KillerRed Assisted Mutagenesis to discover cancer drug resistance genes

KillerRed 辅助诱变发现癌症耐药基因

基本信息

批准号：
8285231
负责人：
Daniel G. Jay
金额：
$ 34.24万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-13 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8285231
关键词：
Acute Address Alleles Antineoplastic Agents Apoptosis Biological Assay Biology Cancer Biology Cell Line Cell physiology Cells Chimeric Proteins Chronic Myeloid Leukemia Clinical Development Diploid Cells Dominant-Negative Mutation Drug effect disorder Drug resistance Elements Exons Expression Library Financial compensation Galactosidase Gene Targeting Genes Genetic Genetic Screening Genome Genomics Haploid Cells Haploidy Imatinib In Situ Individual Infection Insertional Mutagenesis Lead Light Light Cell Malignant Neoplasms Mediating Methotrexate Mutagenesis Mutation P-Glycoprotein Pharmaceutical Preparations Photosensitizing Agents Process Proteins RNA Interference Reagent Research Research Personnel Resistance Route SRC gene Sampling Screening procedure Small Interfering RNA Somatic Cell Technology Testing Time Work abl Oncogene anticancer research authority base cDNA Expression cancer cell cancer therapy chromophore clinically significant cost cost effective gain of function gain of function mutation genetic selection innovation loss of function mutant new technology novel novel strategies overexpression prevent promoter success tool vector

项目摘要

DESCRIPTION (provided by applicant): Genetic approaches have contributed greatly to our understanding of biology, but they are limited in cancer cells because genetic loss-of-function in diploid cells is obscured by expression from the wild type allele. To address this limitation, we propose to develop KillerRed Assisted Mutagenesis (KRAM) to generate an unbiased forward genetic screen for somatic cells to identify proteins required for cancer-relevant processes. There are several components to KRAM: first, enhanced retroviral mutagenesis (ERM) will be used to introduce a regulated promoter/guest exon fusion encoding the photosensitizer gene, KillerRed, randomly throughout the genome. The promoter segment provides overexpression of the fused gene, leading to gain of function; while the KillerRed fusion permits Chromophore-Assisted Light Inactivation (CALI), a light-mediated inactivation technology, to destroy the protein fusion, leading to loss of function. As will be discussed later, in contrast to genetic deletion, we expect photo damage by CALI to exert dominant effects regardless of wild type allele expression. Thus, KRAM will provide for the first time a low cost high throughput approach to address the consequences of loss-of-function and gain-of-function globally in diploid cells. KRAM selection would cost markedly less than RNAi and cDNA expression library screens, as it does not require synthesis of specific reagents for every gene. To develop and test KRAM, we will use it to identify genes that act in methotrexate resistance of chronic myelogenous leukemia (CML), a well-characterized process whose genes are known. We will then use KRAM to study imatinib resistance, an important clinical problem in CML, where we expect to identify new genes in this process. We have assembled a research team whose combined expertise is optimized for the success of the proposed work. The PI pioneered CALI and is the leading authority on this approach and its application to cancer. Dr. Cochran developed the cell lines that will be used to develop KRAM, Dr. Songyang invented ERM screening and Dr. Van Etten is an expert in CML and abl-oncogenes. To establish KRAM, we propose three Aims: 1) optimize KillerRed CALI using ¿-galactosidase and endogenous proteins implicated in drug resistance; 2) develop KRAM and test its ability to select for genes required for methotrexate resistance; and 3) conduct a full-scale KRAM selection to identify new genes important for imatinib resistance and validate them by overexpression and siRNA. Successful completion of these Aims will provide a proof-of-principle for selections that are generally applicable for other cancers and also for other cancer relevant processes such as proliferation, invasiveness and apoptosis. In addition, it will identify and validate new targets to develop drugs that prevent imatinib resistance in CML, which has potentially high clinical significance. As a generalized low cost approach for gain-of-function/loss-of-function selection in somatic cells, KRAM will have wide application across biomedicine and be a transformative technology. PUBLIC HEALTH RELEVANCE: Genetic selections using somatic cancer cells have been limited by high cost and difficulty and additionally, they currently select for loss-of-function or gain-of-function mutations but not both. We propose to develop KillerRed Assisted Mutagenesis (KRAM), which combines insertional mutagenesis to generate gain-of- function mutants and KillerRed CALI, a targeted photo-destruction strategy for loss-of-function, to provide a low cost genetic approach that would have high utility in cancer biology and throughout biomedicine, and thus be potentially transformative.

描述（由适用提供）：遗传方法对我们对生物学的理解有很大贡献，但是它们在癌细胞中受到限制，因为二倍体细胞的遗传功能丧失被野生型等位基因的表达所掩盖。为了解决这一限制，我们建议开发杀伤的辅助诱变（KRAM），以生成一个无偏向的前遗传筛选，以识别体细胞以鉴定与癌症相关的过程所需的蛋白质。 KRAM有几个组成部分：首先，将使用增强的逆转录病毒诱变（ERM）引入调节的启动子/宾客外显子融合编码光敏剂基因，并在整个基因组中随机捕获。启动子段提供了融合基因的过表达，从而导致功能增长。尽管Killerred融合允许染色体辅助光灭活（Cali）（Cali）（一种光介导的灭活技术）破坏了蛋白质融合，从而导致功能丧失。正如稍后将讨论的那样，与遗传缺失相反，我们预计卡利的照片损害会产生显着效应，而不论野生型等位基因表达如何。这就是KRAM首次提供低成本高吞吐量方法，以解决二倍体细胞中全球功能丧失和功能障碍的后果。 KRAM选择的成本明显低于RNAi和cDNA表达式库筛选，因为它不需要为每个基因合成特定试剂。为了开发和测试KRAM，我们将使用它来鉴定以慢性粒细胞性白血病（CML）为甲羟耐药性作用的基因，这是一种已知基因的良好特征化过程。然后，我们将使用KRAM研究伊马替尼抗性，伊马替尼的抗性，这是CML中重要的临床问题，我们希望在此过程中鉴定新基因。我们组建了一个研究团队，其联合专业知识已针对拟议工作的成功进行了优化。 PI率先加油，是这种方法及其在癌症中的应用。 Cochran博士开发了用于开发KRAM的细胞系，Sonyang博士发明了ERM筛查，而Van Etten博士是CML和ABL-Concogenes的专家。为了建立KRAM，我们提出了三个目的：1）使用耐药性实施的 - 半乳糖苷酶和内源性蛋白来优化Killerred Cali； 2）开发KRAM并测试其选择方法二邻抗抗性所需的基因的能力； 3）进行全面的KRAM选择，以确定对伊马替尼抗性重要的新基因，并通过过表达和siRNA对其进行验证。这些目标的成功完成将为通常适用于其他癌症以及其他相关过程（例如增殖，侵入性和凋亡）的选择提供原则证明。此外，它将确定并验证新靶标，以开发可防止CML中伊马替尼抗性的药物，CML具有可能具有很高的临床意义。作为在体细胞中获得功能获得/功能丧失选择的普遍低成本方法，KRAM将在生物医学中具有广泛的应用，并且是一种变革性的技术。公共卫生相关性：使用体细胞细胞的遗传选择受到高成本的限制，并且困难，此外，他们目前选择了功能丧失或功能性突变，但并非两者兼而有之。我们建议开发杀伤的辅助诱变（KRAM），该诱变（KRAM）结合了插入性诱变以产生功能障碍突变体和Killerred Cali（一种针对性的造成功能丧失的造成照相策略）提供了一种低成本的遗传方法，在癌症生物学和整个生物医学中都具有很高的实用性，因此具有很高的实用性。