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人最近开发的慢病毒RNAi构建的人类全基因组文库（统称为“RNAi联盟”（TRC）），该文库使用超过90,000个慢病毒克隆靶向超过18,000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兼容。为了使用该文库实现小型化、高通量、阵列功能丧失筛选，我们的第一个目标将是优化慢病毒感染细胞微阵列（LICMs）的特性，这是我们实验室目前正在开发的一项技术。具体来说，该目标将侧重于通过使用纳米结构表面特征和/或生物相容性水凝胶涂层增加阵列表面官能团的密度来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筛查中的可行性。在我们的最终目标中，我们将把这些系统应用于筛选，用于识别在PTEN缺陷的癌症中合成的致命相互作用，PTEN是一种经常与人类癌症有关的肿瘤抑制基因。具体来说，在这个目标中，我们将使用一个具有四环素诱导PTEN表达的工程U-87人类细胞系，首先鉴定具有PTEN合成致死性的基因，然后在PTEN功能受损的人类癌细胞系上测试这些候选基因，以鉴定在一系列不同的遗传和生理背景下具有合成致死性的基因。该项目的长期目标是：(1)鉴定具有PTEN合成致死作用的基因，从而成为癌症药物开发的靶点；(2)建立一个强大的、广泛使用的阵列筛选平台，以加速功能基因组学的进展。概要描述：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新的、选择性的人类癌症药物靶点。本项目涉及三个部分：拟筛选平台的优化；该平台在全基因组筛选实验中的应用；并利用这个平台系统地识别新的抗癌药物靶点。本研究开发的筛选平台有可能成为整个生物医学研究界高通量筛选实验的启用工具，并且这些系统在癌症中的特殊应用可能会显著加速新的癌症药物的发现。

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.