Abstract B014: NSD3 regulates BRD4-NUT-driven chromatin 3D organization and gene regulation in NUT carcinoma

Abstract BRD4-NUT and NUT variant fusion oncoproteins drive NUT carcinoma, an aggressive squamous cancer with 6.5-month median survival. BRD4-NUT induces de novo tumor formation by reprogramming chromatin organization to promote the expression of pro-growth genes, yet the mechanisms by which BRD4-NUT dysregulates gene expression remain poorly defined. We identify the chromatin regulator NSD3, itself a recurrent NUT fusion partner, as a critical cofactor of BRD4-NUT that regulates chromatin organization and supports NUT carcinoma cell viability. Using CUT&RUN chromatin profiling, we show that NSD3 is highly enriched within BRD4-NUT chromatin ‘megadomains’ where it promotes histone H3K36me2 and stabilizes BRD4-NUT on chromatin. These functions are accompanied by NSD3-dependent stabilization of BRD4-NUT nuclear condensates. Hi-C revealed that loss of NSD3 attenuates BRD4-NUT–dependent chromatin 3D interactions both within (cis) and between (trans) chromosomes. Through its chromatin regulatory functions, NSD3 supports the expression of BRD4-NUT target genes, including MYC and MYC targets. NSD3 depletion reduces proliferation and promotes differentiation of BRD4-NUT+ cells. Our findings establish NSD3 as a cofactor of BRD4-NUT that integrates chromatin organization and transcriptional regulation in NUT carcinoma, highlighting how gene regulatory mechanisms of a wild-type NUT fusion partner contribute to fusion oncoprotein function. Given the recent development of NSD3-targeted inhibitors and degraders, our results provide a rationale for therapeutic strategies that combine NSD3 inhibition with bromodomain and extraterminal (BET) protein inhibitors that target BRD4-NUT. Citation Format: Yi-Hung Chen, Krupa Sampat, Ivaylo D. Yonchev, Catherine E. Hawkins, Christopher Ponne, Kyle P. Eagen. NSD3 regulates BRD4-NUT-driven chromatin 3D organization and gene regulation in NUT carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B014.