KillerRed Assisted Mutagenesis to discover cancer drug resistance genes

描述(申请人提供)：遗传学方法对我们对生物学的理解有很大贡献，但它们仅限于癌细胞，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。为了解决这一局限性，我们建议开发KillerRed辅助突变(Kram)来为体细胞生成一个无偏见的正向遗传筛选，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，增强型逆转录病毒突变(ERM)将被用于在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，导致功能获得；而KillerRed融合允许发色团辅助的光灭活(CALI)，一种光介导的失活技术，破坏蛋白质融合，导致功能丧失。正如后面将讨论的，与基因缺失相反，我们预计CALI的光损伤将发挥显性效应，而不考虑野生型等位基因的表达。因此，Kram将首次提供一种低成本、高吞吐量的方法，在全球范围内解决二倍体细胞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粒细胞白血病(CML)的甲氨蝶呤耐药中起作用的基因，这是一个已经很好描述的过程，其基因是已知的。然后，我们将使用Kram来研究伊马替尼耐药性，这是慢性粒细胞白血病的一个重要临床问题，我们希望在这个过程中识别新的基因。我们已经组建了一个研究小组，他们的综合专业知识得到了优化，以确保拟议工作的成功。PI是CALI的先驱，也是这一方法及其在癌症应用方面的领先权威。科克伦博士开发了将用于开发Kram的细胞系，宋阳博士发明了ERM筛查，Van Etten博士是慢性粒细胞白血病和ABL癌基因方面的专家。为了建立Kram，我们提出了三个目标：1)使用半乳糖苷酶和与耐药有关的内源蛋白来优化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选择以发现与伊马替尼耐药有关的新基因，并通过过表达和siRNA验证它们。这些目标的成功完成将为普遍适用于其他癌症以及其他癌症相关过程(如增殖、侵袭和凋亡)的选择提供原则证明。此外，它还将确定和验证新的靶点，以开发预防慢性粒细胞白血病对伊马替尼耐药的药物，这可能具有很高的临床意义。作为体细胞功能获得/功能丧失选择的一种普遍的低成本方法，Kram将在生物医学中得到广泛的应用，并成为一项革命性的技术。

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.