Project Summary/Abstract The overarching goal of this proposal is to determine how mutations in Nucleophosmin 1 (NPM1) and DNA Methyltransferase 3A (DNMT3A) cooperatively regulate HOX gene expression in the development of acute myeloid leukemia (AML). AML is a hematological malignancy arising from somatic mutations in hematopoietic progenitor cells that lead to their expansion in the bone marrow niche. Approximately 20,000 cases of AML are diagnosed annually in the United States, with a 5-year survival of less than 30%. NPM1 and DNMT3A are the most frequently mutated genes in AML, occurring in 30% and 20% of all cases, respectively. These mutations frequently co-occur, with nearly 70% of NPM1-mutant AML harboring a concomitant DNMT3A mutation. Together, these mutations confer a worse overall prognosis. Despite their established association, the synergistic mechanisms that drive robust malignancy in the presence of both NPM1/DNMT3A mutations are poorly understood, thus preventing the development of effective targeted therapies. Our group and others have shown that these mutations converge on HOX regulation, where high HOX expression maintains leukemic cells in an undifferentiated state. Our group has shown that mutant NPM1 is required for HOX expression in AML. Moreover, DNMT3A mutations have been associated with global changes in methylation, particularly at HOX loci. To better define the mechanism of mutant NPM1, I performed a protein interaction screen that revealed mutant NPM1 interacts with the rixosome, a conserved ribosomal biogenesis complex with additional roles in HOX gene silencing. We hypothesize that, during AML pathogenesis, DNMT3A mutations enhance chromatin accessibility at HOX gene loci, which robustly increases the ability of mutant NPM1 to interfere with rixosome-mediated HOX gene silencing. To test this hypothesis, we will first determine the mechanism of increased HOX expression by mutant NPM1, with specific focus on the rixosome’s role in HOX gene silencing. We will also characterize HOX chromatin organization in NPM1/DNMT3A mutant AML in vitro and in vivo. This proposed work will reveal key molecular mechanisms underlying the most common co-mutations found in AML. As a result, these studies will identify new therapeutic vulnerabilities in NPM1/DNMT3A mutant AML and clear a path for the development of targeted therapies to improve patient outcomes.