Novel Strategies for Treatment of Myeloproliferative Disorders

摘要： Bcr-abl癌蛋白在几乎所有慢性粒细胞白血病(CML)患者中都存在，15%-30% 急性淋巴细胞白血病(ALL)病例。这里我们假设FOXO3a是一种肿瘤 造血细胞中抑制因子和bcr-Abl的表达通过介导 FOXO3a的磷酸化和随后的蛋白酶体降解，从而促进细胞 转型。因此，FOXO3a有望成为治疗骨髓增生性疾病的潜在靶点。 精神错乱。我们基于以下观察结果：1)FOXO3a对BCR-Abl诱导的BCR-Abl表达有调节作用 2)bcr-Abl介导蛋白酶体降解中FOXO3a活性的抑制。 3)-TrCP1 E3连接酶和PP2A磷酸酶可能是FOXO3a的调节因子 4)酶抑制剂对bcr-Abl的抑制常因bcr-abl的突变而导致耐药； 5)靶向下游效应分子的治疗药物可以是有效的抑制剂，不会导致 耐药bcr-abl突变的选择压力；以及6)用高度特异的蛋白酶体治疗 抑制剂Bortezomib可恢复FOXO3a水平，从而降低bcr-abl-abl-1细胞的存活率。 表达细胞和一名bcr-Abl阳性白血病患者。我们的目标是： 目的1：探讨bcr-Abl是否诱导FXO3a转录因子表达下调。 造血细胞是细胞转化的关键。我们将调查FOXO3a的参与 作为肿瘤抑制因子在A)小鼠体内携带表达bcr-Abl和野生型(Wt)或a的造血细胞 构成活性FOXO3a；b)FOXO_3基因缺失或对照小鼠的原代骨髓细胞逆转录病毒 用BCR-Abl单独或与wt或组成活性FOXO3a联合转导；c)在FOXO3a中为空 老鼠。 具体目的2：探讨BCR-Abl对FOXO3a基因调控的分子机制： 癌症治疗的靶点。我们计划研究TrCP1和PP2A在BCR-Abl诱导中的作用 免疫沉淀和免疫印迹分析、定点突变和shRNA对FOXO3a的调控 技巧。我们还计划设计和分析能够稳定FOXO3a的多肽模拟物。 具体目标3.确定高度特异的细胞是否上调FOXO3a 蛋白酶体抑制剂Bortezomib是其治疗效果的关键。作为一种基于FOXO3a的特异性疗法 是远远不现实的，目前针对FOXO3a的治疗策略可能立即转化为 诊所。在这里，我们将研究FOXO3a对介导治疗效果的需求。 硼替佐米治疗bcr-Abl诱导的白血病。研究叙述： FOXO3a是Forkhead转录因子家族的成员，调节关键细胞凋亡的表达 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FOXO3a介导的细胞凋亡逃避和 促进转化的造血细胞存活。在这个提案中，我们将检验假设 FOXO3a是造血细胞的肿瘤抑制因子及其调控bcr-Abl的表达 活性去除这种肿瘤抑制物以促进细胞转化。特别是，我们建议BCR- ABL诱导的FOXO3a可逆磷酸化和蛋白酶体降解的调节将抑制 FOXO3a在表达bcr-Abl基因的造血细胞中的抑瘤活性 细胞，这会导致随后的存活和白血病的发生。

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.