KillerRed Assisted Mutagenesis to discover cancer drug resistance genes

KillerRed 辅助诱变发现癌症耐药基因

• 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
• 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

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（由申请人提供）：遗传方法对我们对生物学的理解做出了巨大贡献，但它们在癌细胞中受到限制，因为二倍体细胞中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KillerRed辅助突变（KRAM），为体细胞生成公正的正向遗传筛选，以识别癌症相关过程所需的蛋白质。KRAM有几个组成部分：首先，增强逆转录病毒突变（enhanced逆转录病毒mutagenesis， ERM）将用于在整个基因组中随机引入编码光敏基因KillerRed的调控启动子/客外显子融合。启动子片段提供融合基因的过表达，导致功能的获得；而KillerRed融合允许发色团辅助光失活（CALI），一种光介导的失活技术，破坏蛋白质融合，导致功能丧失。正如后面将要讨论的，与基因缺失相反，我们预计CALI的光损伤无论野生型等位基因的表达如何，都会发挥显性效应。因此，KRAM将首次提供一种低成本高通量的方法来解决二倍体细胞整体功能丧失和功能获得的后果。KRAM选择的成本明显低于RNAi和cDNA表达文库筛选，因为它不需要为每个基因合成特定的试剂。为了开发和测试KRAM，我们将使用它来识别在慢性髓性白血病（CML）的甲氨蝶呤耐药中起作用的基因，这是一个已知基因的特征过程。然后我们将使用KRAM来研究伊马替尼耐药性，这是CML的一个重要临床问题，我们希望在这个过程中发现新的基因。我们已经组建了一个研究小组，他们的综合专业知识将为拟议工作的成功而优化。PI开创了CALI，是该方法及其应用于癌症的领先权威。Cochran博士开发了将用于开发KRAM的细胞系，Songyang博士发明了ERM筛选，Van Etten博士是CML和abl-癌基因的专家。为了建立KRAM，我们提出了三个目标：1)利用¿-半乳糖苷酶和内源性耐药蛋白优化KillerRed CALI；2)开发KRAM并测试其选择甲氨蝶呤抗性所需基因的能力；3)进行全面的KRAM选择，鉴定对伊马替尼耐药重要的新基因，并通过过表达和siRNA进行验证。这些目标的成功完成将为一般适用于其他癌症以及其他癌症相关过程（如增殖、侵袭性和细胞凋亡）的选择提供原理证明。此外，它将确定和验证新的靶点，以开发防止CML中伊马替尼耐药的药物，这具有潜在的高临床意义。作为一种普遍的低成本体细胞功能获得/功能丧失选择方法，KRAM在生物医学领域具有广泛的应用前景，是一项革命性的技术。