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

描述（由申请人提供）：拟议的研究重点是人Noxa的新型调节机制和存活功能，Noxa是一种促凋亡的“仅BH 3”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，促生存Bcl-2家族成员Mcl-1 L相互作用，以促进细胞色素c释放到胞质溶胶中。Noxa/Mcl-1 L相互作用在免疫系统细胞的存活和死亡中起关键作用，并且Noxa是增殖的人淋巴细胞和造血癌细胞系的葡萄糖剥夺诱导的细胞凋亡所需的。这种仅BH 3蛋白在大多数白血病中稳定且组成型表达。初步观察已经确定，Noxa是磷酸化的丝氨酸残基的激酶，Cdk 5，在营养物质和葡萄糖的存在下，并确定了两个胞质多蛋白颗粒，螯合修饰的蛋白质。研究还表明，修饰的隔离Noxa调节葡萄糖代谢，并具有促生存功能。假设人类Noxa是后免疫调节，它在白血病细胞中发挥促生存和促凋亡的作用，将通过三个具体的目标进行测试。第一个目的是确定后代谢修饰的人Noxa如何在有氧糖酵解和细胞存活中发挥作用。Cdk 5和葡萄糖敏感的PI 3 K/Akt信号通路对Noxa这种新的代谢功能的贡献也将被研究。将纯化和表征含有Noxa的胞质多蛋白颗粒;将确定它们在调节Noxa和有氧糖酵解中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PHLPP 2在激活白血病细胞中的Noxa和凋亡两者中的作用。将Cdk 5的联合收割机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品上进行测试，随后在小鼠异种移植物测定中进行验证。还将测定人Noxa的不可磷酸化的突变体和肽促进细胞凋亡的能力。第三个目的是建立一个条件性转基因小鼠模型，以研究人类Noxa在白血病发生和葡萄糖代谢中的作用。采用Vav启动子-四环素反式激活因子（Vav-tTA）驱动的多西环素阻遏系统，人仅BH 3蛋白将在造血谱系细胞中表达。转基因模型将用于确定人Noxa的表达是否促进白血病发生，蛋白质的持续表达是否是维持白血病表型所需的，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因子BH 3-only蛋白，在造血系统癌症中也具有存活功能。拟议的研究将确定新的信号通路和中间体参与调节Noxa在这些双重和相反的作用，并应导致开发新的治疗策略，针对这种蛋白质在淋巴和骨髓白血病。 公共卫生相关性：拟议的研究旨在调查一个假定的促生存功能的小促凋亡Bcl-2家族蛋白，Noxa，在人类淋巴和骨髓恶性肿瘤。了解这种肿瘤抑制蛋白的调控机制，以及描绘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型合理的治疗策略。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，将建立条件性转基因小鼠模型以研究人Noxa在白血病发生和葡萄糖消耗中的作用。一旦建立，这种小鼠模型也将有助于设计和测试旨在激活白血病中Noxa的肿瘤抑制功能的新疗法。

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.