PROJECT SUMMARY Chromosomal translocations involving Nucleoporin 98 (NUP98) are observed in approximately 5% of pediatric acute myeloid leukemia (AML) and are associated with resistance to therapy and poor patient outcomes (approximately 35% 5 year overall survival). NUP98 rearrangements lead to expression of oncogenic chimeric gene fusions involving the N-terminal region of NUP98 and the C-terminal region of one of over 30 identified partner genes. The partner genes commonly have domains with key functional properties, including homeodomain moieties (e.g. HOXA9) and roles in transcriptional regulation (e.g. NSD1, KDM5A). In complex with other machinery needed for gene regulation, NUP98 fusion oncoproteins (FOs) bind to the promoters of many developmental genes. This leads to changes in chromatin structure, increased expression of target genes, and aberrant hematopoietic self-renewal. Recent leukemia genomic sequencing studies, including those by my laboratory, have shown that NUP98 fusions are commonly accompanied by recurring genetic events in distinct subsets of AML, suggesting the importance of co-alterations in lineage-specific leukemogenesis. This research proposal seeks to elucidate the effects of co-alterations in NUP98-rearranged AML, thus building on previous studies with genetically faithful systems that could present undiscovered therapeutic vulnerabilities in this high- risk leukemia subset. Aim 1 will evaluate the effects of co-altered genes in NUP98 FO-driven leukemogenesis. I will use lentiviral overexpression and CRISPR/Cas9-based approaches to perform gene editing in hematopoietic stem and progenitor cells (HSPCs) to introduce NUP98 FO and/or co-alteration, and I will study the in vitro and in vivo consequences of these alterations using colony forming unit and bone marrow transplantation assays, respectively. Immunophenotyping, pathology, and genomic characterization will be used to determine the role of individual and combined genetic events on distinct disease states. Aim 2 will examine the molecular mechanisms that occur at the transcriptional and epigenetic level when NUP98 FO and relevant co-alterations are expressed alone or in concert. Using RNA sequencing, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), I will integrate the gene expression profiles, DNA binding properties and histone modifications, and regions of activate chromatin, respectively, that accompany expression of NUP98 FO and/or co-alteration. This multiomic approach will provide a robust dataset for the investigation of the gene-regulatory effects of NUP98 fusions and frequent co-alterations as well as for validation of their functional consequences. Together, these studies will uncover lineage-specific and molecular impacts of cooperation between recurrent genetic events and NUP98 rearrangements in AML, revealing novel opportunities for therapeutic exploitation to impr...