NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT–mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain–associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. Implications: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.

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NUT carcinoma (NC) is an extremely aggressive tumor and current treatment regimens offer patients a median survival of six months only. This article reports on the first in vitro studies using immunovirotherapy as a promising therapy option for NC and its feasible combination with BET inhibitors (iBET). Using NC cell lines harboring the BRD4-NUT fusion protein, the cytotoxicity of oncolytic virus talimogene laherparepvec (T-VEC) and the iBET compounds BI894999 and GSK525762 were assessed in vitro in monotherapeutic and combinatorial approaches. Viral replication, marker gene expression, cell proliferation, and IFN-β dependence of T-VEC efficiency were monitored. T-VEC efficiently infected and replicated in NC cell lines and showed strong cytotoxic effects. This implication could be enhanced by iBET treatment following viral infection. Viral replication was not impaired by iBET treatment. In addition, it was shown that pretreatment of NC cells with IFN-β does impede the replication as well as the cytotoxicity of T-VEC. T-VEC was found to show great potential for patients suffering from NC. Of note, when applied in combination with iBETs, a reinforcing influence was observed, leading to an even stronger anti-tumor effect. These findings suggest combining virotherapy with diverse molecular therapeutics for the treatment of NC.