Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1,2,3,4,5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7,8,9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10,11,12,13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.
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We thank Z. Zha for technical assistance with ChIP–seq data analysis. For X-ray crystallography, use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357.
E.J.F., K.F.M., D.H.A., S.R.M., L.Z., M.H.B., G.S.S., L.W., J.P.P., V.S., X. Lin, X.H., X. Lu, T.U., L.T.L., R.J.B., G.M., S.F., J.K.P., D.L., L.A.H., C.S., S.C.P., J.J.N., S.L.F., K.L., L.B., M.T., S.H.R., W.M.K. and Y.S. are employees of AbbVie. C.H.P., D.W. and P.H. were employees of AbbVie at the time of the study. The design, study conduct and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review and approval of the publication.
Peer review information Nature thanks Arul Chinnaiyan, Stefan Knapp, William Pomerantz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, TR-FRET, surface plasmon resonance (SPR) and NanoBRET potency and selectivity of ABBV-744. b, Surface plasmon resonance binding of ABBV-075 and ABBV-744 to BD1 and BD2 domains of BRD4. ABBV-075 binding curves (coloured) with fits to the 1:1 binding model (black). ABBV-744 binds to BD1 with very fast on and off kinetics, therefore a steady-state fit to equilibrium responses was used to determine Biacore affinities. Dissociation of ABBV-744 from BD2 is very slow and therefore binding was profiled using the single-cycle kinetics method. All experiments were repeated once with similar results. c, Binding affinities of ABBV-744 to selected bromodomains for which ABBV-744 exhibited more than 50% inhibition at 1 μM using BromoScan profiling. d, Pharmacokinetic parameters in mice. e, ABBV-744 stability after incubation with various CYP enzymes.
a, Chemical structure of A-083. b, Activity of A-083 across multiple assays. c, Anti-proliferation activity of A-083 across the OncoPanel of Europhin, which consist of 240 cancer cell lines across a broad spectrum of cancer indications. d, Characterization and antiproliferative activities of ABBV-075, ABBV-744 and BD2 and DbBis as described in the literature. e, Antiproliferative activities of ABBV-075 and ABBV-744 against IEC-6 and LNCaP cells and the activities of both compounds in a Mk-CFU assay—an assay that measures the generation of megakaryocytes from mouse haematopoietic stem cells—carried out by Stemcell Technology.
a, Comparison of differentially regulated genes from this study with those reported in the literature using JQ1 and iBET. b, Reduction in MYC and KLK2 protein levels detected by western blot after treatment for 24 h with ABBV-075 (60 nM) or ABBV-744 (90 nM); no effect on AR was found. ABBV-075 but not ABBV-744 increases HEXIM1 protein levels. Representative of n = 3 independent experiments with similar results. For gel source data, see Supplementary Fig. 2. c, Biochemical, biophysical and cellular characteristics of the BD1 inhibitor (BD1i) described in the indicated GSK patent application. Bottom, Expression of KLK2 and MYC in LNCaP cells after 6 h treatment with ABBV-075 (60 nM), ABBV-744 (90 nM), BD1i (200 nM) or ABBV-744 (90 nM) and BD1i (200 nM) was determined by qPCR. Data are mean ± s.d. (n = 3 biologically independent samples) and are representative of n = 2 independent experiments. d, Gene set enrichment analysis of RNA-seq data (n = 2) from LNCaP cells treated with ABBV-075, ABBV-744 or enzalutamide. Statistical significance was determined using a false-discovery rate (FDR) (Benjamini–Hochberg correction) and negative enrichment scores (NES) with q < 0.05 are listed in the table. Venn diagram shows the overlap of enriched hallmarks with each treatment. AR, MYC and E2F gene set enrichment analyses are shown as examples. Source data
a, AR peaks measured by AR ChIP–seq after treatment for 24 h with DHT and DMSO, ABBV-075 or ABBV-744. As a reference, literature-reported changes in AR peaks after JQ-1 treatment were also included. b, BRD4 but not BRD2 or BRD3 had strong dependency scores across all prostate cancer cell lines (left) and was correlated with ABBV-744 sensitivity (right). Dependency scores were obtained from the DepMap portal. Scores less than −0.5 indicate the dependence of a cancer cell line on a given gene. Dots represent the dependency score for an individual cell line. Data are mean ± s.d. across the group. Significance was calculated using unpaired, one-sided Student’s t-tests. ns, not significant. c, BRD4 and AR-binding profile at AR-regulated KLK genes for which ABBV-075 (60 nM) and ABBV-744 (90 nM) in LNCaP cells or JQ-1 (500 nM) in VCAP20 showed similar displacement of BRD4. Loss of AR was more notable after treatment with ABBV-075 and JQ-1 than after treatment with ABBV-744. d, Venn diagram of BRD4–AR peak overlap in LNCaP cells. In total, 43% of AR–BRD4 common regions were located in super-enhancers.
