Increasing evidence suggests a critical role for epigenetic mechanisms in development, stem cell physiology and cancer. Polycomb Repressive Complex 2-mediated trimethylation of histone 3 on lysine residue 27 (H3K27me3) is catalyzed by the enzyme Enhancer of Zeste 2 (EZH2). Overexpression of EZH2 is correlated with clinical cancer progression in patients and forced expression of EZH2 can contribute to transformation of cultured cells. Inhibition of EZH2 by shRNA strategies has been correlated with decreased proliferative potential in several cancer models. Interestingly, haploinsufficiency for EZH2 has recently been described in human B-cell lymphomas. The functional consequences of EZH2 haploinsufficiency on cellular phenotype have not yet been characterized in experimental cancer models. We propose to study the role of Ezh2 in a genetically defined model of acute leukemia mediated by MLL-AF9 that is well characterized in our laboratory. Genetic loss of Ezh2 in homozygous and/or heterozygous settings will allow us to compare the effects of defined dosages of Ezh2 on tumor phenotype, including complete loss of activity. This technical advance will circumvent limitations associated with shRNA technology and will definitively clarify if Ezh2 is essential for AML. Our system will also allow for detailed characterization of chromatin and gene expression changes associated with loss of Ezh2, thus providing important mechanistic insights. Results obtained in the murine system will be compared with results obtained from the analysis of human cell lines and primary patient samples. The specific aims are: 1: Loss of Function and Forced Expression Studies to Define the Role of Ezh2 in Leukemic Self-Renewal 2: Molecular Characterization of Pathways Affected by Loss of Ezh2 in Murine and Human MLL-AF9 Leukemia Cells Dr. Tobias Neff, a pediatric hematology/oncology fellow at Children's Hospital Boston (CHB) has outlined a 5-year career plan that will build upon his background in clinical hematology/oncology and experimental hematology. Under the co-mentorship of Drs. Scott Armstrong and Stuart Orkin, recognized leaders in leukemia, hematopoietic stem cell biology, and transcriptional gene regulation, he seeks to utilize a definitive biologically relevant mouse model and genomic approaches to study the role of Enhancer of Zeste 2 (Ezh2) in acute myeloid leukemia. Dr. Neff will be mentored by an Advisory Committee of internationally recognized experts in the field. Finally, the plan is ideally carried out in the Division of Hematology/Oncology at Children's Hospital Boston, given its distinguished record for training physician-scientists in a rich and collaborative environment.