Role of AML1/ETO in Hematopoiesis and Leukemogenesis

AML 1/ETO融合蛋白与40%的M2亚型急性髓系白血病的发病有关，占人类AML总数的12%。该融合基因位于21号染色体上的AML1基因的氨基端部分(CBFA2)和8号染色体上的ETO基因的几乎完整的编码区。AML1/ETO以显性的负向方式干扰转录因子CBF的功能，可能是由于其能够与异源二聚体转录伙伴CBFβ结合并通过CBF增强子元件抑制转录。缺乏AML1或CBFβ的小鼠缺乏明确的造血功能，这些小鼠在胚胎发育过程中死亡。同样，被设计表达干扰CBF功能的白血病融合蛋白的小鼠死于类似的表型，使AML动物模型的发展复杂化。在逆转录病毒基因传递系统、造血干细胞生物学和免疫缺陷动物发育方面的最新进展使人类和小鼠干细胞过表达感兴趣的基因并利用这些细胞重建受体动物的免疫系统成为可能。本工作的最终目的是建立小鼠急性髓系白血病模型，特别是与AML1/ETO表达相关的AML(特异性靶点1)。将使用一种针对干细胞表达进行优化的小鼠轮状病毒，并将使用IRES元件从相同的mRNA中共表达绿色荧光蛋白，以便于鉴定转导细胞。人类和小鼠干细胞都将用于这些研究，并将选择合适的动物模型，以允许转化细胞在体内生长。体外研究还将确定AML1/ETO过度表达对正常造血的影响(特定目标2)。使用细胞因子和间质层的特定组合，研究人员将确定AML1/ETO表达影响哪一种造血系。AML1/ETO的突变体也将被包括在该系统中，以破译哪些信号通路在体内AML1/ETO诱导的白血病中起重要作用。表达AML1/ETO的人干细胞的mRNA将用于高密度微阵列的差异杂交筛选，以确定AML1/ETO(特异性目标3)调控的靶基因。使用上述检测方法，将评估这些靶基因对AML1/ETO在人类干细胞中的表达所引起的表型的贡献。综上所述，这些数据将提供AML1/ETO在造血和白血病发生中的功能作用的详细信息。AML小动物模型的建立将极大地提高我们开发和测试治疗策略和药物的能力，这些策略和药物可能对AML的治疗有用。

The AML 1/ETO fusion protein is causally implicated in the pathogenesis of 40% of acute myeloid leukemias of the M2 subtype, and accounts for 12% of human AMLs overall. The fusion gene is compromised of the amino-terminal portion of the AML1 (CBFA2) gene on chromosome 21 and the nearly full coding region of ETO gene on chromosome 8. Aml1/Eto interferes with the function of the transcription factor, CBF, in a dominant negative fashion, presumably by its ability to bind to the heterodimeric transcription partner CBF beta and repress transcription through CBF enhancer elements. Mice deficient in AML1 or CBF BETA lack definitive hematopoiesis, and these mice die during embryogenesis. Similarly, mice engineered to express a leukemic fusion protein that interferes with CBF function die from a similar phenotype, complicating the development of an animal model of AML. Recent advances in retroviral gene delivery systems, hematopoietic stem cell biology, and immunodeficient animal development have made it possible to overexpress genes of interest in human and murine stem cells and use these cells to reconstitute the immune system of recipient animals. The ultimate objective of this work is the development of murine model AML, specifically of AML associated with expression of AML1/ETO (Specific Aim 1). A murine retovirus optimized for expression in stem cells will be used, and the green fluorescent protein will be co-expressed from the same mRNA using an IRES element, to facilitate identification of transduced cells. Both human and murine stem cells will be used in these studies, and the appropriate animal model will be chosen to allow the growth of transformed cells in vivo. In vitro studies will also be performed to determine the effects of AML1/ETO over-expression on normal hematopoiesis (Specific Aim 2). Using specific combinations of cytokines and stromal layers, the investigator will determine which hematopoietic lineage is affected by AML1/ETO expression. Mutants of AML1/ETO will also be included in the system, to decipher which signaling pathways are important in AML1/ETO-induced leukemia in vivo. mRNA from human stem cells expressing AML1/ETO will be used for differential hybridization screening of high-density microarrays to identify target genes regulated by AML1/ETO (Specific Aim 3). These target genes will be evaluated for their contribution for the phenotype elicited by expression of AML1/ETO in human stem cells, using the assays mentioned above. Taken together, these data will provide detailed information on the functional role of AML1/ETO in both hematopoeisis and leukemogenesis. The establishment of a small animal model of AML will greatly enhance our ability to develop and test treatment strategies and drugs that may be useful in the therapy of AML.