PROJECT SUMMARY/ABSTRACT An evolutionary conserved developmental program is carefully maintained in hematopoietic stem cells (HSCs). Genetic alterations and epigenetic mechanisms can alter the balance of normal blood development resulting in hematological malignancies. Our laboratory and others have found that the MUSASHI2 (MSI2) RNA binding proteins is highly expressed in the most aggressive cancers and predicts a poor clinical outcome in acute myeloid leukemia (AML) patients. Genetic models have found that MSI2 is required for leukemia stem cell function. Utilizing a new way to identify mRNA targets of RNA binding proteins, we have found that MSI2 activity is increased in leukemia stem cells compared to normal stem and progenitor cells. This surprising finding suggests that RNA binding protein function can be dysregulated beyond just expression differences. We hypothesize that the MSI family of RNA binding protein have differential activity in AML compared to normal cells and that MSI enhances the dysregulated epigenome in AML. We propose two possible mechanisms for this intriguing finding 1) MSI2 associated RBPs compete for MSI2-binding sites and 2) Post- translation modifications can modulate MSI2 activity. Our preliminary data has uncovered that MSI2 can mediate resistance to PRMT5 and that PRMT1 and PRMT5 can directly methylate MSI2. PRMT5 inhibitors are being investigated as therapeutic targets and our proposal suggests a novel link to this pathway and may explain cell context MSI2 activity. Our proposal will utilize new genetic models to characterize MSI2 targets in specific cellular contexts and explore the MSI2 associated program to identify new therapeutic strategies in AML.