Abstract B014: Novel dual BET/p300 bromodomain inhibitors therapeutically inhibit BRD4-NUT driven NUT Carcinomas

Abstract

Abstract Introduction: NUT carcinoma (NC) is an aggressive subtype of squamous carcinoma whose growth is driven by the BRD4-NUT fusion oncoprotein. With a median survival of 6.7 months, NC is perhaps the most aggressive solid tumor in humans, and there exist no effective or standard treatment regimens. BRD4-NUT drives growth by blocking differentiation. BRD4-NUT binds chromatin through the acetyl-histone binding, dual bromodomains (BD) of BRD4. BET inhibitors are highly selective for the BDs of BET proteins and have demonstrated on-target activity in human NC patients, however toxicity has limited efficacy. BRD4-NUT forms hyperacetylated regions, termed megadomains, enriched with the NUT-interacting histone acetyltransferase, p300, that upregulate expression of key oncogenic targets, including MYC. Here we sought to determine the role of p300 in the blockade of differentiation and growth of NC cells. Next, we investigated whether inhibiting both BRD4 and p300, by blocking the BDs of BRD4 and p300 with a novel dual inhibitor class of compounds, termed ‘NEO’ (NEO2734 and NEO1132), might exhibit greater inhibition of NC growth than BET BD inhibition alone. Methods: We utilized in vitro reporter assays to map the minimal essential domain of NUT necessary for its interaction with p300. Using our NC cell derivative, 797TRex, which contains a single genomic FRT recombination site, and constitutively express tetracycline repressor (TR), allows for FLP-recombinase insertion of tet-inducible NUT or p300 constructs. Immunofluorescence, immunoblotting, cell-titer glo (Promega), and nascent RNAseq were used to evaluate effects of transgene induction and treatment with compounds. Tolerability and efficacy of various compounds were evaluated in vivo in a disseminated mouse NC xenograft model using the PER-403 human BRD4-NUT+ cell line. Results: We determined that a putative transcriptional activation domain (TAD1 (NUT390-482)) of NUT and the TAZ2 domain of p300 (p3001721-1837) bind directly. We found that inducing expression of TAD1 or TAZ2 in 797TRex cells results in rapid differentiation and promotes the dispersion of acetylated and p300-enriched BRD4-NUT megadomains/foci, indicating that p300 is required for the blockade of differentiation through its direct interaction with NUT. Preliminary data also suggest TAD1 (NUT390-482) expression in 797TRex cells decreases nascent transcription of BRD4-NUT-mediated oncogenic targets. Furthermore, our findings indicate that NEO compounds inhibit in vitro NC growth with IC50s = 44-225nM, which associated with the induction of squamous differentiation. Most strikingly, the lead clinical NEO compound completely abrogated NC tumor growth in our pilot disseminated xenograft mouse study, which greatly prolonged survival as compared to a lead clinical BET inhibitor (GSK762) or “standard” chemotherapy (cisplatin/etoposide) commonly used for NC. Conclusions: These data support our hypothesis that p300 recruitment is necessary for the oncogenic functions of BRD4-NUT and dual targeting of BET and p300 BDs represents a promising therapeutic option for NC patients. Future studies are necessary to determine the relative contribution of BET versus p300 BD inhibition to preventing NC growth by NEO compounds. Citation Format: Chevaun D Morrison-Smith, Amy DiBona, Erica Walsh, Kristina Danga, Ivona Filic, Tatiana Knox, Adlai Grayson, Michael Cameron, Christopher French. Novel dual BET/p300 bromodomain inhibitors therapeutically inhibit BRD4-NUT driven NUT Carcinomas [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B014. doi:10.1158/1535-7163.TARG-19-B014