Abstract 2740: Mechanisms of phase-separation driven chromatin looping in acute myeloid leukemia

Abstract New therapies are desperately needed for NUP98-fusion-driven Acute Myeloid Leukemia (AML) patients as they exhibit a poor prognosis on existing therapies, with a 5-year overall survival rate of ∼30%. We and others have shown that one fusion, NUP98-HOXA9 drives leukemogenesis through the formation of liquid-liquid phase separated (LLPS) condensates. Specifically, NUP98-HOXA9 binds DNA and pulls bound regions into the nuclear condensates, forming 3D chromatin loops that activate oncogenes' transcription. Mutation of the phenylalanine-glycine (FG) repeats responsible for NUP98-HOXA9 phase separation abolishes the formation of these condensates and inhibits malignant transformation. These findings suggest a new class of chromatin loops, that form via a mechanism distinct from loop extrusion and drive oncogene expression. We set out to characterize the necessary components for the formation and maintenance of these aberrant loops. Using an Auxin Inducible Degron (AID) system in HCT116 cells, we rapidly degraded loop extrusion proteins, CTCF and RAD21, followed by in situ Hi-C to observe impact on loop formation. We observe approximately 8, 000 canonical loops, which are bound by CTCF and lost upon auxin treatment. Additionally, we identify approximately 60 phase-separation driven loops, bound by NUP98-HOXA9 which do not require CTCF. We additionally explore the role of ATP, BRD4, and p300 as requirements for the formation of these loops. Future work includes further characterization of loop maintenance and screening for compounds to disrupt condensate formation with the goal of using a deeper understanding of phase-separation driven loops to create new promising therapeutic avenues for NUP98-fusion-driven AMLs. Citation Format: Tooba Rashid, Zachary Drum, Doris Cruz Alonso, Gelila Petros, Yoseli Quiroga, Douglas Phanstiel. Mechanisms of phase-separation driven chromatin looping in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2740.