A Genome Wide RNAi Vulnerability Map for Myeloma

描述(申请人提供)：拟议研究的中心假设是，对特定基因靶标的全基因组RNAi抑制将识别先前未被识别的调控骨髓瘤细胞生长的关键分子途径，并且这些信息可以在临床上得到积极利用。虽然新的药物如波特佐米、沙利度胺和来那度胺对骨髓瘤有深刻的临床活性，但确切的分子作用点尚不清楚。此外，这些药物都不是治愈药物，识别可利用的新的易损性靶点，以及识别可能在MM中具有未被识别的活性的现有药物制剂，是未来进展的关键目标。由于MM细胞的关键靶点仍然难以捉摸，我们认为，需要进一步的基因组规模分析和创建MM的脆弱性图，以促进我们对这种疾病的全球理解，以及如何进行个性化和优化的治疗。因此，我们在这项建议中的目标是应用先进的功能基因组学策略来识别和快速验证候选基因在多发性骨髓瘤生长和生存中的关键作用，并将这一信息推广到临床。有鉴于此，我们最近在MM细胞系中进行了高通量、基因组范围的RNAi致死性筛选，以确定对人类MM的生存至关重要的激酶。此外，我们还通过在存在这种药物的情况下进行RNAi筛选，确定了使肿瘤细胞对Bortezomib治疗敏感的激酶靶点。最后，我们现在已经完成了在单个MM细胞系中使用和不使用硼替佐米和来那度胺的可药物基因组(7000个基因)的初步筛选。通过之前的其他努力，我们已经拥有了一个由60个MM细胞系组成的广泛的小组，拥有一个关于细胞系和初级患者样本中的基因表达和比较基因组杂交的大型数据库，并随时可以访问患者组织。我们建议在此扩展这些发现，以探索在MM细胞系中更具全球代表性的17,000个基因的高通量、合成致死RNAi筛查，并使用二次筛选、初级患者细胞中的靶标验证以及将我们不同的基因组数据集整合在一起的生物信息处理来验证这些发现。具体地说，我们将通过平行的RNAi来确定沉默基因表达在多大程度上会改变培养的骨髓瘤细胞系中任何一个方向的细胞毒敏感性。然后，我们将把药物基因组学和经典分子生物学应用于细胞系、动物模型和患者样本，以验证这些假定的药物靶点的临床相关性，并确定它们可用作治疗反应的生物标记物的程度。公共卫生相关性：多发性骨髓瘤在任何给定的时间都会影响到美国的5万人。在治疗方面已经取得了重大进展，新的药物如硼替佐米、沙利度胺和来那度胺具有深刻的临床活性。然而，这些药物都不是治愈的，它们的作用方式也不透明。因此，确定可利用的新的脆弱性目标，以及确定可能在MM中具有未被识别的活性的现有药物制剂，是未来进展的关键目标。为了实现这一目标，我们将筛选17,000个能够控制骨髓瘤细胞生长的基因。这种基因组规模的分析和骨髓瘤脆弱性图的创建，是进一步加深我们对这种疾病的全球理解以及如何进行个性化和优化治疗的先决条件。

DESCRIPTION (provided by applicant): The central hypothesis for the proposed research is that genome wide RNAi inhibition of specific gene targets will identify previously unrecognized critical molecular pathways which modulate the growth of Myeloma cells in either direction and that such information can be positively exploited clinically. While newer agents such as bortezomib, thalidomide and lenalidomide have profound clinical activity in myeloma the exact point of molecular action is unknown. Furthermore, none of these drugs is curative and the identification of novel vulnerability targets which can be exploited, together with the identification of existing pharmaceutical agents which may have unrecognized activity in MM is a critical goal for future progress. Since the critical targets of MM cells remain elusive further genomic scale analysis and the creation of a vulnerability map of MM, is in our opinion, required to further our global understanding of this disease and how treatments may be both personalized and optimized. Our objective in this proposal is therefore to apply advanced functional genomic strategies to identify and rapidly validate the critical role of candidate genes in MM growth and survival and advance this information clinically. In that light we have recently conducted high throughput, kinome wide, RNAi lethality screening in MM cell lines to identify kinases essential to the survival of human MM. In addition, we have identified kinase targets that sensitize tumor cells to bortezomib therapy by RNAi screening in the presence of this drug. Finally, we have now completed a preliminary screen of the druggable genome (7000 genes) in a single MM cell line with and without bortezomib and lenalidomide. Through other previous efforts we have an extensive panel of 60 MM cell lines, a large database of both gene expression and comparative genomic hybridization in cell lines and primary patient samples and ready access to patient tissues. We propose here to extend these findings to explore a more globally representative 17,000 gene high throughput, synthetic lethal RNAi screen in MM cell lines and to validate these findings using secondary screening, target validation in primary patient cells, and bioinformatic processing which brings together our disparate genomic datasets. Specifically, we will determine the extent to which silencing gene expression, by parallel RNAi, will modify cytotoxic sensitivity in either direction in cultured Myeloma cell lines. We will then apply pharmacogenomics and classical molecular biology to cell lines, animal models and patient samples to validate the clinical relevance of these putative drug targets and determine the extent to which they can be used as biomarkers of therapeutic response. PUBLIC HEALTH RELEVANCE: Multiple Myeloma affects 50,000 people in the United States at any given time. Major advances have been made in the treatment and newer agents such as bortezomib, thalidomide and lenalidomide have profound clinical activity. However, none of these drugs is curative and the means by which they work opaque. Thus identification of novel vulnerability targets which can be exploited, together with the identification of existing pharmaceutical agents which may have unrecognized activity in MM is a critical goal for future progress. To achieve this we will screen 17,000 genes for there ability to control myeloma cell growth. This genomic scale analysis and the creation of a vulnerability map of Myeloma, is a prerequisite to further our global understanding of this disease and how treatments may be both personalized and optimized.