Molecular and Cellular Characterization of Myeloproliferative Disease

描述(申请人提供)：骨髓增生性疾病(MPD)代表血液和骨髓中髓系细胞的异常增殖，被认为是一种“干细胞疾病”。虽然MPD的病理分类最近已经明确，但其发病的分子机制还不是很清楚。大多数被诊断为MPD的人要么死于进行性红细胞减少症(出血或感染)的并发症，要么死于转化为急性白血病。因此，我们必须确定治疗MPD的替代方法。我们建立了CREB转基因小鼠，在其中cAMP反应元件结合蛋白(CREB)在髓系中表达。这些小鼠出现单核细胞增多症、脾肿大和MPD。在骨髓集落分析中，我们观察到CREB转基因小鼠的骨髓随着第三次复制而增加了增殖、生长因子独立性和原始细胞转化。用CREB转基因小鼠骨髓移植后，骨髓植入增强，干细胞数量增加。CREB在MPD患者的造血细胞中高表达。在这个方案中，我们假设CREB转基因小鼠是人类MPD的模型，并且CREB在调节干细胞自我更新和可能向急性白血病转化方面发挥作用。为了验证这些假设，我们将：1)鉴定hMRP8-CREB转基因小鼠的MPD表型；2)鉴定CREB在造血干细胞中过表达对生物学和细胞的影响；3)鉴定CREB与其他癌基因在MPD中的协同作用。在特定的目标1中，我们将研究CREB转基因小鼠中MPD的时间进程、免疫表型、克隆形成和发展。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。我们将在分化的不同阶段用CREB逆转录病毒或慢病毒转导骨髓祖细胞，检测CREB在体外和体内对干细胞增殖和分化的影响。由于30%的MPD患者存在ras基因突变，我们将从带有CREB逆转录病毒的K-RasG12D小鼠身上转移骨髓，并检测克隆形成、免疫表型、植入以及向AML转化的可能性。这些 研究将对导致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.