A Genome Wide RNAi Vulnerability Map for Myeloma

描述（由申请人提供）：拟议研究的中心假设是，对特定基因靶点的全基因组RNAi抑制将识别以前未识别的关键分子途径，这些分子途径在任一方向上调节骨髓瘤细胞的生长，并且这些信息可以在临床上积极利用。虽然较新的药物如硼替佐米、沙利度胺和来那度胺在骨髓瘤中具有深刻的临床活性，但分子作用的确切点尚不清楚。此外，这些药物中没有一种是治愈性的，并且鉴定可以利用的新的脆弱性靶点，以及鉴定可能在MM中具有未识别活性的现有药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，因此我们认为需要进一步的基因组规模分析和创建MM的脆弱性图谱，以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。因此，我们在这项提案中的目标是应用先进的功能基因组策略来识别和快速验证候选基因在MM生长和生存中的关键作用，并在临床上推进这一信息。鉴于此，我们最近在MM细胞系中进行了高通量、激酶组范围的RNAi致死性筛选，以鉴定对人MM存活至关重要的激酶。此外，我们还鉴定了在硼替佐米治疗存在下通过RNAi筛选使肿瘤细胞对硼替佐米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用基因组（7000个基因）的初步筛选。通过其他先前的努力，我们有一个广泛的小组60 MM细胞系，一个大型数据库的基因表达和比较基因组杂交的细胞系和原代患者样本和准备访问患者组织。我们在这里建议扩展这些发现，以探索更具全球代表性的17，000个基因高通量，MM细胞系中的合成致死RNAi筛选，并使用二次筛选，原代患者细胞中的靶标验证和生物信息学处理来验证这些发现，这些生物信息学处理将我们不同的基因组数据集结合在一起。具体来说，我们将确定通过平行RNAi沉默基因表达将在培养的骨髓瘤细胞系中以任一方向改变细胞毒性敏感性的程度。然后，我们将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，以验证这些推定的药物靶点的临床相关性，并确定它们可用作治疗反应生物标志物的程度。公共卫生相关性：多发性骨髓瘤在美国任何时候都影响着5万人。在治疗方面已经取得了重大进展，并且较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，这些药物中没有一种是治愈性的，而且它们的作用方式也不透明。因此，鉴定可以利用的新的脆弱性靶点，以及鉴定可能在MM中具有未识别活性的现有药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个基因，以控制骨髓瘤细胞生长的能力。这种基因组规模分析和创建骨髓瘤脆弱性地图是进一步了解这种疾病以及如何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.