A platform for genome-wide discovery of synthetic lethal interactions in cancer

描述(由申请人提供)：我们建议开发一个平台，以加速发现人类癌症中的合成致命相互作用。这项工作利用了我们实验室和其他实验室最近开发的人类全基因组慢病毒RNAi结构文库(统称为“RNAi联盟”(TRC))，该文库使用90,000多个慢病毒克隆来靶向18,000多个人类基因，产生稳定的靶基因抑制，并且与分裂和非分裂细胞都兼容。为了利用这个文库实现小型化、高通量、阵列化的功能丧失屏幕，我们的首要目标将是优化慢病毒感染细胞微阵列(LICM)的性能，这是我们实验室目前正在开发的一项技术。具体地说，这一目标将侧重于通过使用纳米结构表面特征和/或生物兼容水凝胶涂层来增加阵列表面上的官能团密度，从而优化沉积在每个微阵列特征上的病毒数量。在我们的第二个目标中，我们将制造全基因组LICM，并证明它们在大规模功能丧失屏幕上的可行性。在我们的最终目标中，我们将把这些系统应用于一种筛查，该筛查将用于识别PTEN基因缺失的癌症中的合成致命相互作用。PTEN是一种肿瘤抑制基因，经常与人类癌症有关。具体地说，在这个目标中，我们将使用四环素可诱导表达PTEN的工程U-87人类细胞系来首先识别与PTEN合成致命的基因，然后在一组PTEN功能受损的人类癌细胞系上测试这些候选基因，以识别在一系列不同的遗传和生理背景下合成致命的基因。该项目的长期目标是：(1)确定与PTEN合成致死的基因，从而成为抗癌药物开发的靶点；(2)建立一个强大的、广泛使用的阵列筛选平台，以加快功能基因组学的进展。行业描述：我们建议开发一个高通量筛选平台，该平台将允许快速和系统地识别针对人类癌症的新的、选择性的药物靶点。该项目涉及三个部分：拟议的筛选平台的优化；该平台在全基因组筛选实验中的应用；以及利用该平台系统地识别新的癌症药物靶点。这项研究中开发的筛查平台有可能成为整个生物医学研究界高通量筛查实验的使能工具，这些系统在癌症方面的特殊应用可能会显著加快新抗癌药物的发现。

DESCRIPTION (provided by applicant): We propose the development of a platform to accelerate the discovery of synthetic lethal interactions in human cancers. This work makes use of a human genome-wide library of lentiviral RNAi constructs recently developed by our lab and others (collectively termed "The RNAi Consortium" (TRC)) that targets over 18,000 human genes using over 90,000 lentiviral clones, yields stable suppression of target genes, and is compatible with both dividing and non-dividing cells. To enable miniaturized, high-throughput, arrayed loss- of-function screens with this library, our first aim will be to optimize the properties of lentivirus-infected cell microarrays (LICMs), a technology currently under development in our laboratory. Specifically, this aim will focus on optimizing the number of viruses deposited on each microarray feature by increasing the density of functional groups on the array surface using nanostructured surface features and/or biocompatible hydrogel coatings. In our second aim, we will fabricate whole-genome LICMs and demonstrate their feasibility toward large-scale loss-of-function screens. In our final aim, we will apply these systems to a screen that will be used to identify synthetic lethal interactions in cancers harboring deficiencies in PTEN, a tumor suppressor gene that is frequently implicated in human cancers. Specifically, in this aim we will use an engineered U-87 human cell line with tetracycline inducible PTEN expression to first identify genes that are synthetic lethal with PTEN, then test these candidate genes on a panel of human cancer cell lines with impaired PTEN function to identify those genes which are synthetic lethal in a range of diverse genetic and physiological contexts. The long-term objectives of this project are: (1) to identify genes which are synthetic lethal with PTEN, and thus targets for cancer drug development, and (2) to establish a robust, broadly useful arrayed screening platform to accelerate progress in functional genomics. Lay description: We propose to develop a high-throughput screening platform which will allow for the rapid and systematic identification of new, selective drug targets for human cancers. This project involves three components: the optimization of the proposed screening platform; the application of this platform to genome-wide screening experiments; and the use of this platform to systematically identify new cancer drug targets. The screening platform developed in this study has the potential to become an enabling tool for high-throughput screening experiments throughout the biomedical research community, and the particular application of these systems to cancer may significantly accelerate the discovery of new cancer drugs.