PROJECT SUMMARY There has been considerable progress in the last few years in the characterization of molecular alterations in childhood cancer. Many defining genomic abnormalities as well as transcriptional and signaling networks involved in specific sub-types of pediatric cancers have been identified. However, despite these impressive advances, safe and effective therapies for most pediatric cancers are lacking. Leukemias are the leading cause of cancer-linked mortality in children and for many pediatric leukemias, therapies that can selectively eliminate cancer cells with few undesirable side effects have been elusive. Therefore, there is an urgent clinically unmet need to develop non-toxic and targeted therapies for pediatric leukemia. Our proposal is focused on leukemias with rearrangements of the AF10 gene. These rearrangements are observed in acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL) and other sub-types of leukemia in children and adults. AF10-rearranged (AF10-R) leukemias can be considered prototypical models of pediatric cancers in which chimeric fusion oncoproteins or altered transcription factors drive extensive self- renewal and impaired differentiation of cancer cells. Our recent analysis of data from over 1,000 pediatric AML patients demonstrates that AF10 gene fusions are much more common than generally appreciated (representing 6.5% of pediatric AML) and constitute one of the highest risk-groups in terms of resistance to primary therapies and relapse rates. Our detailed mechanistic studies demonstrate that the AF10 fusion protein CALM-AF10 activates a number of pathways linked to stem cell self-renewal and inflammatory signaling, many of which are independently associated with poor outcomes in AML. In this proposal, we will use a series of orthogonal methodologies, including proteomic, genomic, epigenomic and detailed mechanistic studies in both human and mouse AML models, to determine how the CALM-AF10 fusion protein causes leukemogenesis. The successful execution of the proposed aims will advance our efforts towards finding newer targeted therapies for AF10-R AML with fewer unwanted toxicities. Results from our studies also have the potential to significantly influence treatment strategies in several other sub-types of pediatric leukemias driven by related mechanisms.