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.
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