AML1-ETO (aka RUNX1-ETO and RUNX1-MTG8) is a DNA binding transcription factor encoded by a fusion gene generated from t(8;21)(q22;q22). This chromosomal translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in over 10% of all cases. Our own studies and those of others have demonstrated that AML1-ETO plays an important role in the development of leukemia. However, it is not sufficient by itself for leukemia development. We demonstrated that one isoform of t(8;21) fusion protein AML1-ETO9a, which lacks 178 amino acids comprising the C-terminus of the full length AML1- ETO, is highly leukemogenic in a mouse model of retrovirus vector mediated transduction-hematopoietic cell transplantation. Using combined gene expression profiling and promoter occupancy profiling, we have identified a group of direct target genes of AML1-ETO9a in leukemia initiating cells and revealed the therapeutic potential of targeting the JAK-STAT signaling pathway in treatment of the t(8;21) AML. One interesting target is the tumor suppressor gene Ras association domain family member 2 (RASSF2). Aberrant transcriptional repression of RASSF2 is a frequently occurring event in many cancer types, however its involvement in leukemia development remains unexamined. More recently, it has been shown that at the single cell level t(8;21)+ leukemia stem cells at diagnosis have an over 100 fold higher amount of AML1-ETO transcript compared with t(8;21)+ hematopoietic stem cells in remission. Interestingly, no corresponding increase in wild type AML1 transcript is observed. These findings lead to the hypothesis that the enhanced alterations of the AML1-ETO target gene expression via the increased amount of t(8;21) fusion protein is critical for development of the t(8:21)+ AML. Understanding the regulation of AML1-ETO expression and the associated effect of its target genes may provide valuable insight into the molecular mechanisms of leukemogenesis and contribute to rational therapeutic drug design for treating related malignancies. Furthermore, a highly reproducible germline transmitted mouse model of the t(8;21) AML will be valuable for mechanistic studies and therapeutic evaluations. We have developed three specific aims to test this hypothesis. Specific Aim #1 will analyze the effect of t(8;21) on leukemogenesis and hematopoiesis using a new model of AML1-ETO9a transgenic mice. Specific Aim #2 will study the effect of t(8;21) on leukemogenesis by characterizing its direct target RASSF2. Specific Aim #3 will examine the molecular mechanisms of increased AML1-ETO expression during leukemia development.