a, HOMER motifs enriched in super-enhancers in which ABBV-744 and ABBV-075 (common) displaced BRD4 or super-enhancers in which only ABBV-075 displaced BRD4 (exclusive), n = 1. Statistics were derived using FDR (Benjamini–Hochberg correction) and q values are shown. b, Upstream regulators for differentially expressed genes (n = 2) associated with ABBV-744 and ABBV-075 BRD4-displaced super-enhancers compared with ABBV-075-exclusive super-enhancers (n = 1), as analysed by ingenuity pathway analysis. AR, E2F1 and MYC all associated with common BRD4-displaced super-enhancers. c, Gene track examples of differential displacement pattern for ABBV-744 and ABBV-075 commonly sensitive (ACPP) or ABBV-075 exclusive (ZG16B).
a, LNCaP cells were treated for 16 h with DHT in the presence of vehicle, ABBV-744 (90 nM) or ABBV-075 (60 nM) with or without trichostatin A (TSA) (0.5 μg ml−1). AR immunoprecipitation (IP) using nuclear extracts pulled down BRD4 in trichostatin-A- and DHT-treated samples. ABBV-744 and ABBV-075 blocked BRD4 co-immunoprecipitation with AR. Fold change values from densitometry analysis are listed below the BRD4 blot, in which a 1.9-fold increase in the AR:BRD4 immunocomplex was measured in the trichostatin-A- and vehicle-treated lane compared with 0.87 or 0.88 after treatment with ABBV-744 or ABBV-075, respectively. Western blot of 2% immunoprecipitation input revealed no change in nuclear protein levels after inhibitor treatment. b, LNCaP cells were treated for 16 h with DHT in the presence of vehicle, ABBV-744 (90 nM) or ABBV-075 (60 nM). CDK9 or BRD4 immunoprecipitation using nuclear extracts pulled down BRD4 or GATA2, which is not blocked by treatment with ABBV-744. c, LNCaP cells were treated for 16 h with DHT in the presence of vehicle, ABBV-744 (90 nM) or ABBV-075 (60 nM). CDK9 or cyclin T1 immunoprecipitation using nuclear extracts pulled down HEXIM1, which is not blocked or enhanced by treatment with ABBV-744. d, Alignment of a KXXK motif in H4, AR and the lack of this motif in AR-V7. e, Cooperative interaction of BD1 and BD2 of BRD4 with acetylated AR at BRD4–AR co-occupied super-enhancers may underlie sensitivity to ABBV-744. a–c, Results are representative of n > 2 independent experiments. For a–c gel source data, see Supplementary Fig. 2.
a, ABBV-075 but not ABBV-744 induces a robust dose-dependent increase of senescent (β-galactosidase-positive) 22RV1 cells after 7 days of treatment. Data are mean ± s.d. (n = 3 biological replicates) and are representative of n = 2 independent experiments. b, Scatter plot of gene expression changes (n = 2) caused by ABBV-075 (60 nM) or ABBV-744 (90 nM) treatment for 24 h in DHT-stimulated 22RV1 cells. Statistical analysis of fold change (FC) > 2.0, P < 0.01 was conducted using the DESeq2 method. c, Split violin representation of DHT-regulated compared with all differentially expressed genes in 22RV1 from RNA-seq as shown in b. The long solid line represents the mean fold change. The small lines represent individual data points. The dotted line represents the overall average. Statistical significance between all versus DHT was determined by two-tailed unpaired Student’s t-test and P < 0.01 by DESeq2. ABBV-075 affects both DHT and a broad distribution of genes, whereas ABBV-744 has a more limited effect on both DHT-stimulated genes and overall. d, ABBV-075 but not ABBV-744 negatively regulated the androgen response in 22RV1 cells as shown by gene set enrichment analysis. NES > 2.0, q < 0.05 calculated using FDR (Benjamini–Hochberg correction). e, H3K27Ac and BRD4 ChIP–seq heat maps at transcription start sites and enhancers in 22RV1 cells. f, ABBV-744 less effectively displaces BRD4 from super-enhancers in the resistant 22RV1 cell line compared with sensitive LNCaP cells.
a, Sprague-Dawley rats (n = 3 animals per group) were treated daily with vehicle, ABBV-075 (3 mg kg−1) or ABBV-744 (30 mg kg−1) for 14 days, and platelet counts were determined using the standard method. Efficacious exposure levels of ABBV-075 (1 mg kg−1) and ABBV-744 (4.7 mg kg−1) in mice and exposure levels associated with the indicated doses of each compound in rats were determined in separate pharmacokinetic studies using different animals (n = 3 animals per group). b, Antitumour activity of well-known BET inhibitors in the xenograft model in which LNCaP cells were implanted in the mouse flank. JQ-1 and iBET-762 were administered at their respective MTD. RVX-208 was administered at its maximal achievable dose. Data are mean ± s.e.m. of tumour size for each treatment group (n = 6). WL, maximum weight loss relative to initial value; FD, found dead. c, Efficacy comparison of BET inhibitors in the LNCaP model. d, e, Mice bearing LNCaP tumours (d; n = 9 per group) or OPM2 tumours (e; n = 10 per group) were treated with vehicle or ABBV-075 using oral gavage at the indicated amounts for 21 days (PO, QDX21). Data are mean ± s.e.m. of tumour size for each treatment group. Source data
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Faivre, E.J., McDaniel, K.F., Albert, D.H. et al. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer. Nature 578, 306–310 (2020). https://doi.org/10.1038/s41586-020-1930-8
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