Novel Strategies for Treatment of Myeloproliferative Disorders

治疗骨髓增生性疾病的新策略

• 批准号: 8271290
• 负责人: ROYA KHOSRAVI-FAR
• 金额: $ 34.22万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-07 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8271290
• 关键词: A Mouse; Acute Lymphocytic Leukemia; Apoptosis; Apoptotic; Bone Marrow Cells; Bortezomib; Carcinoma; Cell Culture Techniques; Cell Death Inhibition; Cell Nucleus; Cell Survival; Cell physiology; Cells; Chromosomal translocation; Chronic Myeloid Leukemia; Clinic; Clinical; Cytoprotection; DNA Damage; Degradation Pathway; Disease; Down-Regulation; FDA approved; Family; Family member; Genes; Growth; Hematopoietic; Hematopoietic stem cells; Immunoblot Analysis; Immunoprecipitation; Inhibition of Cell Proliferation; Knockout Mice; Malignant Neoplasms; Mediating; Mediator of activation protein; Molecular; Mus; Mutation; Myeloproliferative disease; Oncogene Proteins; Oxidative Stress; Pathogenesis; Patients; Peptides; Peripheral Blood Lymphocyte; Philadelphia Chromosome; Philadelphia Chromosome Negative Chronic Myelogenous Leukemia; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Proteasome Inhibitor; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Recycling; Regulation; Research; Residual state; Resistance; Resistance development; Role; Signal Pathway; Site-Directed Mutagenesis; Spleen; Techniques; Testing; Therapeutic; Therapeutic Effect; Translations; Tumor Suppressor Proteins; Ubiquitination; Up-Regulation; abl Oncogene; abstracting; base; cell transformation; clinically relevant; combat; cytokine; design; forkhead protein; inhibitor/antagonist; kinase inhibitor; leukemia; leukemogenesis; member; metaplastic cell transformation; mimetics; mouse model; multicatalytic endopeptidase complex; novel strategies; pressure; prevent; response; small hairpin RNA; therapeutic target; transcription factor; tumor; tumorigenesis; ubiquitin-protein ligase

Abstract: The Bcr-Abl oncoprotein is present in nearly all chronic myelogenous leukemia (CML) patients and 15-30% of acute lymphoblastic leukemia (ALL) cases. Here we hypothesize that FOXO3a serves as a tumor suppressor in hematopoietic cells and that Bcr-Abl expression removes this suppressive function by mediating the phosphorylation and subsequent proteasomal degradation of FoxO3a, thereby promoting cellular transformation. Thus, FOXO3a represents a potential therapeutic target for the treatment of myeloproliferative disorders. We base this hypothesis on the observations that 1) FOXO3a functions to regulate Bcr-Abl-induced evasion of apoptosis; 2) Bcr-Abl mediates the inhibition of FOXO3a activity in a proteasome degradation- dependent manner; 3) a ¿-TrCP1 E3 ligase and a PP2A phosphatase are putative regulators of FOXO3a stability; 4) inhibition of Bcr-Abl by kinase inhibitors frequently leads to resistance, due to mutations of Bcr-Abl; 5) therapeutics that target the downstream effector molecules can be effective inhibitors that will not result in selection pressure for resistant Bcr-Abl mutations; and 6) treatment with the highly specific proteasome inhibitor, bortezomib, restores levels of FOXO3a and consequently decreases the survival of Bcr-Abl- expressing cells and a Bcr-Abl-positive leukemia patient. Our Aims are: Aim 1: To determine whether Bcr-Abl-induced down-regulation of the FOXO3a transcription factor in hematopoietic cells is critical for cellular transformation. We will investigate the involvement of FOXO3a as a tumor suppressor in A) mice bearing hematopoietic cells expressing Bcr-Abl and a wild-type (wt) or a constitutively active FOXO3a; B) primary bone marrow cells from FoxO3-null or control mice retrovirally transduced with Bcr-Abl alone or in combination with wt or constitutively active FOXO3a; C) in FoxO3a null mice. Specific Aim 2: To investigate the molecular mechanism for Bcr-Abl-induced regulation of FOXO3a: targets for cancer therapeutics. We plan to investigate the role of ¿-TrCP1 and PP2A in Bcr-Abl-induced regulation of FOXO3a by immunoprecipitation and immunoblot analysis, site-directed mutagenesis and shRNA techniques. We also plan to design and analyze peptide mimetics that can stabilize FOXO3a. Specific Aim 3. To determine whether upregulation of FOXO3a by the highly the highly specific proteasome inhibitor bortezomib is critical for its therapeutic effect. As a specific FOXO3a-based therapy is far from reality, current therapeutic strategies that target FOXO3a could have immediate translation to the clinic. Here, we will investigate the requirement of FOXO3a for mediating the therapeutic efficiency of bortezomib in Bcr-Abl-induced leukemia. Research Narrative: FOXO3a, a member of the Forkhead transcription factor family, regulates the expression of key apoptotic genes and is thought to be a tumor suppressor. We and others have established that Bcr-Abl, the primary cause of Chronic Myelogenous Leukemia (CML), inhibits FOXO3a to mediate evasion of apoptosis and to promote the survival of the transformed hematopoietic cells. In this proposal, we will test the hypothesis that FOXO3a is a tumor suppressor in hematopoietic cells and Bcr-Abl-expression via regulation of FOXO3a activity removes this tumor suppressor to promote cellular transformation. In particular, we propose that Bcr- Abl-induced regulation of the reversible phosphorylation and proteasomal degradation of FOXO3a will inhibit tumor-suppressor activity of FOXO3a in Bcr-Abl-expressing hematopoietic cells, potentially hematopoietic stem cells, and this leads to subsequent survival and leukemogenesis.

