Bet Bromodomain Inhibitors Antagonize Brd4-Mediator Complexes To Undermine The Acute Myeloid Leukemia Cell State

Acute myeloid leukemia (AML) is a hematologic malignancy with a 5-year survival rate of under 30%. We recently identified the bromodomain and extraterminal (BET) protein Brd4 as a therapeutic target in AML, and several trials are currently evaluating the clinical utility of BET inhibitors for this disease. BET inhibitors displace Brd4 from chromatin and subsequently reduce the expression of key oncogenes, such as Myc, leading to AML blast differentiation and cell death. However, the mechanism by which Brd4 maintains oncogene expression in AML is still unclear. We hypothesized that Brd4 functions by working with other coactivators in AML to promote expression of oncogenes. One such coactivator is the Mediator complex, which is comprised of ~30 subunits and directly contacts RNA Polymerase II to regulate its function. Initial purifications of Mediator from mammalian cells identified Brd4 as an associated factor. Using ChIP-seq analysis in AML cells, we show that Brd4 and Mediator closely co-localize across the genome. Moreover, chemical inhibition of Brd4 results in the displacement of Mediator from enhancer and promoter regions across the genome, an effect that occurrs within 30 minutes of treatment. While the genome-wide loss of Mediator occupancy is approximately 2-fold, a subset of promoters and enhancers exhibit dramatic loss of Mediator occupancy. Importantly, these regions are disproportionately associated with genes related to leukemia biology but only modestly overlap with super-enhancers. In addition, we show that shRNA-based knockdown of several Mediator subunits phenocopies the transcriptional and cellular effects of Brd4 inhibition without affecting levels of Brd4 in the cell. These effects include downregulation of Myc expression, induction of myeloid differentiation, and reduction of P-TEFb recruitment to chromatin. These findings support a model in which Brd4 functions in concert with the Mediator complex to maintain oncogenic gene expression programs in leukemia and shed light on the mechanisms of action underlying a promising new class of therapeutics for AML.