Role of MELK in glioblastomas

描述(申请人提供)：恶性胶质母细胞瘤(GBM)是最常见和最致命的脑肿瘤，目前还没有明确的治疗方法。最近的研究发现，在胶质母细胞瘤中有一小部分具有干细胞样特征的细胞(1-10%)。我们发现母体胚胎亮氨酸拉链酶(Melk)是正常造血祖细胞和神经前体细胞的标志物。此外，我们和其他人已经将Melk与肿瘤生长联系在一起，并建议将其作为治疗GBM的靶点。尽管大量的体外数据表明，与正常大脑相比，Melk在GBM中选择性上调，但到目前为止，还没有体内证据表明Melk的表达是肿瘤启动细胞的标志，或证明其在整个肿瘤中的普遍表达。在这项提案中，我们将研究Melk是否在GBM中发挥作用，以及Melk的表达是否在体内丰富了肿瘤启动细胞。我们将利用Melk-GFP启动子-报告基因的优势，通过流式细胞仪前瞻性地分离Melk-High/-Low细胞，并将它们以有限的稀释度移植到受体脑中，以确定它们的致瘤能力(Aim1)。我们将确定体内肿瘤发生是否需要Melk催化功能。(目标2)。我们将研究胚系切除Melk催化功能(3MELK小鼠)是否消除/减少INK4/Arf-Null/EGFRvIII小鼠模型中的实验性胶质母细胞瘤。在发展过程中，各种机制有可能弥补MELK的不足。为了解决这个问题，我们将使用移植方法来确定使用shRNA敲除Melk表达是否会抑制体内GBM的形成。最后，我们将使用原代人GBM在体外测试Melk的小分子抑制剂。我们将在原代人类GBM系中验证现有的6种铅熔体抑制剂，并使用组合/药物化学来优化最有效的铅熔体抑制剂。对原发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：哪些基因代表了有希望的治疗靶点？确认一种特定的蛋白质是一个相关和现实的治疗目标，从字面上讲是一个生死攸关的决定。鉴于Gleevec、Iressa、Tarceva和其他小分子激酶催化活性抑制剂的成功，Melk作为激酶为药物开发提供了一个巨大的机会。尽管我们和其他人以前曾建议将Melk作为治疗GBM的独特药物靶点，但到目前为止还没有体内验证。这项建议的意义在于验证Melk，特别是其催化活性，在使用Melk候选小分子抑制剂的动物模型和原代人类GBM系培养中，都是针对GBM的药物靶点。 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，目前还没有明确的治疗方法。对原发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：哪些基因代表了有希望的治疗靶点？这项建议的目标是验证Melk作为治疗GBM的药物靶点，无论是在动物模型中使用遗传方法，还是在原代人类GBM系中使用Melk候选小分子抑制剂进行培养。

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.