Hijacking the BRD4-NUT fusion oncoprotein to activate programmed cell death

Fusion oncogenes account for about 10-20% of all cancer drivers and generally result from in-frame recombination with specific partners. Cancers driven by fusion oncogenes often occur in young people whose genomes are relatively free of other mutations, providing a singular reliance on the fusion and its underlying mechanisms. Despite this focus, fusion oncogenes - particularly those that arise from transcription factors - have been difficult to target therapeutically. We demonstrate in this study that the fusion oncoprotein BRD4-NUT can be used to specifically activate programmed cell death in an aggressive form of squamous cancer, NUT carcinoma (NC). NC is caused by a translocation of the NUTM1 gene downstream of the BRD4 gene. Transcription of the fusion gene is therefore controlled by the BRD4 regulatory region, leading to high-level expression of the fusion oncoprotein. The NUT protein is normally restricted to the testes, where it is essential for histone hyperacetylation, histone removal and replacement by protamines. NC currently has a mean‬‭ survival of 6-9 months and no approved treatments. We reasoned that the oncogenic program of BRD4-NUT could be reprogrammed to activate a synthetic death circuit. To selectively inhibit NC, we developed bifunctional molecules that target the BRD4-NUT fusion, BRD4 and PARP. We call the new class of molecules DD-CIPs for DNA Damage-Chemical Inducers of Proximity. The most potent member of this new class (TWQ-184) kills NC cell lines (10-15, SW and 14169) at concentrations between 0.74 nM and 6 nM. TWQ-184 forms a ternary complex with BRD4-NUT and PARP, resulting in cell death. P300 inhibitors partially rescue NC cells from TWQ-184-induced apoptosis, suggesting that transcription at PARP-bound DNA breaks driven by BRD4-NUT is required to initiate cell death. TWQ-184 induces gammaH2AX deposition and ATM activation within 24 hours, suggesting that PARP-bound single-strand breaks are converted to lethal double-strand breaks in the cancer cells. Exposure to TWQ-184 for one hour gives near-complete NC death at 72 hours, suggesting that intermittent dosing might be effective. TWQ-184 additionally exhibits high specificity for the malignant cells, evidenced by the substantial therapeutic windows between NC cell lines and healthy human lymphocytes and fibroblasts. Based on our current findings, the compound's high efficacy in the cancer cells and low toxicity in healthy cells offer a promising approach to treat NC. This strategy for hijacking a fusion oncoprotein to activate transcriptionally coupled DNA damage-mediated cell death could be applicable to other transcriptional fusion oncogenes.‬‬‬