Molecular and Cellular Characterization of Myeloproliferative Disease

描述（由申请人提供）：骨髓增生性疾病（MPD）代表血液和骨髓中骨髓细胞的异常增殖，被认为是一种“干细胞疾病”。“虽然MPD的病理分类最近已经确定，但疾病的分子发病机制还不清楚。大多数被诊断患有MPD的个体死于进行性血细胞减少症（出血或感染）的并发症或转化为急性白血病。因此，我们必须确定治疗MPD的替代方法。我们产生了CREB转基因小鼠，其中cAMP反应元件结合蛋白（CREB）在髓系中表达。这些小鼠发生单核细胞增多症、脾肿大和MPD。在骨髓集落测定中，我们观察到CREB转基因小鼠骨髓三次再铺板的增殖、生长因子独立性和原始细胞转化增加。CREB转基因小鼠骨髓移植可增强骨髓植入和增加干细胞数量。CREB在MPD患者的造血细胞中过表达。在这个提议中，我们假设CREB转基因小鼠是人类MPD的模型，CREB在调节干细胞自我更新和可能转化为急性白血病中起作用。为了验证这些假设，我们将：1）表征hMRP 8-CREB转基因小鼠中的MPD表型; 2）表征CREB在造血干细胞中过表达的生物学和细胞效应; 3）表征CREB与MPD中其他癌基因的协同作用。在具体目标1中，我们将研究CREB转基因小鼠中MPD随时间的时程、免疫表型、集落形成和发展。我们还将把我们的发现与人类疾病联系起来。在特定目标2中，我们将研究CREB在原代干细胞中过表达的表型。我们将CREB逆转录病毒或慢病毒转染不同分化阶段的骨髓祖细胞，并在体外和体内研究CREB对干细胞增殖和分化的影响。由于30%的MPD患者有ras突变，我们将用CREB逆转录病毒感染K-rasG 12 D小鼠的骨髓，并检查集落形成、免疫表型、植入和向AML的潜在转化。这些 研究将为MPD的分子途径提供新的见解。

DESCRIPTION (provided by applicant): Myeloproliferative disease (MPD) represents abnormal proliferation of myeloid cells in the blood and bone marrow, and is considered to be a "stem cell disorder." Although the pathologic classification of MPD has been recently defined, the molecular pathogenesis of disease is not well understood. Most individuals diagnosed with MPD succumb to their disease from either complications of progressive cytopenias (bleeding or infections) or transformation to acute leukemia. Therefore, it is imperative that we identify alternative approaches to treat MPD. We generated CREB transgenic mice in which the cAMP Response Element Binding protein (CREB) is expressed in the myeloid lineage. These mice develop monocytosis, splenomegaly, and MPD. In bone marrow colony assays, we observed increased proliferation, growth factor independence, and blast transformation with tertiary replating of bone marrow from CREB transgenic mice. Bone marrow transplantation with CREB transgenic mouse bone marrow result in enhanced myeloid engraftment and increased numbers of stem cells. CREB is overexpressed in hematopoietic cells from patients with MPD. In this proposal, we hypothesize that the CREB transgenic mouse is a model for human MPD and that CREB plays a role in regulating stem cell self-renewal and possibly transformation to acute leukemia. To test these hypotheses, we will: 1) characterize the MPD phenotype in hMRP8-CREB transgenic mice; 2) characterize the biological and cellular effects of CREB overexpression in hematopoietic stem cells; and 3) characterize the cooperation of CREB with other oncogenes in MPD. In Specific Aim 1, we will study the time course, immunophenotype, colony formation, and development of MPD in CREB transgenic mice over time. We will also correlate our findings with human disease. In Specific Aim 2, we will investigate the phenotype of CREB overexpression in primary stem cells. We will transduce bone marrow progenitor cells with CREB retrovirus or lentivirus at different stages of differentiation and examine the effects of CREB on stem cell proliferation and differentiation in vitro and in vivo. Since 30% of patients with MPD have ras mutations, we will transduce bone marrow from K-rasG12D mice with CREB retrovirus and examine colony formation, immunophenotype, engraftment, and potential transformation to AML. These studies will provide new insights into the molecular pathways leading to MPD.