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: 8083459
• 负责人: AMEETA KELEKAR
• 金额: $ 29.91万
• 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/8083459
• 关键词: 3-Dimensional; Apoptosis; Apoptotic; B-Lymphocytes; 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. PUBLIC HEALTH RELEVANCE: The proposed research is aimed at investigating a putative pro-survival function for a small pro-apoptotic Bcl-2 family protein, Noxa, in human lymphoid and myeloid malignancies. Understanding the regulatory mechanisms that keep this tumor suppressor protein in check, as well as delineating the signaling pathways that activate its pro- apoptotic function, will aid in the design of novel and rational therapeutic strategies that target this multifunctional protein. The proposed research will use both established cell lines as well as patient cancer cells to carry out the studies. Additionally, a conditional transgenic mouse model will be established to investigate the role of human Noxa in leukemogenesis and glucose consumption. Once established, this mouse model will also be useful in designing and testing of novel therapies aimed at activating the tumor suppressor function of Noxa in leukemias.

描述（由申请人提供）：拟议的研究重点是人 Noxa 的新型调节机制和生存功能，Noxa 是一种促凋亡的“仅 BH3”Bcl-2 家族蛋白，迅速成为血液恶性肿瘤的重要治疗靶点。活化的 Noxa 与其结合伴侣（促存活 Bcl-2 家族成员 Mcl-1L）相互作用，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生存和死亡中发挥着至关重要的作用，并且 Noxa 是葡萄糖剥夺诱导的增殖性人类淋巴细胞和造血癌细胞系细胞凋亡所必需的。这种仅 BH3 的蛋白在大多数白血病中稳定且组成型表达。初步观察已确定，在营养物和葡萄糖存在的情况下，Noxa 在丝氨酸残基上被激酶 Cdk5 磷酸化，并确定了两种胞质多蛋白颗粒，可隔离修饰的蛋白。研究还表明，经过修饰、隔离的 Noxa 可调节葡萄糖代谢并具有促生存功能。人类 Noxa 受到翻译后调节并且在白血病细胞中发挥促生存和促凋亡作用的假设将通过三个具体目标进行测试。第一个目标是确定翻译后修饰的人类 Noxa 如何在有氧糖酵解和细胞存活中发挥作用。还将研究 Cdk5 和葡萄糖敏感的 PI3K/Akt 信号通路对 Noxa 这种新型代谢功能的贡献。含有Noxa的胞质多蛋白颗粒将被纯化和表征；它们的功能相关性以及在调节 Noxa 和有氧糖酵解中的作用将被确定。第二个目标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。肿瘤抑制磷酸酶 PHLPP2 在激活白血病细胞 Noxa 和细胞凋亡中的作用将被确定。将 Cdk5 特异性抑制剂与糖酵解抑制剂相结合的新策略将在白血病细胞系和患者样本上进行测试，并随后在小鼠异种移植试验中进行验证。还将确定人Noxa的不可磷酸化突变体和肽促进细胞凋亡的能力。第三个目标是开发条件转基因小鼠模型，以研究人类 Noxa 在白血病发生和葡萄糖代谢中的作用。人类 BH3-only 蛋白将在造血谱系细胞中表达，采用 Vav 启动子-四环素反式激活子 (Vav-tTA) 驱动的多西环素抑制系统。该转基因模型将用于确定人 Noxa 的表达是否促进白血病发生、维持白血病表型是否需要该蛋白的持续表达以及淋巴细胞中 Noxa 的组成型表达是否改变其代谢葡萄糖以产生能量的能力。初步研究表明，Noxa 是一种典型的肿瘤抑制蛋白 BH3-only 蛋白，在造血系统癌症中也具有生存功能。拟议的研究将确定参与调节 Noxa 这些双重和相反作用的新信号通路和中间体，并应导致开发针对淋巴和髓性白血病中的这种蛋白质的新治疗策略。 公共健康相关性：拟议的研究旨在调查一种小型促凋亡 Bcl-2 家族蛋白 Noxa 在人类淋巴和骨髓恶性肿瘤中的推定促生存功能。了解控制这种肿瘤抑制蛋白的调节机制，以及描述激活其促凋亡功能的信号通路，将有助于设计针对这种多功能蛋白的新颖且合理的治疗策略。拟议的研究将使用已建立的细胞系以及患者癌细胞来进行研究。此外，还将建立条件转基因小鼠模型来研究人Noxa在白血病发生和葡萄糖消耗中的作用。一旦建立，该小鼠模型也将可用于设计和测试旨在激活 Noxa 在白血病中的肿瘤抑制功能的新疗法。