MOLECULAR ANALYSIS OF TRANSFORMATION IN TEL/PDGFR MICE

在既存骨髓增生异常综合征的背景下发生的急性白血病对治疗特别耐药。这些疾病的唯一治愈性治疗是骨髓移植，这种治疗仍然有毒，仅适用于一部分患者。 慢性粒单核细胞白血病（CMML）是一种骨髓增生异常综合征，其特征是单核细胞增多、可变骨髓纤维化和进展为急性白血病。 t（5; 12）与CMML相关，并导致融合TEL的氨基末端与PDGF β R的细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表征。 在体外和COS细胞中，TEL/PDGF β R组成性酪氨酸磷酸化并形成由TEL指向（PNT）结构域介导的寡聚体。 在初步结果中，候选人描述了使用TEL/PDGF β R开发的两种造血系统恶性肿瘤动物模型的开发。 移植了通过逆转录病毒介导的基因转移引入了TEL/PDGF β R的小鼠骨髓（BMT）的小鼠发生快速致命的骨髓增殖综合征。 此外，具有通过免疫球蛋白重链启动子-增强子（EmuVHP）靶向淋巴区室的TEL/PDGF β R的转基因小鼠发展成淋巴细胞性淋巴瘤。该建议的重点是增加我们对这些小鼠模型中TEL/PDGFbetaR转化的分子机制的理解。 具体目标是：1）表征体内由TEL/PDGFbetaR激活的信号通路，2）确定激活的信号通路与小鼠中造血恶性肿瘤发展的相关性，和3）鉴定与造血细胞转化相关的TEL/PDGFbetaR的下游靶点。 一系列TEL/PDGF β R的信号突变体将用于重复BMT实验，以确定体内转化的关键残基。 使用免疫共沉淀和凝胶迁移试验，分析Ras和JAK/STAT途径中信号分子的活性。 最后，DNA微阵列技术将用于检测培养细胞中TEL/PDGFbetaR及其突变体下游靶点的基因组表达。 这些实验将提供对TEL/PDGF β R转化机制的详细了解，并将为开发合理的治疗方法以治愈或阻止CMML的进展提供基础。

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.