Abstract
The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.
Highlights
BRD4 is a member of the bromodomain and extraterminal (BET) family of chromatin reader proteins
Additional biophysical characterization by analytical ultracentrifugation and small angle X-ray scattering demonstrated that AZD5153 can bind bivalently, namely, that one molecule of AZD5153 binds and ligates two bromodomains in BRD4, in contrast to other reported BET inhibitors such as JQ1 and I-BET762 that bind monovalently with one molecule binding to each bromodomain in BRD4
To confirm the in cell binding mode of AZD5153, we utilized a NanoBRET assay to monitor the interaction of BRD4 with chromatin using Halo-tagged histone H3 and a Nanoluciferase fusion protein with either the Bromodomain 1 construct (BD1) domain alone (BD1-BRD4) or a full-length BRD4 construct (FL-BRD4) (Figure 1B) [24]
Summary
BRD4 is a member of the bromodomain and extraterminal (BET) family of chromatin reader proteins. The discovery of lysine-mimetic small molecules capable of disrupting the chromatin binding activity of BET bromodomains was a crucial step towards uncovering the cancer supporting activity of BRD4 [10,11,12]. In hematologic malignancies such as AML, MM and DLBCL, the misregulated expression of hematopoietic transcription factors is important. MYC deregulation in MM can occur through gene translocation, amplification and overexpression [14, 15] In this setting BRD4 inhibition disrupts MYC activity and results in MM cell cycle arrest and senescence [16]. Based on this and other work, several drug candidates targeting BRD4/BET have progressed into clinical trials to treat Nut Midline Carcinoma (NMC), AML, Myelodysplastic Syndromes (MDS), MM, DLBCL, Glioblastoma Multiforme, and other solid tumors
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