Novel Strategies for Treatment of Myeloproliferative Disorders

描述(申请人提供)：bcr-abl癌蛋白存在于几乎所有慢性粒细胞白血病(CML)患者和15%-30%的急性淋巴细胞白血病(ALL)患者中。在这里，我们假设FOXO3a在造血细胞中作为肿瘤抑制因子，BCR-Abl的表达通过介导FOXO3a的磷酸化和随后的蛋白酶体降解来消除这种抑制功能，从而促进细胞转化。因此，FOXO3a是治疗骨髓增生性疾病的潜在靶点。我们基于以下观察结果：1)FOXO3a功能调节BCR-Abl诱导的细胞凋亡逃避；2)BCR-Abl以蛋白酶体降解依赖的方式介导FOXO3a活性的抑制；3)α-TrCP1 E3连接酶和PP2A磷酸酶是FOXO3a稳定性的假定调节因子；4)由于bcr-abl的突变，抑制bcr-abl经常导致耐药性；5)针对下游效应分子的治疗可以是有效的抑制剂，不会导致耐药的bcr-abl突变；6)用高度特异的蛋白酶体抑制剂Bortezomib治疗，可以恢复FOXO3a的水平，从而降低bcr-Abl表达细胞和bcr-Abl阳性白血病患者的存活率。我们的目标是：目的1：确定BCR-Abl诱导的造血细胞中FOXO3a转录因子的下调是否在细胞转化中起关键作用。我们将研究FOXO3a作为肿瘤抑制因子在A)表达bcr-Abl和野生型(Wt)或固有活性FOXO3a造血细胞的小鼠中的作用；b)单独或联合逆转录病毒转导BCR-Abl或联合表达野生型(Wt)或固有活性FOXO3a的FOXO3a缺失小鼠的原代骨髓细胞；c)FOXO3a缺失小鼠。具体目的2：探讨BCR-Abl对FOXO3a基因调控的分子机制：肿瘤治疗靶点。我们计划通过免疫沉淀和免疫印迹分析、定点突变和shRNA技术来研究-TrCP1和PP2A在BCR-Abl诱导的FOXO3a调控中的作用。我们还计划设计和分析能够稳定FOXO3a的多肽模拟物。具体目的3.确定高度特异的蛋白酶体抑制剂Bortezomib上调FOXO3a是否对其疗效起关键作用。由于一种基于FOXO3a的特定疗法还远未实现，目前针对FOXO3a的治疗策略可能会立即转化为临床。在这里，我们将研究FOXO3a在BCR-Abl诱导的白血病中介导Bortezomib疗效的必要性。与公共健康相关：FOXO3a是Forkhead转录因子家族的成员，调节关键的凋亡基因的表达，被认为是一种肿瘤抑制因子。我们和其他人已经确定，BCR-Abl是慢性粒细胞白血病(CML)的主要原因，它抑制FOXO3a，以介导逃避凋亡，并促进转化的造血细胞的存活。在这个提案中，我们将检验FOXO3a是造血细胞中的肿瘤抑制因子的假设，并且通过调节FOXO3a的活性来表达bcr-Abl-消除这种肿瘤抑制因子以促进细胞转化。特别是，我们认为BCR-Abl诱导的FOXO3a可逆磷酸化和蛋白酶体降解的调节将抑制表达BCR-Abl的造血细胞中FOXO3a的肿瘤抑制活性，这可能导致随后的存活和白血病发生。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.