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Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer

Subjects

Abstract

Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. Progression to CRPC after androgen ablation therapy is predominantly driven by deregulated androgen receptor (AR) signalling1,2,3. Despite the success of recently approved therapies targeting AR signalling, such as abiraterone4,5,6 and second-generation anti-androgens including MDV3100 (also known as enzalutamide)7,8, durable responses are limited, presumably owing to acquired resistance. Recently, JQ1 and I-BET762 two selective small-molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit anti-proliferative effects in a range of malignancies9,10,11,12. Here we show that AR-signalling-competent human CRPC cell lines are preferentially sensitive to bromodomain and extraterminal (BET) inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ1 (refs 11, 13). Like the direct AR antagonist MDV3100, JQ1 disrupted AR recruitment to target gene loci. By contrast with MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR-mediated gene transcription, including induction of the TMPRSS2-ERG gene fusion and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft mouse models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.

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Figure 1: Prostate cancer cell lines with intact androgen signalling are sensitive to BET bromodomain inhibition.
Figure 2: Physical association of the N-terminal domain of AR with BRD4 and its disruption by BET bromodomain inhibition.
Figure 3: BET bromodomain inhibition disrupts AR and BRD4 binding to target loci.
Figure 4: BET bromodomain inhibition blocks CRPC in vivo.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Gene expression and ChIP-seq coordinates have been deposited in the Gene Expression Omnibus under accession number GSE55064.

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Acknowledgements

We thank A. Paliakov, T. Barrette, Y. Qiao, P. Vats, R. Stender, X. Jiang, M. Pranithi and S. Han for technical assistance; C. Kumar-Sinha, M. Dhanasekaran, N. Palanisamy and P. Kunju for helpful discussions; J. Athanikar and K. Giles for critically reading the manuscript and submission of documents. This work was supported by a Movember-Prostate Cancer Foundation Challenge Award and in part by the Early Detection Research Network (UO1 CA111275) and the NCI Prostate SPORE (P50CA69568) to A.M.C. A.M.C. is also supported by the Doris Duke Charitable Foundation, American Cancer Society, and A. Alfred-Taubman Institute. I.A.A. and R.A. are supported by a PCF Young Investigator Award.

Author information

Authors and Affiliations

Authors

Contributions

I.A.A. and A.M.C. conceived the study and the experiments. I.A.A. performed the experiments with assistance from V.L.D., R.M., S.D., C.E. and X.J.; X.W. carried out in vitro interaction studies; M.C., R.M. and I.A.A. analysed microarray data; J.E.-W., K.W.-R. and F.Y.F performed mouse xenograft studies; Y.-M.W. generated ChIP-seq libraries and X.C. performed the sequencing; M.C., R.Y., M.K.I. and Z.S.Q. performed ChIP-seq analysis with input from I.A.A.; S.W. provided compounds. I.A.A. and A.M.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Arul M. Chinnaiyan.

Ethics declarations

Competing interests

A.M.C. serves as an advisor to Hologic, Life Technologies and Ventana/Roche and has formally served as an advisor to GSK and Constellation Pharmaceuticals. A.M.C. and S.W. are co-founders of Oncofusion Therapeutics, which is developing novel BET bromodomain inhibitors.

Extended data figures and tables

Extended Data Figure 1 BET bromodomain inhibitor JQ1 blocks cell growth, induces apoptosis and transcriptionally suppresses anti-apoptotic factor BCL-xl without affecting BRD2/3/4 proteins.

