Uncovering the leukemogenic mechanism of non-canonical NUP98 fusion oncoproteins

Gene translocations involving NUP98 are associated with diverse pediatric leukemias, including acute myeloid leukemia (AML), with poor patient outcomes. The resulting gene fusions encode fusion oncoproteins (FOs) linking the disordered, phase-separation (PS) prone phenylalanine-glycine (FG) repeats of the NUP98 N-terminus to C-terminal DNA/chromatin-binding domains from >30 transcriptional/epigenetic regulators. NUP98 FOs form biomolecular condensates (puncta) that drive aberrant gene expression and transform hematopoietic stem and progenitor cells (HSPCs), promoting leukemogenesis. Interestingly, the C-terminal of the NUP98-LNP1 FO lacks a known DNA/chromatin-binding domain and is predicted to be disordered, yet this so-called “non-canonical” FO recapitulates the leukemogenic phenotype of other NUP98 FOs in HSPCs and in mice. Live cell fluorescence microscopy imaging showed that NUP98-LNP1 forms nuclear puncta that are larger and more sparce than those formed by other NUP98 FOs. Intriguingly, the parent LNP1 protein displays diffuse localization in cells. Our observations raise questions as to how NUP98-LNP1 alters gene expression and transforms HSPCs. We hypothesize that the C-terminal LNP1 region within the NUP98-LNP1 FO interacts with chromatin through an unknown mechanism, enabling the NUP98 FG-rich region to promote puncta formation and recruitment of the transcriptional machinery to drive aberrant gene expression and cell transformation. To test this hypothesis, I will determine whether NUP98-LNP1 induces the hallmark HOX gene expression program associated with AML in HEK293T cells and HSPCs. Further, I will determine protein interaction partners of NUP98-LNP1 by developing biochemical procedures to isolate NUP98-LNP1-driven puncta. Finally, I will identify genomic sites bound by NUP98-LNP1. Following these studies, I will extend my studies to a second non-canonical NUP98 FO, NUP98-RAP1GDS1, whose folded C-terminal interacts with GTPases. The proposed studies will uncover how non-canonical NUP98 FOs drive aberrant gene expression, expanding our knowledge of the role of PS in leukemogenesis by NUP98 FOs.