Role of MELK in glioblastomas

MELK 在胶质母细胞瘤中的作用

• 批准号: 7794961
• 负责人: ALEXEY V TERSKIKH
• 金额: $ 43.09万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7794961
• 关键词: Ablation; Address; Alleles; Animal Model; Biological Assay; Brain; Brain Neoplasms; Candidate Disease Gene; Catalytic Domain; Cell Separation; Cells; Cessation of life; Chronic Myeloid Leukemia; Collaborations; Computer Simulation; Data; Development; Drug Delivery Systems; Embryo; Erlotinib; Gefitinib; Genes; Genetic; Germ Lines; Gleevec; Glioblastoma; Goals; Growth; Hand; Hematopoietic; Human; In Vitro; Knowledge; Laboratories; Lead; Leucine Zippers; Life; Light; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mammary Tumorigenesis; Methods; Molecular; Monoclonal Antibodies; Mus; Patients; Pharmaceutical Chemistry; Phosphotransferases; Play; Population; Primary Neoplasm; Protein Kinase; Proteins; RNA Interference; Reagent; Reporter; Role; Series; Small Interfering RNA; Solid; Stem cells; Structure; Testing; Therapeutic; Transplantation; Tumor Cell Line; Tumor Markers; Tumor Stem Cells; Tumorigenicity; Undifferentiated; Validation; Work; antitumor drug; base; cancer type; cell type; combinatorial; drug development; epidermal growth factor receptor VIII; established cell line; human monoclonal antibodies; in vitro Assay; in vivo; inhibitor/antagonist; kinase inhibitor; knock-down; knockout animal; mouse model; mutant; neoplastic cell; nerve stem cell; promoter; public health relevance; research study; small hairpin RNA; small molecule; stem; success; therapeutic target; tool; tumor; tumor growth; tumorigenesis

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.

描述（由申请人提供）：恶性胶质瘤母细胞瘤（GBMs）是最常见和致命的脑肿瘤，迄今尚无明确的治疗方法。最近的研究发现，胶质母细胞瘤中有一小部分细胞（1-10%）具有干细胞样特征。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记物。此外，我们和其他人认为MELK与肿瘤生长有关，并提出它是GBMs的治疗靶点。尽管大量的体外实验数据表明，与正常大脑相比，GBM中MELK的表达有选择性上调，但到目前为止，还没有体内证据表明MELK表达是肿瘤启动细胞的标志，也没有证据表明其在整个肿瘤中的普遍表达。在本课题中，我们将研究MELK是否在GBM中发挥作用，以及MELK是否在体内肿瘤启动细胞中表达丰富。我们将利用MELK-GFP启动子报告子，通过FACS前瞻性地分离melk -高/低细胞，并将其以有限的稀释度移植到受体脑中，以确定其肿瘤启动能力（Aim1）。我们将确定MELK的催化功能是否需要体内肿瘤发生。(目标2)。我们将在Ink4/Arf-null，/EGFRvIII小鼠模型中研究种系消融MELK催化功能（3MELK小鼠）是否消除/减少实验性胶质母细胞瘤。有可能在开发过程中有各种机制弥补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抑制MELK表达是否会抑制体内GBM的形成。最后，我们将在体外用原代人GBMs测试MELK的小分子抑制剂。我们将在原发性人GBM细胞系中验证6种现有的MELK先导抑制剂，并使用组合/药物化学优化最有效的MELK抑制剂。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？验证一个特定的蛋白质是一个相关的和现实的治疗目标是一个生死攸关的决定。鉴于格列卫、易瑞沙、特罗凯等激酶催化活性小分子抑制剂的成功，MELK作为激酶为药物开发提供了巨大的机会。虽然我们和其他人之前提出MELK作为GBM治疗的独特药物靶点，但迄今为止还没有得到体内验证。该建议的意义在于验证MELK，特别是其催化活性，作为GBMs的药物靶点，在动物模型中使用遗传方法，在培养的初级人类GBM系中使用候选的MELK小分子抑制剂。