a, Cell viability curves for the six prostate lines treated with JQ1. N = 6 wells of a 96-well plate per condition. b, BET bromodomain proteins are ubiquitously expressed in prostate cell lines. AR and MYC protein levels are also shown. GAPDH serves as a loading control. c, Knockdown of BET bromodomain proteins attenuates cell proliferation and invasion. qRT–PCR analyses of BRD2, BRD3 or BRD4 in VCaP cells transfected with short interfering RNA (siRNA) against their respective transcript or non-targeting (NT) siRNA. Data show mean ± s.e.m. (n = 3) from one of three independent experiments. d, VCaP- and LNCaP-cell proliferation after indicated gene knockdown. 20,000 cells were seeded in 24-well plates 24 h post-transfection with siRNAs and counted on day 0, 2, 4 and 6 (n = 3) by Coulter counter. Data show mean ± s.e.m. e, VCaP- and LNCaP-cell invasion (n = 6) after indicated gene knockdown. JQ1 was used at 500 nM. f, Cell cycle analysis of JQ1-treated prostate cell lines (after 48 h treatment with JQ1). Data represent three independent experiments. g, Induction of apoptosis as determined by appearance of cleaved PARP (cPARP) in VCaP prostate cancer cells by JQ1. GAPDH served as a loading control. h, Immunoblot demonstrating an increase in cPARP and decrease in BCL-xl in all three AR-positive cell lines compared with AR-negative PC3 cells upon JQ1 treatment. i, Relative BCL-xl mRNA levels as determined by TaqMan qPCR in JQ1-treated cells. Data show mean ± s.e.m. (n = 3) from one of three independent experiments. j, ChIP-seq data depicting loss of BRD2/3/4 recruitment to the BCL-xl promoter upon JQ1 treatment in VCaP cells. The genome browser representation of BRD2/3/4 binding events on the BCL-xl promoter region. The y-axis denotes reads per million per base pair (r.p.m. bp−1), the x-axis denotes the genomic position. The bottom panel depicts the H3K27ac mark on the same promoter region in VCaP cells. k, Colony formation assays of prostate cell lines. Cells were cultured in the presence or absence of 100 and 500 nM of JQ1 for 12 days followed by staining (top) and quantification (bottom; mean ± s.e.m. n = 6). Representative photographs of crystal violet stained colonies (except for VCaP) used for quantification are shown. l, BET bromodomain inhibitor JQ1 does not affect its target proteins. qRT–PCR analyses of BRD2, BRD3 and BRD4 in prostate cancer cell line panel treated with two different concentrations of JQ1 for 24 h. Data show mean ± s.e.m. (n = 3) from one of the three independent experiments. m, Immunoblot analysis of BRD proteins in prostate cell line panel treated with JQ1 for 48hrs. GAPDH serves as a loading control. Asterisks in b and m indicate non-specific band. Representative blots shown are from triplicate biological experiment. NS, not significant; *P ≤ 0.01; **P ≤ 0.001 by two-tailed Student’s t-test.

Extended Data Figure 2 Effect of JQ1 on AR target genes and on MYC transcription.

a, qRT–PCR analysis of indicated genes in LNCaP and 22RV1 cells treated with varying concentrations of JQ1 for 24 h. Data show mean ± s.e.m. (n = 3) from one of two independent experiments. b, Immunoblot analysis of AR and PSA in a panel of prostate cancer cells after treatment with two different doses of JQ1. GAPDH serves as a loading control. c, ERG and PSA are transcriptional targets of JQ1. Proteasome inhibitor bortezomib does not rescue ERG and PSA levels in JQ1-treated VCaP cells. Immunoblot analyses of ERG and PSA in VCaP and PSA in LNCaP cells treated with JQ1 followed by incubation with bortezomib as indicated. MYC, known to be degraded by proteasome, was used as a positive control for bortezomib treatment. GAPDH serves as a loading control. d, GSEA showing loss of MYC signature (four-gene set) in AR-positive VCaP, LNCaP and 22RV1 cells but not AR-negative DU145 cells after JQ1 treatment. Size, number of genes in each set; NES, normalized enrichment score. P and false discovery rate (FDR) q values indicate statistical significance. e, qRT–PCR and immunoblot analysis of MYC in JQ1-treated prostate cancer cells. Data show mean ± s.e.m. (n = 3) from one of two independent experiments. f, g, Time-course qRT–PCR and immunoblot analysis of MYC in AR-positive VCaP, LNCaP and 22RV1 cells after JQ1 treatment. h, Cyclohexamide (translation inhibitor) treatment does not enhance JQ1-mediated loss of MYC protein, ruling out post-translational degradation of MYC by JQ1. Time-course immunoblot analysis of MYC in VCaP, LNCaP and 22RV1 cells treated with cyclohexamide or cyclohexamide plus JQ1 as indicated. Representative blots from two independent experiments are shown. i, GAPDH-normalized MYC protein levels are shown. Band intensities from d were determined by ImageJ and the plots were generated using GraphPad Prism. j, MYC knockdown does not affect cell invasion. Box plot shows invasion of VCaP cells transfected with siNT or siMYC. Inset shows the image of invaded VCaP cells (n = 6). Right, qRT–PCR of MYC upon siRNA transfection. Data show mean ± s.e.m. from one of three independent experiments. k, Exogenous MYC introduction does not rescue JQ1-mediated cell growth inhibition. Cells were infected with control adeno-LacZ or adeno-MYC virus. Equal numbers of cells were plated 24 h after infection and treated with 500 nM JQ1 or I-BET762. Cells were counted (n = 3 wells) and plotted; day 0 of drug treatment was set at 100%. Data show mean ± s.e.m. from one of four independent experiments. l, Immunblot analysis depicts overexpression of MYC in adeno-MYC infected cells on day 0 and day 7 of the experiment. GAPDH serves as a loading control. *P ≤ 0.05; **P ≤ 0.005 by two-tailed Student’s t-test.

