MOLECULAR ANALYSIS OF TRANSFORMATION IN TEL/PDGFR MICE

在先前存在的骨髓增生异常综合征的背景下出现的急性白血病尤其对治疗具有抵抗力。这些疾病的唯一治愈方法是骨髓移植，这种治疗仍然是有毒的，只对一小部分患者有效。慢性粒单核细胞白血病(CMML)是一种以单核细胞增多、可变的骨髓纤维化和进展为急性白血病为特征的骨髓增生异常综合征。T(5；12)与CMML相关，导致融合TEL氨基末端和PDGFbetaR胞内部分的嵌合蛋白的表达。我们实验室克隆了TEL/PDGFbetaR融合基因，并对其体外特性进行了广泛的研究。TEL/PDGFbetaR在体外和COS细胞中被结构性酪氨酸磷酸化，并形成由TEL指向结构域(PNT)介导的寡聚体。在初步结果中，候选人描述了使用TEL/PDGFbetaR建立的两种血液系统恶性肿瘤动物模型的发展。通过逆转录病毒介导的基因转移导入TEL/PDGFbetaR的小鼠骨髓(BMT)移植后，小鼠出现了一种快速致死性的骨髓增殖性综合征。此外，通过免疫球蛋白重链启动子增强子(EmuVHP)靶向淋巴室的TEL/PDGFbetaR转基因小鼠会发生淋巴母细胞性淋巴瘤。这项建议的重点是增加我们对TEL/PDGFbetaR在这些小鼠模型中转化的分子机制的理解。其具体目的是：1)研究TEL/PDGFbetaR在体内激活的信号通路；2)确定激活的信号通路与小鼠恶性血液病发生发展的相关性；3)确定TEL/PDGFbetaR下游与造血细胞转化相关的靶点。TEL/PDGFbetaR的一系列信号突变体将用于重复的BMT实验，以确定对体内转化至关重要的残基。使用免疫共沉淀和凝胶位移分析，将分析信号分子在RAS和JAK/STAT通路中的活性。最后，将利用DNA微阵列技术检测TEL/PDGFbetaR及其突变体下游靶标在培养细胞中的基因组表达。这些实验将提供对TEL/PDGFbetaR转化机制的详细了解，并将为开发治疗或阻止CMML进展的合理疗法提供基础。

Acute leukemia arising in the setting of a preexisting myelodysplastic syndrome is especially resistant to treatment. The only curative treatment for these disorders is bone marrow transplantation, a treatment that remains toxic and available only to a subset of patients. Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by monocytosis, variable bone marrow fibrosis, and progression to acute leukemia. The t(5;12) is associated with CMML and results in the expression of a chimeric protein fusing the amino-terminus of TEL with the intracellular portion of the PDGFbetaR. Our lab cloned the TEL/PDGFbetaR fusion gene and has extensively characterized its in vitro properties. TEL/PDGFbetaR is constitutively tyrosine phosphorylated and forms oligomers mediated by the TEL pointed (PNT) domain in vitro and in COS cells. In Preliminary Results the candidate describes the development of two animal models of hematopoietic malignancy developed using TEL/PDGFbetaR. Mice transplanted with murine bone marrow (BMT) with TEL/PDGFbetaR introduced by retrovirally mediated gene transfer develop a rapidly fatal myeloproliferative syndrome. Furthermore, transgenic mice that have TEL/PDGFbetaR targeted to the lymphoid compartment by the immunoglobulin heavy chain promoter-enhancer (EmuVHP) develop lymphoblastic lymphomas. The focus of this proposal is to increase our understanding of the molecular mechanisms underlying transformation by TEL/PDGFbetaR in these murine models. The specific aims are: 1) To characterize the signaling pathways activated by TEL/PDGFbetaR in vivo, 2) To determine the relevance of activated signaling pathways to the development of hematopoietic malignancy in mice, and 3) To identify the downstream targets of TEL/PDGFbetaR relevant to the transformation of hematopoietic cells. A series of signaling mutants of TEL/PDGFbetaR will be used in repeat BMT experiments to determine residues critical for in vivo transformation. Using coimmunoprecipitation and gel shift assays, the activity of signaling molecules in the Ras and JAK/STAT pathways will be analyzed. Lastly, DNA microarray technology will be used to examine genomic expression of downstream targets of TEL/PDGFbetaR and its mutants in cultured cells. These experiments will provide a detailed understanding of the mechanisms of transformation by TEL/PDGFbetaR, and will provide a basis for the development of rational therapies to cure or halt the progression of CMML.