Role of a pro-apoptotic Bcl-2 protein in the survival and death of leukemia cells

促凋亡 Bcl-2 蛋白在白血病细胞存活和死亡中的作用

• 批准号: 8460563
• 负责人: AMEETA KELEKAR
• 金额: $ 30.65万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-08 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460563
• 关键词: 3-Dimensional; Apoptosis; Apoptotic; B-Lymphocytes; BCL-2 Protein; BCL2 gene; Binding; Biological Assay; Cancer cell line; Cell Death; Cell Line; Cell Lineage; Cell Survival; Cells; Cessation of life; Complex; Consumption; Coupled; Cyclin-Dependent Kinases; Cytosol; DNA Damage; Development; Doxycycline; Enzymes; Family member; Generations; Glucose; Glycolysis; Goals; Hematologic Neoplasms; Hematopoietic; Human; Hypoxia; Immune system; Jurkat Cells; Knowledge; Lead; Lymphoblastic Leukemia; Lymphocyte; Malignant lymphoid neoplasm; Mass Spectrum Analysis; Metabolic; Modification; Mus; Myeloid Leukemia; Myeloproliferative disease; Normal Cell; Noxae; Nutrient; PMAIP1 gene; Patients; Peptides; Phenotype; Phosphoglycerate Kinase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Post-Translational Regulation; Production; Proliferating; Protein Dephosphorylation; Protein Family; Proteins; Protocols documentation; Regulation; Research; Research Proposals; Role; Sampling; Serine; Signal Pathway; Signal Transduction; Stimulus; Stress; System; T-Lymphocyte; Testing; Therapeutic; Trans-Activators; Transgenic Mice; Transgenic Model; Transgenic Organisms; Tumor Suppressor Proteins; Xenograft procedure; aerobic glycolysis; cancer cell; cell type; cytochrome c; deprivation; design; established cell line; glucose metabolism; inhibitor/antagonist; leukemia; leukemogenesis; mouse model; mutant; novel; novel strategies; novel therapeutics; particle; pro-apoptotic protein; promoter; protein complex; protein expression; protein function; protein phosphatase 2C; response; therapeutic target

DESCRIPTION (provided by applicant): The proposed research focuses on a novel regulatory mechanism and survival function for human Noxa, a pro- apoptotic 'BH3-only' Bcl-2 family protein, fast emerging as an important therapeutic target in hematological malignancies. Activated Noxa interacts with its binding partner, pro-survival Bcl-2 family member Mcl-1L, to facilitate the release of cytochrome c into the cytosol. Noxa/Mcl-1L interactions play a crucial role in the survival and death of cells of the immune system and Noxa is required for glucose deprivation-induced apoptosis of proliferating human lymphocytes and hematopoietic cancer cell lines. This BH3-only protein is stably and constitutively expressed in a majority of leukemias. Preliminary observations have determined that Noxa is phosphorylated on a serine residue by the kinase, Cdk5, in the presence of nutrients and glucose and have identified two cytosolic multi-protein particles that sequester the modified protein. Studies also suggest that modified, sequestered Noxa regulates glucose metabolism and has a pro-survival function. The hypothesis that human Noxa is post-translationally regulated and that it plays both pro-survival and pro-apoptotic roles in leukemia cells will be tested through three specific aims. The first aim is directed at determining how post-translationally modified human Noxa plays a role in aerobic glycolysis and cell survival. The contribution of Cdk5 and the glucose-sensitive PI3K/Akt signaling pathway to this novel metabolic function of Noxa will also be investigated. The cytosolic multi-protein particles containing Noxa will be purified and characterized; their functional relevance and role in regulating Noxa and aerobic glycolysis will be determined. The second aim is directed at investigating apoptotic stimuli and signaling pathway/s that lead to the activation of Noxa pro-apoptotic function in leukemia cells. The role of the tumor suppressor phosphatase, PHLPP2, in activating both Noxa and apoptosis in leukemia cells will be determined. Novel strategies that combine specific inhibitors of Cdk5 with inhibitors of glycolysis will be tested on leukemia cell lines and patient samples and later validated in murine xenograft assays. The ability of non- phosphorylatable mutants and peptides of human Noxa to promote apoptosis will also be determined. The third aim is directed at developing a conditional transgenic mouse model to investigate the role of human Noxa in leukemogenesis and glucose metabolism. The human BH3-only protein will be expressed in hematopoietic lineage cells, employing the Vav promoter-tetracyline transactivator (Vav-tTA)-driven doxycycline repressible system. The transgenic model will be utilized for determining whether expression of human Noxa promotes leukemogenesis, whether sustained expression of the protein is required for maintaining the leukemic phenotype, and whether constitutive expression of Noxa in lymphocytes alters their ability to metabolize glucose for energy generation. Preliminary studies show that Noxa, a canonical tumor suppressor BH3-only protein, also has a survival function in hematopoietic cancers. The proposed research will identify novel signaling pathways and intermediates involved in regulating Noxa in these dual and opposing roles, and should lead to the development of new therapeutic strategies for targeting this protein in lymphoid and myeloid leukemias.