Extended Data Figure 3 Physical association of AR with BRD4 and its disruption by BET bromodomain inhibitor.

a, LNCaP nuclear extract was fractionated on a Superose-6 column and AR, BRD4 and RNA Pol II were analysed by immunoblot analysis. b, c, Representative sensorgrams for AR–RNF2, RAS–BRD4(BD1–BD2) and RNF2–BRD4(BD1–BD2) interactions by an OctetRED biolayer interferometry. Real-time binding was measured by immobilizing biotinylated AR, RAS or RNF2 proteins separately on a streptavidin biosensor and subsequent interaction with varying concentrations of analyte proteins (RNF2 or BRD4(BD1–BD2)) individually. Immobilized RAS or RNF2 biosensors did not bind with BRD4, indicating that the AR–BRD4 interaction is specific. Representative sensorgrams from 4–6 independent experiment are shown. d– f, In vitro binding analysis of AR and indicated domains of BRD4. Equal amounts of in vitro translated full-length Halo–AR protein and GST–BRD4 domains were combined and immunoprecipitated using Halo beads followed by immunoblot analysis with anti-GST antibody. g, JQ1 disrupts the endogenous AR–BRD4 interaction. VCaP cells were treated with JQ1 for 6 h followed by immunoprecipitation and immunoblot analysis as in Fig. 2b.

Extended Data Figure 4 Changes in genome-wide enrichment profiles of BRD proteins in response to bromodomain inhibitors.

a, Table showing high-throughput sequencing read information for ChIP libraries of BRD2, BRD3, BRD4, AR, RNA Pol II, ERG, H3K27ac and IgG performed for this study. b, ChIP-seq was performed using BRD2, BRD3 and BRD4 antibodies in VCaP cells treated with DMSO, JQ1 or I-BET762 for 12 h. Genome-wide distribution of BRD2, BRD3 and BRD4 enriched sites. Highly significant peaks (see Methods) show relatively high overlap. A large majority of sites are occupied by at least two BRD proteins. BRD2 and BRD3 have the most similar localization pattern. c, BRD proteins show varying degrees of overlap. Shown is the ratio of sites occupied by either protein alone (unique) or co-occupied with another BRD-family protein (overlap). BRD4 shows the largest number of unique peaks. d, BET inhibitors JQ1 and I-BET762 attenuate recruitment of BRD proteins from chromatin. Enrichment levels for each protein were normalized to the median enrichment in vehicle-treated cells. BRD2 and BRD3 proteins show similar responses to both inhibitors, whereas BRD4 is more potently evicted by JQ1. e, BET bromodomain inhibitors deplete target proteins from genomic regions with or without AR. Mean enrichment levels within each subpanel were normalized to the maximum mean enrichment in vehicle-treated cells.

Extended Data Figure 5 Influence of JQ1 and anti-androgens on genome-wide recruitment of AR and their effect on DHT-induced AR target gene expression.

a, Two independent biological replicates of AR ChIP-seq experiments in VCaP cells show high correlation of normalized enrichment levels (see Methods) in the majority of treatment conditions. R2 values for each biological duplicate are shown. b, Mean enrichment (coverage) profiles are similar between biological replicates and different between treatment conditions, indicating that no adverse changes in enrichment levels are observed between the replicates. c, Bar graph showing total number of AR peaks for VCaP-treated cells. The genome-wide individual peaks for AR yielded the highest number of peaks for DHT (35,390) whereas vehicle control cells showed only 13,874 peaks. However, the number of peaks for AR was 23,961, 18,264 and 32,212 in the presence of JQ1, MDV3100 and bicalutamide, respectively. d, Heat map representation of AR binding peaks in different treatment groups. Genomic target regions are rank-ordered based on the level of AR enrichment at each androgen response elements (ARE) within −1 kb and +1 kb flanking the genomic region. e, Venn diagram illustrating the overlap of AR-bound genes between different treatment groups. f, AR–BRD4 binding on KLK3 and FASN upstream regions. Genome browser representation of AR and BRD4 binding events on a putative enhancer and super-enhancer of AR-regulated KLK3 and FASN gene, respectively. The y-axis denotes reads per million per base pair (r.p.m. bp−1), the x-axis denotes the genomic position with a scale bar on top right. g, Expression of AR target genes in the presence of JQ1, MDV3100 or bicalutamide. Heat maps for VCaP and LNCaP cells treated with DHT (10 nM), DHT plus JQ1 (0.5 µM), DHT plus MDV3100 (10 µM) and DHT plus bicalutamide (25 µM). Red arrows indicate well-characterized AR target genes. h, qRT–PCR analysis of AR-regulated genes in the VCaP and LNCaP treated cells. To directly compare JQ1 and MDV3100 in blocking AR signalling, cells were treated with varying concentrations of JQ1 or MDV3100 followed by DHT treatment and analysed for AR targets. The reduction in DHT-induced gene expression was observed for JQ1 even at 100–250 nM whereas MDV3100 showed a marginal reduction at 10 µM, demonstrating the higher efficacy of JQ1 in blocking AR target gene expression. Data show mean ± s.e.m. (n = 3) from one of two independent experiments.

