Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer

Abstract

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Figure 1.

(A-B) Docking studies of THQ analogs of 1. (C) Synthetic route to aryl substituted THQ and BETi BD binding affinities for derived analogs. Reagents and conditions: (a) TFA, CH₂Cl₂, quantitative; (b) R₁-Br, K₂CO₃, BrettPhos Palladacycle Gen. 3, BrettPhos, THF 100 °C.

Figure 2.

Biophysical characterization of SJ018. (A) Chemical structure, biophysical parameters and growth inhibition data. (B) SPR analysis with BRD2-BD2. (C) BROMOscan analysis using 1 μM SJ018. (D) ITC analysis with BRD2-BD2. (E) Comparison of BETi-modulated FRAP in U2OS cells using GFP labelled BRD4. Upper panel depicts photobleaching recovery in nucleus after a pulse of laser light (area circled in yellow). Lower panel indicates quantitation of FRAP datasets following incubation of cells with SJ018 (gray), JQ1 (gold), or DMSO (blue).

Figure 3.

Microarray studies comparing the changes in gene expression induced after 3h by increasing doses of SJ018 or JQ1 across three pediatric cancer cell lines (MV4–11, Kelly and HDMB03). (A) Venn diagrams depicting the number of significant changes in gene expression and the number of transcripts modulated over the dose range (FDR<0.05). (B) Scatterplots comparing all gene changes between the two drugs. All datasets were normalized to gene expression obtained from treatment of the relevant cell line with DMSO.

Figure 4.

ChIP-seq experiments identifying areas of active chromatin in MV4–11 cells following 3h treatment with either SJ018 or (+)-JQ1. (A) Number of H3K27ac sites perturbed by SJ018 or JQ1 at 25 nM and 1 μM. (B) Histograms indicating that both SJ018 and JQ1 exhibit consistent, but more pronounced changes in H3K27ac activated and repressed sites at 1 μM versus 25 nM. (C) ChIP-seq H3K27ac read enrichment plot at the NT5C2 gene locus where chromatin was found to be selectively activated by JQ1. (D) ChIP-seq H3K27ac read enrichment plots at the LINC01565 and RPN1 gene loci where chromatin was found to be selectively repressed by SJ018.

Figure 5.

Crystal structures of the BRD2 complexes with SJ432. (A) SJ432 chemical structure and indicated selectivity for BD2 with BRD2 and BRD4. (B) BRD2-BD1: SJ432 (orange). (C) BRD2-BD2: SJ432 (blue). (D) BRD2-BD1: acetylated histone H4 peptide (magenta, PDB 2DVQ). (E) BRD2-BD2: acetylated histone H4 peptide (green, PDB 2E3K). (F) Overlay of the BRD2-BD1:SJ432 complex (orange/yellow) and BRD2-BD2 (blue/cyan).

Figure 6.

Biological activity of SJ432. (A) Western analysis indicating loss of C-MYC protein in SK-N-SH cells after 16hr exposure to either SJ432 or JQ1. (B) Quantitation of C-MYC levels from A. (C) Modulation of MYC targets by SJ432 in SK-N-AS cells. (D) Upregulation of HEXIM1 by SJ432 or JQ1 in SK-N-AS cells. (E) Loss of BRD4, and to a lesser extent BRD2, following exposure of cells to SJ432 or JQ1. (F) Quantitation of BRD4 and BRD2 protein levels from panel E.

Figure 7.

In vivo activity of SJ432. In all experiments, 10 mice were used per group. (A) Flank SK-N-AS tumor volumes in cohorts of mice treated with increasing daily doses of SJ432 (5, 10 or 15 mg/kg). Statistical significance versus control tumors is indicated by asterisks (blue asterisk – 15 mg/kg vs control; red asterisk – 10 mg/kg vs control; green asterisk – 5 mg/kg vs control). (B) Comparison of tumor volumes of animals bearing SK-N-AS xenografts following treatment with either JQ1 (50 mg/kg; blue line) or SJ432 (15 mg/kg; red line). Asterisks above the data points indicate statistical significance as compared to control animals (red asterisk – 15 mg/kg SJ432 vs control; blue asterisk – JQ1 vs control). Asterisks (red) below the data points indicate statistical significance between JQ1- and SJ432-treated animals. (C) Kaplan Meier curves demonstrating increased survival of animals bearing SK-N-AS tumors treated with SJ432 (15 mg/kg; red line) as compared to JQ1 (50 mg/kg; blue line), or control mice (black line). Median survivals were 22.5, 26 and 31 days for control, JQ1-treated, and SJ432-treated mice, respectively. Significance for these data sets using log rank test were as follows: SJ432 vs control – p < 0.0007; JQ1 vs control – p = 0.067; SJ432 vs JQ1 – p = 0.0791. (D) Weights of mice from the study presented in panels B and C. (E) Western analyses indicating the levels of C-MYC, HEXIM1, BRD4 and BRD2 protein in SK-N-AS xenografts harvested either 4h or 24 h after SJ432 dosing. C – Control, untreated tumors. (F) Quantitation of the levels of protein in panel E. Data was corrected for gel loading differences using β-actin expression.