NEO2734

Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain–selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition.^[1]

NUT carcinoma patient cell lines (TC-797, 14169, 10-15, and PER-403) are plated in T-25 flasks and grown in DMEM (797) containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. Cells are treated with either 500 nM OTX-015, 1 μM GNE-781, combination of 62.5 nM GNE-781 and 62.5 nM OTX-015, 1 μM NEO2734, or the equivalent volume of DMSO (0.025 %). At 24H, 2 × 10⁶ cells are spun at 500 × g for 5 minutes at 4 ℃ and washed twice with PBS. Pellets are resuspended in 1 ml of cold PBS and added dropwise while gently vortexing to 9 ml 70 % ethanol in a 15 ml polypropylene centrifuge tube. Fixed cells are then frozen at –20 ℃ overnight. The next day, cells are centrifuged at 500 × g for 10 minutes at 4 ℃ and washed twice with 3 ml of cold PBS. Cells are resuspended in 400 μl of propidium iodide staining solution (0.2 mg/ml RNAse A, 0.02 mg/ml propidium iodide, 0.1 % Triton-X in PBS) and incubated for 15 minutes at 37 ℃.

In three disseminated NMC xenograft models, NEO2734 provides greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or “standard” chemotherapy.^[1]