摘要： Bcr-Abl癌蛋白存在于几乎所有的慢性粒细胞白血病（CML）患者中，15-30%的CML患者存在Bcr-Abl癌蛋白。 急性淋巴细胞白血病（ALL）病例。在这里，我们假设FOXO 3a作为肿瘤 Bcr-Abl表达通过介导造血细胞中抑制因子而消除了这种抑制功能， FoxO 3a的磷酸化和随后的蛋白酶体降解，从而促进细胞 转型因此，FOXO 3a代表了治疗骨髓增生性疾病的潜在治疗靶点。 紊乱我们基于以下观察结果提出了这一假设：1）FOXO 3a的功能是调节Bcr-Abl诱导的Bcr-Abl诱导的细胞凋亡。 逃避凋亡; 2）Bcr-Abl介导蛋白酶体降解中FOXO 3a活性的抑制- 依赖性方式; 3）一个<$-TrCP 1 E3连接酶和一个PP 2A磷酸酶是FOXO 3a的假定调节剂 稳定性; 4）由于Bcr-Abl的突变，激酶抑制剂对Bcr-Abl的抑制经常导致抗性; 5)靶向下游效应分子的治疗剂可以是有效的抑制剂 抗性Bcr-Abl突变的选择压力;和6）用高度特异性蛋白酶体处理 抑制剂硼替佐米可恢复FOXO 3a的水平，从而降低Bcr-Abl的存活率。 表达细胞和Bcr-Abl阳性白血病患者。我们的目标是： 目的1：确定Bcr-Abl是否诱导FOXO 3a转录因子下调。 造血细胞对于细胞转化至关重要。我们将调查FOXO 3a是否参与 作为肿瘤抑制因子，在A）携带表达Bcr-Abl的造血细胞和野生型（wt）或 组成型活性FOXO 3 a; B）来自FoxO 3缺失或对照小鼠的原代骨髓细胞， 用Bcr-Abl单独或与wt或组成型活性FOXO 3a组合转导; C）在FoxO 3a缺失中 小鼠 具体目的2：研究Bcr-Abl诱导FOXO 3a调节的分子机制： 癌症治疗的靶点。我们计划研究<$-TrCP 1和PP 2A在Bcr-Abl诱导的细胞凋亡中的作用。 通过免疫沉淀和免疫印迹分析、定点突变和shRNA调控FOXO 3a 技术.我们还计划设计和分析可以稳定FOXO 3a的肽模拟物。 具体目标3。为了确定FOXO 3a是否被高度特异性的 蛋白酶体抑制剂硼替佐米对其治疗效果至关重要。作为一种基于FOXO 3a的特异性疗法 这与现实相去甚远，目前针对FOXO 3a的治疗策略可能会立即转化为 诊所在这里，我们将研究FOXO 3a介导的治疗效率的要求， 硼替佐米治疗Bcr-Abl诱导的白血病研究叙述： FOXO 3a是Forkhead转录因子家族的成员，调节关键的凋亡基因的表达。 基因，被认为是一种肿瘤抑制因子。我们和其他人已经确定，Bcr-Abl，主要的 慢性粒细胞白血病（CML）的病因，抑制FOXO 3a介导细胞凋亡的逃避， 促进转化的造血细胞的存活。在本提案中，我们将检验以下假设： FOXO 3a是造血细胞中的肿瘤抑制因子，通过FOXO 3a调节Bcr-Abl表达 活性去除这种肿瘤抑制因子以促进细胞转化。特别是，我们建议Bcr- FOXO 3a的可逆磷酸化和蛋白酶体降解的调节将抑制FOXO 3a的表达。 FOXO 3a在表达Bcr-Abl的造血细胞中的肿瘤抑制活性， 细胞，这导致随后的生存和白血病。