PROJECT SUMMARY/ABSTRACT Rearrangement of NUP98 gene (NUP98-r) is recurrent in leukemias such as acute myeloid leukemia (AML). Patients with NUP98-r show poor prognosis and therapy failure. Most NUP98-r partners (>30 identified from patients) are a DNA-binding domain of transcription factor (TF; e.g. HOXA9) or a histone-binding motif such as Plant Homeodomain (PHD), suggesting chromatin deregulation as an oncogenic mechanism. NUP98-r fusions invariably retain Phenylalanine-Glycine (FG) repeats, termed intrinsically disordered region (IDR), from NUP98. How unstructured IDR contributes to oncogenesis remains elusive. Our studies of NUP98-HOXA9, an AML NUP98-TF chimera, unveil an essential requirement of NUP98’s IDR for liquid-liquid phase separation (LLPS). We also show that IDR and LLPS are critical for the much-enhanced genome binding by NUP98-HOXA9 and for long-distance chromatin looping between oncogene promoters and enhancers, leading to development of aggressive AML in mice. Our unpublished preliminary studies of other recurrent leukemic fusions (namely, NUP98-PHD fusions and MSI2-HOXA9, a leukemia-related chimera formed by fusing a less-studied IDR of an RNA-binding protein with HOXA9’s DNA-binding domain) all point to involvements of IDR and LLPS for oncogenesis. Thus, we hypothesize that, due to aberrant genic fusions, a number of leukemia-related onco- TFs and chromatin factors acquire a phase-separation-inducing IDR to establish LLPS, which confers chimera a much more enhanced ability in genomic targeting; consequently, an oncogenic gene-expression program is over-activated while aberrant chromatin loops are formed between oncogene promoters and enhancers, which drives formation of aggressive leukemias. Dissection of the mechanisms underlying the IDR- and phase- separation-mediated aberrant genome organization and oncogene activation in cancer cells shall provide new and paradigm-shifting views as for how aggressive cancer develops, implicative of potentially new treatments in future. Towards this goal, we will further characterize the role for the un-studied IDR (that of MSI2) in establishing LLPS in vitro and in cells (Aim 1A) and will use primary human hematopoietic stem/progenitor cells (HSPCs) and derived cells to define roles of IDR and LLPS in regulating genomic targeting (1B), the target gene expression (1C), and leukemic transformation in vitro/vivo (1D) by various fusions (NUP98-PHD and MSI2/NUP98-HOXA9). LLPS-indued chromatin looping is CTCF-independent and represents a previously unstudied mechanism underlying 3D chromatin organization. We will further define the 3D chromatin structure alterations caused by various NUP98-r and MSI2-HOXA9 fusions in disease-relevant cells (Aim 2A), define the molecular mechanisms driving formation/maintenance of LLPS-dependent loops (2B), and determine the impact of LLPS DNA loops on the sustained activation of oncogenes by using a novel CRISPR/dCas9-IDR fusion strategy (2C). As phase-separati...