Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation

Abstract

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.

Fig. 1. Selectivity profile of I-BET151, iBET-BD1 (GSK778) and iBET-BD2 (GSK046).

(A) Schematic of the BET Bromodomain proteins and chemical structures. (B) Compound binding to the individual bromodomains of BD1 (orange) and BD2 (cyan) of BET tandem bromodomains in TR-FRET assays. (C) X-ray crystal structure of I-BET151 in BRD2 BD1 (magenta, PDB 4A1G)⁽¹⁹⁾ superimposed on the structure of iBET-BD1 in BRD4 BD1 (orange, PDB 6SWN). (D) iBET-BD1 bound to BRD4 BD1 (dark orange/white, PDB 6SWN) and BRD2 BD2 (light orange/blue, PDB 6SWO) highlighting differences in the BC loop. (E) iBET-BD2 bound to BRD2 BD2 (light blue, PDB 6SWP) and BRD4 BD1 (cyan, PDB 6SWQ). In BRD2 BD2, the inhibitor’s benzyl and cyclohexane rings pack against Pro430 and His433, which adopts a single “in” conformation. In BRD4 BD1, iBET-BD2 makes no significant contacts with the corresponding Asp144 and Lys141 sidechains, and the space occupied by the His433 sidechain in BRD2 BD2 is filled by an ethane-1,2-diol molecule from the crystallisation buffer.

Fig. 2. BET-BD1 inhibition phenocopies pan-BET inhibition.

(A) IC50 assays performed at 72 hrs in MDA-453 and (B) MOLM-13 cells following incubation with a range of doses of iBET-BD1, iBET-BD2 or I-BET151. Data points represent Mean (n= 3 cell culture replicates) plotted as a representative from 3 independent experiments. (C) Cell cycle analysis of MV4;11 cells treated with DMSO, I-BET151 (1000nM), iBET-BD1 (1000nM), or iBET-BD2 (1000nM). (D) Apoptosis assay performed on MOLM13 cells treated with DMSO, I-BET151 (1000nM), iBET-BD1 (1000nM), or iBET-BD2 (1000nM). (E) Kaplan–Meier curve of vehicle-and drug-treated C57BL/6 mice transplanted with 1 ⨯ 10⁶ MLL-AF9 leukemic cells. Treatment commenced at day 9 with bi-daily intraperitoneal injections at 15mg/kg of I-BET151, iBET-BD1, iBET-BD2, or Vehicle. n = 6 mice per group. P values were calculated with the two-tailed log-rank test. (F) Heatmap of differential gene expression from SLAM-seq in THP-1 cells. (G) Average profile of BRD4 ChIP-seq signal at Typical enhancers (TE) and at (H) super-enhancers after treatment with Vehicle (DMSO), I-BET151, iBET-BD1, and iBET-BD2. (I) Genome browser view of the MYC super-enhancer in THP-1 cells showing the occupancy of BRD2, BRD3, and BRD4 after treatment with Vehicle (DMSO), iBET-BD1, iBET-BD2 and I-BET151.

Fig. 3. BET-BD2 is required for activation of IFNg target genes.

(A) FACS analysis of MHC-I expression using antibodies against HLA-A/B/C in K562 cells following stimulation with IFNg (10ng/ml) and treatment with DMSO, iBET-BD1, iBET-BD2 or I-BET151 for 48 hrs. (B) Hierarchical clustering heatmap from SLAM-seq data in K562 cells showing upregulated genes following IFNg treatment for 6 hrs. (C) Scatter plot of differential expression of all genes (grey) and genes significantly upregulated by IFNg (red) in K562 cells, scatter plot and histogram shown for DMSO, iBET-BD1, iBET-BD2 and I-BET151. (D) Average profile plot of BRD2, BRD3 and BRD4 at genomic loci where H3K27ac levels increase following stimulation with IFNg in K562 cells for 6hrs.

Fig. 4. iBET-BD2 has immunomodulatory activity.

(A) schematic of auxin-inducible degradation strategy for BRD4. Blast^R (Blasticidin resistance) linked in-frame with T2A self-cleaving peptide to FLAG-mAID-BRD4. A separate viral vector was used to express CMV promoter driving expression of OsTIR1-IRES-Puro resistance. (B) FACS analysis of HLA/B/C expression in K562 cells pretreated with DMSO, Auxin (IAA) or I-BET151 for 6hrs followed by stimulation with IFNg (10ng/ml) for 48 hrs and 48 hr incubation with compounds. (C) qRT-PCR analysis of MYC expression in K562 cells following treatment with I-BET151 or IAA (Auxin) for 6hrs. Data shown represent the mean ± SD (n=3) (D) qRT-PCR analysis of TAP1 expression in K562 cells before and after IFNg treatment (6 hours) and/or either IAA (Auxin), IBET151, or DMSO (Vehicle control) for 7 hours (1hr pretreatment), Data shown represent the mean (n=3) +/- SD, ** p>0.01. (E) Compound effects on cellular proliferation and (F) cytokine production in anti-CD3/CD28-stimulated human primary CD4⁺ T cells. Data represent the mean ± SEM (n = 4) (G-H) Efficacy of compounds reducing KLH-induced antibody responses (IgM) in mice. N, naїve (n = 4); V, vehicle; iBET-BD1 (15 mg/kg, s.c., BID), iBET-BD2 (40 mg/kg, s.c., QD), I-BET151 (15 mg/kg, s.c., QD). Data shown as the mean ± SEM (n = 10). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to determine statistical significance compared with respective vehicle controls (***P<0.001, **P<0.005 vs V(QD); ⁺⁺ P<0.005, ⁺ P<0.01 vs V(BID)).

Fig. 5. GSK620 is efficacious in preclinical models of immuno-inflammation.

(A) Chemical structure of the BD2-selective BET inhibitor in vivo tool GSK620. (B) Compound binding to the individual tandem bromodomains (BD1 (orange) and BD2(cyan)) of BRD2, 3, 4 and T determined using TR-FRET. Data are the mean ± SEM (n = 12-21). (C) Phylogenetic tree of bromodomain family demonstrating preferential compound binding for the BD2 domains of the BET family of proteins using the BROMOscan bromodomain competition binding assay. Red dots represent K_D values. (D) Mouse imiquimod (IMQ)-induced psoriasis study design. (E) Histology H&P (Hematoxylin-Phloxin) staining of skin sections (F) GSK620 (20mg/kg, p.o., QD) reduces the psoriasis score, (G) the epidermal thickness and (H) the expression levels of inflammatory genes in skin biopsies. V, vehicle; Vani,vanicream; Apremilast (20 mg/kg, p.o., BID). Data represent the mean ± SEM (n = 10). One-way ANOVA followed by Dunnett’s multiple comparison test (***P<0.0001 vs V + IMQ).