A Bromodomain-Swap Allele Demonstrates That On-Target Chemical Inhibition Of Brd9 Limits The Proliferation Of Acute Myeloid Leukemia Cells

Recent studies have revealed vital roles of SWI/SNF complexes in leukemia and a variety of other cancers, making this chromatin remodeler a candidate drug target in human malignancy. Chemical modulation of SWI/SNF activity, however, remains to be achieved. Given the success of pharmacological bromodomain inhibition, we evaluated the role of bromodomain-carrying SWI/SNF subunits and identified Bromodomain-containing protein 9 (BRD9) as critical for the growth of Acute Myeloid Leukemia (AML). In AML cells, BRD9 binds the enhancer of the MYC proto-oncogene and sustains MYC transcription, rapid cell proliferation, as well as a block in differentiation. Based on these observations, we derived a small-molecule inhibitor of the BRD9 bromodomain, which partially displaces BRD9 from MYC enhancer elements and selectively suppresses the proliferation of mouse and human AML cell lines. Given the known role of other bromodomains, namely those of BRD4, in leukemia growth, ruling out potential off-target activity of our BRD9 inhibitor was critical. Traditionally, bromodomain inhibitor selectivity is tested using in vitro binding assays that examine a subset of other bromodomains. To sample the entire space of potential off-target proteins, we sought an in-cell selectivity assay. To this end, we engineered a bromodomain-swap allele of BRD9, which retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the anti-proliferative effects of our BRD9 inhibitor, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. Our study provides the first evidence for a role of BRD9 in cancer and further highlights a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells. Citation Format: Anja F. Hohmann, Laetitia J. Martin, Jessica Minder, Jae-Seok Roe, Junwei Shi, Steffen Steurer, Gerd Bader, Darryl McConnell, Mark Pearson, Thomas Gerstberger, Teresa Gottschamel, Diane Thompson, Yutaka Suzuki, Manfred Koegl, Christopher Vakoc. A bromodomain-swap allele demonstrates that on-target chemical inhibition of BRD9 limits the proliferation of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-206.