Role of NSD3 in Fusion Oncoprotein-Driven 3D Genome Reorganization

Fusion oncoprotein transcription factors such as BRD4-NUT reprogram chromatin organization to activate oncogenic transcriptional programs, yet the mechanisms by which they reshape gene regulatory architecture remain poorly understood. BRD4-NUT and NUT variant fusions drive NUT carcinoma, an aggressive squamous malignancy with a median survival of only 6.5 months. Although BRD4-NUT induces de novo tumor formation by reorganizing chromatin to activate pro-growth transcriptional programs, the chromatin-level mechanisms underlying this process have not been established. Here, we identify the chromatin regulator NSD3—also a recurrent NUT fusion partner—as a critical cofactor required for fusion oncoprotein-mediated chromatin reorganization and transcriptional regulation. Using CUT&RUN chromatin profiling, we show that NSD3 is highly enriched within BRD4-NUT chromatin “megadomains”, where it deposits histone H3K36me2 and stabilizes BRD4-NUT binding to chromatin. NSD3 also promotes the stability of BRD4-NUT nuclear condensates. Hi-C analysis reveals that loss of NSD3 attenuates the long-range chromatin interactions that link BRD4-NUT megadomains both within (cis) and between (trans) chromosomes, demonstrating that NSD3 is required for 3D genome reorganization by a fusion oncoprotein. Through these chromatin regulatory functions, NSD3 sustains expression of BRD4-NUT target genes, including MYC and MYC-regulated transcriptional networks. NSD3 depletion reduces proliferation and promotes differentiation of BRD4-NUT+ cells. Together, these findings identify NSD3 as a mechanistic cofactor co-opted by a fusion oncoprotein to integrate chromatin architecture, 3D genome topology, and transcriptional activation. This work reveals a generalizable model in which wild-type chromatin regulators are hijacked by fusion oncoproteins to establish oncogenic transcriptional and gene regulatory states, and it nominates NSD3 as an actionable vulnerability for therapeutic strategies targeting fusion-driven cancers. Citation Format: Yi-Hung Chen, Krupa Sampat, Ivaylo D. Yonchev, Catherine E. Hawkins, Christopher Ponne, Kyle P. Eagen. NSD3 is a critical cofactor that enables fusion oncoprotein-driven 3D genome reorganization and activation of oncogenic transcriptional programs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4064.