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) are a promising treatment for NC that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy are limited. Here, we demonstrated 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. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NC cells. CDKN2A was identified as the only gene among all taz-derepressed genes to confer resistance to taz 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 pre-clinical models, combined taz and BETi synergistically blocked tumor growth and prolonged survival of NC-xenografted mice, with complete remission without relapse in one cohort. 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.