种情况称为合成致死性。在突变KRas背景下鉴定合成致死关系的方法尚未完全成功;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KRas系统中进行的适当验证。该项目将寻求使用KRAS突变型肺腺癌PDX来解决这些弱点，这些PDX携带最常见的突变等位基因（G12 C（3），G12 D（1），G12 V（1）和G13 D（1））以及野生型KRAS。第一个目的是基于多西环素诱导型Cas9核酸酶PDX系的开发和体内CRISPR/Cas9筛选。具有不同KRAS突变状态的肺腺癌PDX将用诱导型Cas9慢病毒转导，并选择用于表达组成型报告基因以获得纯的转导细胞群。这些细胞系将用靶向19，050个人类基因的慢病毒sgRNA库转导，并异种移植到幼稚小鼠中。在建立肿瘤后，切除每个肿瘤的一部分，然后通过随意的多西环素加药食物诱导Cas9表达。将在基线和体内选择数周后在多个肿瘤中比较sgRNA频率。第二个目标使用正交方法在肺腺癌PDX中基于多西环素诱导的shRNA进行靶向验证，并连续测量血液中分泌的荧光素酶作为体内克隆动力学的标志物。在正交方法中，将用慢病毒构建体独立地转导肺腺癌PDX，所述慢病毒构建体可诱导表达两种分泌型内切酶之一和对照或基因靶向shRNA。肿瘤建立后，将通过多西环素诱导荧光素酶和shRNA共表达，并通过频繁收集少量外周血测量亚克隆肿瘤群体动力学。预期来自这些研究的候选物包括1）适合于PDX转导和阳性选择的多西环素诱导型Cas9慢病毒载体; 2）代表携带这些构建体的最常见KRas突变等位基因的肺腺癌PDX系; 3）在KRAS突变的背景下用于合成致死相互作用的候选物; 4）用于在携带最常见的KRAS突变等位基因的肺腺癌PDX中交叉验证我们自己的合成致死候选物和KRAS合成致死网络的那些的新平台。

 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.