Treatment with BETi reduces BRD4 occupancy on the enhancers of RUNX1 and in AML cells, and knockdown of BRD4 depletes RUNX1 expression and induces apoptosis of AML cells. (A) Ranked ordering of super-enhancers analysis in OCI-AML5 cells (left). Total H3K27Ac ChIP-Seq signal in units of reads per million in enhancer regions for all enhancers in OCI-AML5. Enhancers are ranked by increasing H3K27Ac ChIP-Seq signal. ChIP-Seq signal density plots for H3K27Ac and ATAC-Seq accessibility profiles in the RUNX1 locus of OCI-AML5 cells (right). The locations of the P1 and P2 promoter, +24 kb enhancer, P2 proximal enhancer (corresponds to the mouse +110 enhancer reported by Marsman et al62 ), and the intronic super-enhancer are noted. (B) Reporter constructs containing the RUNX1 P2 promoter and/or a portion of the +24 kb enhancer were transfected into HEK293 cells utilizing polyethylenimine (PEI) and incubated for 48 hours. Relative luciferase activity for each construct was quantified and is reported as total luminescence. *P < .05 compared to P2 promoter or +24 kb enhancer-alone transfected cells. (C) ChIP qPCR of BRD4, c-Myc, p300, PU.1, and pSer2-RNAP2 occupancy in the RUNX1 +24 kb enhancer after treatment with 1000 nM of OTX015 or 250 nM of ARV-771 for 16 hours in OCI-AML5 cells. (D) Relative mRNA expression of RUNX1, MYC, PU.1, HEXIM1, and p21 in OCI-AML5 cells treated with ARV-771 or OTX015 for 4 hours. Expression of each mRNA was normalized to GAPDH and to the untreated control cells. (E) Immunoblot analysis of OCI-AML5 cells transduced with sh NT or BRD4 shRNAs and incubated for 72 hours. (F) Percent apoptosis induced in OCI-AML5 cells transduced with sh NT or BRD4 shRNA for 72 hours.