描述（由申请人提供）：拟议的研究着重于人类NOXA的新型调节机制和生存功能，这是一种可凋亡的“仅BH3”的BCl-2家族蛋白，快速成为血液恶性肿瘤中重要的治疗靶点。活化的NOXA与其结合伴侣促生物苏子Bcl-2家族成员MCL-1L相互作用，以促进细胞色素C释放到细胞质中。 NOXA/MCL-1L相互作用在免疫系统细胞的存活和死亡中起着至关重要的作用，而NOXA对于葡萄糖剥夺引起的人类淋巴细胞和造血性癌细胞细胞系的凋亡需要。这种仅BH3蛋白在大多数白血病中都稳定且组成型。初步观察结果确定，在营养和葡萄糖的存在下，激酶CDK5在丝氨酸残基上磷酸化，并确定了两个隔离修饰蛋白的胞质多蛋白质颗粒。研究还表明，经过修改的，隔离的NOXA调节葡萄糖代谢，并具有促生存功能。人类NOXA受到后翻译的假设，它将通过三个特定的目的来测试白血病细胞中的促生存和促凋亡角色的假设。第一个目的旨在确定翻译后修饰的人NOXA如何在有氧糖酵解和细胞存活中起作用。还将研究CDK5和葡萄糖敏感的PI3K/AKT信号通路对NOXA的新代谢功能的贡献。含有NOXA的胞质多蛋白颗粒将被纯化和表征。将确定它们在调节NOXA和有氧糖酵解的功能相关性和作用。第二个目的是研究导致白血病细胞中NOXA促凋亡功能激活的凋亡刺激和信号通路。将确定肿瘤抑制磷酸酶PHLPP2在激活白血病细胞中NOXA和凋亡中的作用。将CDK5与糖酵解抑制剂结合的新型策略将在白血病细胞系和患者样品上进行测试，然后在鼠异种移植分析中进行验证。也将确定人NOXA促进凋亡的非磷酸化突变体和肽的能力。第三个目标是开发有条件的转基因小鼠模型，以研究人NOXA在白血病发生和葡萄糖代谢中的作用。仅使用VAV启动子四环素反式激活剂（VAV-TTA）驱动的多西环素抑制系统，仅在造血谱系细胞中表达人类BH3蛋白。将利用转基因模型来确定人NOXA的表达是否促进白血病，是否需要持续的蛋白质表达来维持白血病表型，以及NOXA在淋巴细胞中NOXA的本构表达是否会改变其将葡萄糖代谢葡萄糖代谢产生的能力。初步研究表明，NOXA是一种仅限抑制BH3蛋白的规范性肿瘤，在造血癌中也具有生存功能。拟议的研究将确定在这些双重和相反的角色中调节NOXA的新型信号通路和中间体，并应导致发展新的治疗策略，以靶向该蛋白质在淋巴类和髓样白血病中。