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. 2018 Jul 31;115(31):7949-7954.
doi: 10.1073/pnas.1720000115. Epub 2018 Jul 16.

Spatially Constrained Tandem Bromodomain Inhibition Bolsters Sustained Repression of BRD4 Transcriptional Activity for TNBC Cell Growth

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Spatially Constrained Tandem Bromodomain Inhibition Bolsters Sustained Repression of BRD4 Transcriptional Activity for TNBC Cell Growth

Chunyan Ren et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.

Keywords: BRD4; TNBC; bivalent BET inhibitors; drug discovery; gene transcription.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Biophysical characterization of bivalent BrD inhibition of the BET proteins. (A) Domain organization of BRD4 and depiction of bivalent BET inhibitors. (B) Binding affinity of BrD inhibitors to BET BrD proteins as determined by an FP assay. The BET BrD proteins used are BRD4-BD1 (amino acids 44–168), BRD4-BD2 (amino acids 347–460), BRD4-BD1/2 (amino acids 44–477), BRD4-BD1/2_N140A (amino acids 44–477), BRD4-BD1/2_N433A (amino acids 44–477), BRD2-BD1/2 (amino acids 73–473), and BRD3-BD1/BD2 (amino acids 24–434). The chemical composition and length (depicted by number of atoms) for the linker are indicated for bivalent BET inhibitors. (C) Dose-dependent effects of BET inhibitors on transcriptional expression of IL-6 in MDA-MB-231 cells treated with the BrD inhibitors as indicated for 2 h. Results represent at least three independent experiments and error bars denote SEM. (D) Effects of BrD inhibitor binding on protein conformation of BRD4 BD1 alone or tandem BD1/BD2, as assessed by aMW of the protein determined by DLS. Measurements of UV, scattering intensity, and refractive index change are color-coded in green, red, and blue, respectively.
Fig. 2.
Fig. 2.
Structural insights into bivalent BET inhibitor binding to BRD4. (A) Crystal structure of MS645 (yellow)/BRD4 BD1 dimer complex, shown in ribbon and space-filled surface depictions. MS645 is color-coded by atom type and its electron density with Sigma-A-weighted 2mFo-DFc map. (B) Crystal structure of MS660 (orange)/BRD4 BD1 dimer complex, shown in ribbon and space-filled surface depictions. MS660 is color-coded by atom type and its electron density with Sigma-A-weighted 2mFo-DFc map. (C and D) Structural insights of MS645 or MS660 linker recognition at the domain–domain interface in the BRD4 BD1 dimer. Note that two symmetry-related protomers in the BD1 dimer are denoted as A and B′ as two molecules in two different crystallographic unit cells. (E) Structural model of MS645 bound to the BD1–BD2 of BRD4, built based on the crystal structure of the MS645/BRD4 BD1 dimer.
Fig. 3.
Fig. 3.
Superior cellular efficacy of MS645 in cancer-cell growth inhibition. (A) Persistent transcriptional repression of IL-6 by MS645 over other BET inhibitors in a washout study of MDA-MB-231 cells. The cells were treated with a BET inhibitor (1 μM) or DMSO for 2 h then washed with fresh medium twice and cultured for time periods as indicated. The mRNA level of IL-6 was measured after compound-imposed transcriptional inhibition. The data are plotted from one representative experiment and error bars represent SD of technical repeats. (B) Effects of the BET inhibitors of MS645, MS660, MS688, and JQ1 on protein stability assessed in a cellular thermal shift assay and shown by a representative set of Western blot analyses of BRD4. (C) Effects of MS645, MS660, JQ1, and MS417 on cell growth inhibition of cancer and noncancer cell lines, as assessed in an MTT assay. IC50 values are listed in a table. Results presented in B and C were all from at least three independent experiments and error bars designate SEM.
Fig. 4.
Fig. 4.
RNA-seq analysis of modulation of gene transcription by MS645 vs. JQ1. (A) Global changes in gene transcription of MDA-MB-231 cells after MS645 or JQ1 treatment at 50 or 500 nM, as shown in log twofold change scale. mRNA was collected for RNA-seq analysis from the MDA-MB-231 cells after treatment with DMSO, MS645, or JQ1 for 18 h. (B) Venn diagram analysis showing genes with over twofold changes after MS645 or JQ1 treatment as indicated. (C) Representative RNA-seq tracks highlighting the profoundly superior activity of MS645 over JQ1 in down-regulation of transcriptional expression of a select number of genes that are important for oncogenesis. The BRD4 ChIP-seq tracks shown as reference for BRD4 target genes were generated with MCF7 breast cancer cells and are available from the Gene Expression Omnibus database (accession no. GSE55921) (35).
Fig. 5.
Fig. 5.
Control of TNBC cell proliferation by bivalent BET inhibitors. (A) Time-course measurements of mRNA levels of CDK6 and RAD51 in HCC1806 cells after MS645 treatment. (B) Effects of MS645 on expression of proteins important for cell-cycle control in HCC1806 cells, as assessed by Western blot analysis. (C) ChIP analysis of effects of MS645 vs. JQ treatment on BRD4 dissociation from target genes. Results were from at least three independent experiments and error bars designate SEM. (D) Coimmunoprecipitation experiment showing that MS645, but not JQ1 or MS417, dissociates BRD4 from its interaction with MED1 (Upper) or transcription factor YY1 (Lower). (E) Schematic illustration of the mechanism of action of bivalent BET BrD inhibitor MS645 in blocking BRD4 activity in gene transcriptional activation: MS645 binding to the tandem BD1–BD2 of BRD4 exerts spatially constrained inhibition of BRD4, which locks BRD4 in an inactive state and blocks it from binding to effect proteins, leading to a sustained down-regulation of gene transcription. Note that the study cannot exclude the possibility of MS645 inhibition of other BET proteins or engaging in a bimolecular target protein inhibition in cells.

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