Role of MELK in glioblastomas

描述（由申请方提供）：恶性胶质母细胞瘤（GBM）是最常见和最致命的脑肿瘤，迄今为止尚无明确的治疗方法。最近的研究在胶质母细胞瘤中发现了一小群具有干细胞样特征的细胞（1-10%）。我们鉴定了母胚亮氨酸拉链激酶（MELK）作为正常造血和神经祖细胞的标志物。此外，我们和其他人已经将MELK与肿瘤生长联系起来，并提出将其作为GBM的治疗靶点。 尽管大量体外数据表明MELK在GBM中与正常脑相比选择性上调，但迄今为止没有体内证据可用于确立MELK表达作为肿瘤起始细胞的标志物或记录其在整个肿瘤中的普遍表达。在这个提议中，我们将研究MELK是否在GBM中起作用以及MELK表达是否在体内富集肿瘤起始细胞。我们将利用MELK-GFP启动子-报告基因，通过FACS前瞻性地分离MELK-高/-低细胞，并将其以有限稀释度移植到受体脑中，以确定其肿瘤起始能力（Aim 1）。 我们将确定MELK催化功能是否是体内肿瘤发生所必需的。(Aim 2）。我们将研究MELK催化功能的种系消融（3 MELK小鼠）是否消除/减少Ink 4/Arf-null/EGFRvIII小鼠模型中的实验性胶质母细胞瘤。可能有各种机制在发育过程中弥补MELK的缺乏。为了解决这个问题，我们将使用移植方法来确定使用shRNA敲低MELK表达是否会抑制体内GBM形成。最后，我们将使用原代人GBM在体外测试MELK的小分子抑制剂。我们将在原代人GBM系中验证6种现有的主要MELK抑制剂，并使用组合/药物化学优化最有效的MELK抑制剂。 原发性肿瘤的表达分析确定了数百个候选基因。剩下的关键问题是：哪些基因代表有希望的治疗靶点？验证一种特定的蛋白质是一个相关的和现实的治疗目标，实际上是一个生死攸关的决定。鉴于格列卫、易瑞沙、特罗凯和其他激酶催化活性的小分子抑制剂的成功，MELK作为激酶为药物开发提供了一个巨大的机会。尽管我们和其他人先前提出MELK作为GBM治疗的独特药物靶点，但迄今为止尚未证实体内验证。该提议的意义在于验证MELK，特别是其催化活性，作为GBM的药物靶标，在使用遗传方法的动物模型中和在使用MELK的候选小分子抑制剂的培养物中的原代人GBM系中。 公共卫生相关性：恶性胶质母细胞瘤（GBM）是最常见和最致命的脑肿瘤，迄今尚无明确的治疗方法。原发性肿瘤的表达分析确定了数百个候选基因。剩下的关键问题是：哪些基因代表有希望的治疗靶点？本提案的目的是在使用遗传方法的动物模型和使用MELK的候选小分子抑制剂的培养物中的原代人GBM系中验证MELK作为GBM的药物靶标。

DESCRIPTION (provided by applicant): Malignant gliomablastomas (GBMs) are the most common and fatal brain tumors with no definitive treatment available to date. Recent studies identified a small population of cells (1-10%) with stem- like features in glioblastomas. We identified the Maternal Embryonic Leucine zipper Kinase (MELK) as a marker for normal hematopoietic and neural progenitors. In addition, we and others have implicated MELK in tumor growth and proposed it as a therapeutic target for GBMs. Despite the solid body of in vitro data suggesting that MELK is selectively upregulated in GBM compared to normal brain, no in vivo evidence is available to date establishing MELK expression as a marker for tumor-initiating cells or documenting its ubiquitous expression throughout the tumor. In this proposal we will investigate whether MELK plays role in GBM and whether MELK expression enriches for tumor-initiating cells in vivo. We will take advantage of MELK-GFP promoter-reporter to prospectively isolate MELK-high/-low cells by FACS and transplant them at limiting dilutions in recipient brain to determine their tumor-initiating capacity (Aim1). We will determine whether MELK catalytic function is required for tumorigenesis in vivo. (Aim 2). We will investigate whether the germ line ablation of MELK catalytic function (3MELK mice) eliminates/reduces experimental glioblastomas in the Ink4/Arf-null,/EGFRvIII mouse model. It is possible that various mechanisms compensate for the lack of MELK during development. To address this issue we will use a transplantation approach to determine whether knocking down MELK expression using shRNA will inhibit GBM formation in vivo. Finally, we will test small molecule inhibitors of MELK in vitro using primary human GBMs. We will validate 6 existing lead MELK inhibitors in primary human GBM lines and optimize the most potent MELK inhibitors using combinatorial/medicinal chemistry. The expression analysis of primary tumors identified hundreds of gene candidates. The critical remaining question is: which genes represent promising therapeutic targets? Validation of a particular protein to be a relevant and realistic target for treatment is literally a life-and-death decision. Given the success of Gleevec, Iressa, Tarceva, and other small molecule inhibitors of kinase catalytic activity, MELK as kinase presents a formidable opportunity for drug development. Although we and others previously proposed MELK as a unique drug target for GBM treatment, no in vivo validation has been demonstrated to date. The significance of this proposal is in validating MELK, in particular its catalytic activity, as a drug target for GBMs both in animal models using a genetic approach and in primary human GBM lines in culture using candidate small molecule inhibitors of MELK. PUBLIC HEALTH RELEVANCE: Malignant gliomablastomas (GBMs) are the most common and fatal brain tumors with no definitive treatment available to date. The expression analysis of primary tumors identified hundreds of gene candidates. The critical remaining question is: which genes represent promising therapeutic targets? The goal of this proposal is to validating MELK as a drug target for GBMs both in animal models using a genetic approach and in primary human GBM lines in culture using candidate small molecule inhibitors of MELK.