PROJECT SUMMARY B-acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. The protein USO1 is specifically overexpressed in B-acute lymphoblastic leukemia (B-ALL) with translocation t(4;11) MLL-AF4, which portends a dismal prognosis. This subtype of B-ALL, derived from a primitive hematopoietic progenitor cell, is particularly difficult to treat, even with the recently described, and generally successful, antibody- and cell-based therapies that target the CD19 antigen. USO1 is known to be upregulated in other cancer types, and regulates cell survival/proliferation in multiple cancer types. Studies of the cell biological role of USO1 have shown it be a of importance in vesicular trafficking, and recent high-throughput studies have shown that USO1 is an RNA binding protein in some cellular systems- suggesting that this protein may exhibit a novel function connecting post-transcriptional gene regulation to vesicular processing. In this proof-of-concept grant, we hypothesize that (1) USO1 plays a pathogenetic role in MLL-translocated leukemogenesis and that (2) USO1 is an RNA binding protein. In this grant, we will explore the roles of USO1 in cancer using loss-of-function genetic models in a novel in vivo system to study MLL-AF4-driven leukemia. Additionally, we will perform biochemical cross-linking and RNA immunoprecipitation assays to determine if USO1 binds to RNA in B-ALL cell lines. The three aims proposed are independent, self-contained, but also have significant synergy. Together, the successful completion of the aims will uncover whether USO1 plays a pathogenetic role in leukemia, and whether its function involves RNA-based mechanisms. These studies are uniquely suited to the R03 mechanism, which supports small research projects that can be carried out in a short time period with limited resources. However, these studies will also lay the groundwork for novel diagnostic, prognostic and therapeutic strategies in B-ALL.