Project Abstract Acute myeloid leukemia (AML) is an aggressive, heterogeneous disease and is characterized by the expansion of immature myeloid progenitors and the inhibition of myeloid differentiation. AML arises when the complex process of blood formation, known as hematopoiesis, goes wrong. While progress has been made to develop new treatments and therapies for AML, little progress has been made in the past decades. The Musashi (MSI) family of proteins, MSI1 and MSI2 are RNA-binding proteins (RBPs) and are responsible for post- transcriptional regulation of genes in stem cell compartments. However, MSI2 plays a prevailing role to MSI1 in the blood. Here, MSI2 maintains hematopoietic stem cells (HSCs) and determines lineage bias. MSI2 has been implicated in a variety of solid tumors and leukemias suggesting targeting it could be a beneficial cancer therapy. Previous studies demonstrate MSI2 expression is a negative prognostic marker for AML and myelodysplastic syndromes (MDS) as upregulation of MSI2 correlates with poor prognosis and worse clinical outcomes for patients. MSI2 cooperates with the fusion protein BCR-ABL1 during leukemogenesis, potentiating HSC transformation in the presence of other mutations. MSI2 is also able to sustain the mixed-lineage leukemia (MLL) stem cell regulatory program through interaction with Hoxa9, Myc, and Ikzf2 mRNAs. Deletion of MSI2 decreases LSC survival and delays leukemia initiation and progression in vivo. Many studies demonstrate the variety of responsibilities MSI2 has in regulating translation in HSCs and leukemic stem cells, but there remains a significant gap in knowledge as to how MSI2 function is regulated. The proposed project aims will define how MSI2 function changes when different post- translational modifications are added. Our preliminary data establishes protein arginine methyltransferases (PRMT) 1 and 5 can methylate MSI2, and in a genome wide CRISPR screen, MSI2 was the top driver of resistance to PRMT5 inhibition. The outlined proposal will expand our understanding of MSI2 function in normal and malignant stem cell regulatory programs to inform design of novel therapeutic strategies to treat AML.