EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes.

NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. SIGNIFICANCE: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes.

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT's ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE: Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

Efficacy of Oncolytic Herpes Simplex Virus T-VEC Combined with BET Inhibitors as an Innovative Therapy Approach for NUT Carcinoma.

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-ß 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-ß 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.

Chemical Screen Identifies Diverse and Novel Histone Deacetylase Inhibitors as Repressors of NUT Function: Implications for NUT Carcinoma Pathogenesis and Treatment.

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.