Fig 2: BRD4 methylation at K99 inhibits translation.(A to D) Translation assay (SUnSET). (A) MDA-MB-231 control and CRISPR SETD6 KO (two independent gRNAs) cells. (B) MDA-MB-231 CRISPR SETD6 KO cells with or without overexpressed HA SETD6. (C) MDA-MB-231 cells stably expressing empty plasmid, Flag BRD4 wild type, or Flag BRD4 K99R (1-477aa). (D) MDA-MB-231 wild-type cells overexpressing empty plasmid, Flag BRD4 wild type, or Flag BRD4 K99R (long isoform). Cells were incubated with puromycin and then cell extracts were submitted to Western blot to detect protein synthesis using anti-puromycin antibody. (E) Polysome profiling. Monosome and polysome fractions were extracted from MDA-MB-231 cells stably expressing empty plasmid, Flag BRD4 wild type, or Flag BRD4 K99R (1-477aa). The fractions and total cell lysates, served as control, were submitted to Western blot analysis with anti-Flag antibody. Actin served as loading control. Phosphorylated S6 (p-S6) served as control for polysome profiling method.

Fig 3: BRD4 K99me does not affect its binding to acetylated H4.(A and B) MDA-MB-231 wild-type cells were transfected as indicated and treated with TSA for 4 hours. Chromatin fractions were immunoprecipitated with indicated antibodies, followed by Western blot analysis with anti–tetra-acetylated H4 antibody. (C) Left: Topology of the H4K5ac/K8ac (H4 residues 1 to 11) peptide binding to BRD4/BD1. The peptide termini are annotated and the location of K99 is highlighted in red [Protein Data Bank (PDB) ID: 3UVW (34)]. Middle: Crystal structure of the complex of BRD4/BD1 with an H4 (1 to 11) K5ac/K8ac peptide (PDB: 3UVW) shown in ribbon and stick representation. Residues that initiate contact are annotated and K99 is shown in red (same orientation as on the left). Right: Binding of H4 (1 to 11) K5ac/K8ac onto the surface of BRD4/BD1. Protein charge distribution is plotted on the surface of BRD4/BD1, highlighting the extensive electrostatic nature of the interaction. Location of K99 is highlighted in red. (D) ChIP assay for MDA-MB-231 wild-type cells overexpressing Flag BRD4 wild type or Flag BRD4 K99R (1-477aa) using FLAG-M2 (left) or U293 antibody (right). Graphs show percent input of the quantified DNA. Error bars are SEM. Statistical analysis was performed for three experimental repeats using Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: BET inhibitor and degrader treatment downregulates genes required for MCC lineage specification. (A) Volcano plot of top differentially expressed genes compared to DMSO (lfc > 0.6, P < .05). Microarray analysis was performed following 3 hours of treatment with DMSO, BETi-211 (250 nM), thalidomide (10 nM), or BETd-246 (10 nM). (B) MCC47 and MCC29+ cells were treated for 3 and 24 hours. Untreated cells were included as an additional control. Western blot shows Atoh1 protein with on-target degradation of Brd4 by BETd-246. MCC47 cells were transfected with siRNA for 96 hours. (c) Left: RT-qPCR for ATOH1 mRNA and corresponding Western blot for Atoh1 protein show efficacy of siRNA knockdown in MCC47 cells. Right: Cell viability following ATOH1 siRNA transfection in MCC47 cells. (D) Left: RT-qPCR for MYB mRNA and corresponding Western blot for cMyb protein show efficacy of siRNA knockdown in MCC47 cells. Right: Cell viability following MYB siRNA transfection in MCC47 cells. All P values were determined using paired t tests. All error bars represent mean ± SD.

Fig 5: Human MCC cell lines are sensitive to BET degrader BETd-246. (A) Heat map of IC50 values for OTX-015, BETi-211, and BETd-246 (nM). (B) MCC47 and MKL1+ cells were treated with DMSO, BETi-211 (250 nM), thalidomide (10 nM), or BETd-246 (10 nM) for 24 hours. Western blot shows on-target effect of BETd-246 by degradation of Brd4 and Brd2 protein and appearance of cleaved PARP with BETd-246 and BETi-211 treatment. (C) Cell viability following BRD4 siRNA transfection in MCC47 and MKL1+ cells. NT, nontargeting. Error bars represent mean ± SD. (D) Tumor volume in mm3 and weight in grams between treatment groups following 16 days of treatment. Mice were treated with OTX-015 (100 mg/kg, PO), BETd-260 (5 mg/kg, IV), or vehicle. Error bars represent mean ± SEM.