NanoBRET NLuc-BRD4(D1) Target Engagement. In-cell NanoBRET target engagement assays were performed on HEK293T cells according to transiently expressing a BRD4(D1)-NLuc fusion, according to protocols and materials purchased from the Promega BET Target engagement Kit (Cat. N2131). Briefly, HEK293T cells were seeded into a T25-Corning TC-treated flask (Corning, CLS431082) at 200,000 cells/well in DMEM containing 10% FBS (Cat. A5256701) and 100 μg/mL of penicillin/streptomycin (Thermo, Cat. 15140122). Separately, a transfection cocktail containing 1 μL of NLuc-BRD4(D1) (Promega, 1ug/uL), 9 μL of transfection carrier DNA (Promega, 1 ug/uL), 30 μL of FuGENE (Promega), and 1 mL of OptiMEM media. This transfection cocktail was allowed to equilibrate for 10 min before adding to HEK293T cells, which were then allowed to incubate at 37 °C overnight with 5% CO2. The next day, cells were removed from the flask with 0.05% trypsin (Thermo Fisher Cat.# 25300), diluted in culture medium, and centrifuged at 5000 rpm for 4 min before being resuspended in OptiMEM media (Gibco, Cat.# 11-058-021) at a density of 200,000 cells/mL. These cells were then plated into a TC-treated white 384well plate (Corning, 34 μL/well) as technical triplicates including a no-tracer control. The tracer solution was prepared by diluting the 20x tracer (Promega) with 4 parts (v/v) of dilution buffer (Promega), to which the tracer was then added to the cells (2 μL/well). Cells were dosed by preparing a 10x stock of compound in OptiMEM (0.1% DMSO) and adding 4 μL/well to the transfected cells. The cells were allowed to incubate at 37 °C with 5% CO2 for 2 h before a NanoGlo substrate cocktail containing NanoGlo Substrate (Promega) and an Extracellular Luciferase inhibitor (Promega). The donor and the acceptor emissions were measured immediately at 450 nm and 610 nm, respectively, using a Tecan Spark. The mBRET Ratio was calculated using the following equation: