Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain “reader” modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common ‘core’ transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain ‘super-enhancers’, recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

Tenascin-C, an extracellular matrix molecule of the tumor-specific microenvironment, counteracts the tumor cell proliferation–suppressing effect of fibronectin by blocking the integrin α₅β₁/syndecan-4 complex. This causes cell rounding and stimulates tumor cell proliferation. Tenascin-C also stimulates endothelin receptor type A (EDNRA) expression. Here, we investigated whether signaling through endothelin receptors affects tenascin-C–induced cell rounding. We observed that endothelin receptor type B (EDNRB) activation inhibited cell rounding by tenascin-C and induced spreading by restoring expression and function of focal adhesion kinase (FAK), paxillin, RhoA, and tropomyosin-1 (TM1) via activation of epidermal growth factor receptor, phospholipase C, c-Jun NH₂-terminal kinase, and the phosphatidylinositol 3-kinase pathway. In contrast to EDNRB, signaling through EDNRA induced cell rounding, which correlated with FAK inhibition and TM1 and RhoA protein destabilization in the presence of tenascin-C. This occurred in a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase–dependent manner. Thus, tumorigenesis might be enhanced by tenascin-C involving EDNRA signaling. Inhibition of tenascin-C in combination with blocking both endothelin receptors could present a strategy for sensitization of cancer and endothelial cells toward anoikis. [Cancer Res 2007;67(13):6163–73]