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

描述（由申请人提供）：拟研究的重点是人类Noxa的新调控机制和生存功能，Noxa是一种促凋亡的“仅bh3”Bcl-2家族蛋白，迅速成为血液系统恶性肿瘤的重要治疗靶点。激活的Noxa与其结合伙伴，促生存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起着至关重要的作用，并且Noxa是葡萄糖剥夺诱导增殖的人淋巴细胞和造血癌细胞系凋亡所必需的。这种仅bh3蛋白在大多数白血病中稳定且组成性地表达。初步观察已经确定，在营养物质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，并且已经确定了两个隔离修饰蛋白的细胞质多蛋白颗粒。研究还表明，修饰的、隔离的Noxa调节葡萄糖代谢，并具有促进生存的功能。人类Noxa是翻译后调控的假设，它在白血病细胞中发挥促生存和促凋亡的作用，将通过三个特定的目的来验证。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。Cdk5和葡萄糖敏感的PI3K/Akt信号通路对Noxa这种新的代谢功能的贡献也将被研究。将对含有Noxa的胞质多蛋白颗粒进行纯化和表征；它们在调节Noxa和有氧糖酵解中的功能相关性和作用将被确定。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和凋亡中的作用将被确定。结合特异性Cdk5抑制剂和糖酵解抑制剂的新策略将在白血病细胞系和患者样本上进行测试，随后在小鼠异种移植试验中得到验证。人类Noxa的非磷酸化突变体和肽促进细胞凋亡的能力也将被确定。第三个目标是开发一种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-四环素反激活因子（Vav- tta）驱动的强力霉素抑制系统。该转基因模型将用于确定人类Noxa的表达是否促进白血病的发生，是否需要持续表达该蛋白来维持白血病表型，以及Noxa在淋巴细胞中的组成性表达是否会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BH3-only蛋白，在造血肿瘤中也具有存活功能。这项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，并应导致新的治疗策略的发展，针对该蛋白在淋巴和髓性白血病。

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.