Aberrant NUP98 condensates activate leukemogenic genes via discrete genomic engagement

NUP98 FG repeats trigger hematopoietic malignancies when aberrantly fused to various DNA/chromatin binding domains. However, how these heterogeneous NUP98 fusions drive oncogenesis remains unclear. Here we fuse different NUP98 FG repeat compositions to dCas9 and establish that NUP98 FG repeats are sufficient to activate human genes via cooperative activity at human endogenous promoters. Further, we find that NUP98-FG repeats spontaneously undergo liquid-liquid phase separation and co-condensate with other transcriptional coactivators in human cells, which we show is indispensable for transcriptional activation. We also define a threshold of NUP98 FG repeat numbers required for biomolecular condensation and gene activation that is consistent with NUP98 FG repeat lengths observed across clinical cohorts. Using ChIP-seq and live cell imaging, we further demonstrate that although NUP98 FG repeats can robustly shuttle into cell nuclei, engagement with the human genome is directed solely through fusion partner. Interestingly, two frequent oncogenic fusions, NUP98-HOXA9 and NUP98-KDM5A exhibit discrete genomic engagement landscapes and gene-regulatory modes in that NUP98-HOXA9 initiates strong transcription via intragenic super enhancer-like binding and increased intrachromosomal contacts, while NUP98-KDM5A activates genes via strict localization to promoters. Additionally, in primary human HSCs, NUP98-HOXA9 and NUP98-KDM5A globally dysregulate transcription in a largely non-overlapping manner. Nevertheless, both oncogenic fusion proteins directly drive the inappropriate activation of core leukemia-associated oncogenes, which occurs coincident with the downregulation of genes associated with HSC lineage commitment. Altogether, our studies point toward a conserved core set of potential therapeutic targets driving NUP98-associated cancers and clarify the transcriptional and oncogenic role of aberrant NUP98 condensates.