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: 8529476
• 负责人: Daniel G. Jay
• 金额: $ 32.18万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8529476
• 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; 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; screening; 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.

描述（由申请人提供）：遗传方法极大地促进了我们对生物学的理解，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 辅助诱变 (KRAM)，为体细胞进行无偏正向遗传筛选，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，增强型逆转录病毒诱变 (ERM) 将用于在整个基因组中随机引入编码光敏剂基因 KillerRed 的受调控启动子/客体外显子融合。启动子片段提供融合基因的过度表达，从而获得功能； KillerRed 融合允许发色团辅助光灭活 (CALI)（一种光介导灭活技术）破坏蛋白质融合，导致功能丧失。正如稍后将讨论的，与基因删除相比，我们预计 CALI 的光损伤将发挥显着效应，无论野生型等位基因表达如何。因此，KRAM 将首次提供一种低成本高通量方法来解决二倍体细胞中全局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，我们将使用它来识别在慢性粒细胞白血病 (CML) 的甲氨蝶呤耐药性中发挥作用的基因，这是一个已明确描述的过程，其基因是已知的。然后，我们将使用 KRAM 研究伊马替尼耐药性，这是 CML 中的一个重要临床问题，我们希望在此过程中识别新基因。我们组建了一个研究团队，其综合专业知识针对拟议工作的成功进行了优化。 PI 首创了 CALI，并且是该方法及其在癌症中的应用的领先权威。 Cochran 博士开发了用于开发 KRAM 的细胞系，Songyang 博士发明了 ERM 筛查，Van Etten 博士是 CML 和 abl 癌基因方面的专家。为了建立 KRAM，我们提出了三个目标：1）使用 β-半乳糖苷酶和涉及耐药性的内源蛋白优化 KillerRed CALI； 2) 开发KRAM并测试其选择甲氨蝶呤耐药所需基因的能力； 3) 进行全面的 KRAM 选择，以确定对伊马替尼耐药性重要的新基因，并通过过表达和 siRNA 对其进行验证。成功完成这些目标将为通常适用于其他癌症以及其他癌症相关过程（例如增殖、侵袭和凋亡）的选择提供原理证明。此外，还将确定和验证新靶点，以开发预防 CML 伊马替尼耐药的药物，这具有潜在的高度临床意义。作为体细胞功能获得/功能丧失选择的通用低成本方法，KRAM 将在生物医学领域得到广泛应用，并成为一项变革性技术。