Novel Strategies for Treatment of Myeloproliferative Disorders

描述（由申请人提供）：Bcr-Abl癌蛋白存在于几乎所有的慢性粒细胞白血病（CML）患者和15-30%的急性淋巴细胞白血病（all）病例中。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代表了治疗骨髓增生性疾病的潜在治疗靶点。我们基于以下观察得出这一假设：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) a¿- trcp1e3连接酶和PP2A磷酸酶被认为是FOXO3a稳定性的调节因子；4)由于Bcr-Abl的突变，激酶抑制剂对Bcr-Abl的抑制经常导致耐药性；5)靶向下游效应分子的治疗方法可能是有效的抑制剂，不会导致耐药Bcr-Abl突变的选择压力；6)使用高特异性蛋白酶体抑制剂硼替佐米治疗可恢复FOXO3a水平，从而降低表达Bcr-Abl的细胞和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FOXO3a转录因子的下调是否对细胞转化至关重要。我们将研究FOXO3a在造血细胞表达Bcr-Abl和野生型（wt）或组成型活性FOXO3a的a)小鼠中作为肿瘤抑制因子的作用；B)用Bcr-Abl单独或与wt或组成型活性FOXO3a联合逆转录转导foxo3缺失或对照小鼠的原代骨髓细胞；C) FoxO3a缺失小鼠。特异性目的2：探讨bcr - abl诱导的FOXO3a：靶点调控癌症治疗的分子机制。我们计划通过免疫沉淀和免疫印迹分析、定点诱变和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可以稳定FOXO3a的肽模拟物。具体目标3。确定高特异性蛋白酶体抑制剂硼替佐米是否上调FOXO3a对其治疗效果至关重要。由于基于FOXO3a的特异性治疗还远未实现，目前针对FOXO3a的治疗策略可以立即转化为临床。在这里，我们将研究FOXO3a在bcr - abl诱导的白血病中介导硼替佐米治疗效果的需要。公共卫生相关性：FOXO3a是Forkhead转录因子家族的一员，调节关键凋亡基因的表达，被认为是一种肿瘤抑制因子。我们和其他人已经确定Bcr-Abl，慢性髓性白血病（CML）的主要原因，抑制FOXO3a介导细胞凋亡逃避和促进转化的造血细胞的存活。在本提案中，我们将验证FOXO3a在造血细胞中是肿瘤抑制因子的假设，bcr - abl的表达通过调节FOXO3a的活性去除这种肿瘤抑制因子，促进细胞转化。特别是，我们提出Bcr- abl诱导的FOXO3a可逆磷酸化和蛋白酶体降解的调控将抑制表达Bcr- abl的造血细胞（可能是造血干细胞）中FOXO3a的肿瘤抑制活性，从而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.