Extended Data Figure 6 Effect of JQ1 on the TMPRSS2-ERG loci and ERG-mediated transcription in VCaP cells.

a, Genome browser representation of RNA Pol II binding events within the ERG gene body. The y-axis denotes reads per million mapped reads per base pair (r.p.m. bp−1), the x-axis denotes the genomic position and the black arrow indicates the region involved in the TMPRSS2-ERG fusion. b, As in a, AR and BRD4 binding on the promoter of the ERG 5′-fusion partner TMPRSS2 in VCaP cells. Note the reduced RNA Pol II and AR–BRD4 recruitment levels in DHT plus JQ1 tracks for the ERG gene body and TMPRSS2 promoter respectively. c, High reproducibility of ERG ChIP-seq experiments. Biological replicates of ERG ChIP-seq experiments show very high correlation of normalized enrichment levels (see Methods) in the JQ1- and DMSO-treated conditions. d, Significant changes in ERG levels upon JQ1 treatment at ERG-binding sites in the proximity of gene loci. Changes in ERG enrichment levels were assessed using DESeq2. Statistically significant differences were observed for ERG gain and ERG loss. Significant ERG gains are associated with quantitatively modest changes in enrichment level. On the other hand, significant ERG losses are associated with greater changes in enrichment levels. Individual number of peaks for each panel is shown. e, Genome browser representation of ERG-binding events on bona fide ERG-activating target genes. The y-axis denotes reads per million per base pair (r.p.m. bp−1), the x-axis denotes the genomic position. f, Genome browser representation of ERG-binding events on ERG-repressed target genes. g, TaqMan qRT–PCR analysis of ERG-activated genes in VCaP cells after JQ1 treatment. h, TaqMan QRT–PCR analysis of ERG-repressed genes in VCaP cells after JQ1 treatment. Data represent mean ± standard deviation (s.d.) (n = 3) from one of two independent experiments. *P ≤ 0.05; **P ≤ 0.005, ***P ≤ 0.0005 by two-tailed Student’s t-test.

Extended Data Figure 7 BET bromodomain inhibitors reverse ERG-mediated functions in an isogenic cell line system.

a, b, qRT–PCR and immunoblot showing overexpression of ERG in RWPE and PC3 prostate cell lines. Data represent mean ± s.e.m. (n = 3). c, BET inhibitors block ERG-induced RWPE and PC3 cell invasion. RWPE and PC3 cells stably expressing either LacZ or ERG were treated with DMSO (n = 4), 500 nM JQ1 (n = 4) or I-BET762 (n = 4) for 24 h before plating in Matrigel-coated Boyden chambers. After 48 h cell invasion was quantified. Left, representative photomicrographs of invaded cells are shown with a 100 μm scale bar (lower Boyden chamber stained with crystal violet). Right, bar graph shows fold change in cell invasion, with DMSO-treated LacZ-expressing cells set to 1. Data represent mean ± s.e.m. from one of three independent experiments. d, BET inhibitors reverse ERG-induced gene transcription. GSEA of the ERG target gene signature (see Methods) in RWPE cells overexpressing ERG (RWPE-ERG) and PC3-ERG cells treated with JQ1 or I-BET762 (500 nM) for 24 h. ERG-induced genes are repressed by JQ1 or I-BET762 treatment. e, GSEA using a random gene set shows no significant positive or negative enrichment by JQ1 or I-BET762 treatment in RWPE-ERG and PC3-ERG cells. NS, not significant; ***P ≤ 0.0001 by two-tailed Student’s t-test.

Extended Data Figure 8 JQ1 inhibits ETS (ERG/ETV1) factors that regulate MYC expression in VCaP and LNCaP cells.

a, Genome browser representation of ERG- and ETV1-binding events on the MYC distal enhancer42. JQ1 treatment in VCaP cells reduces ERG enrichment, as shown in two independent ERG ChIP-seq experiments. The y axis denotes reads per million per base pair (r.p.m. bp−1), the x axis denotes the genomic position. LNCaP ETV1 ChIP-seq data are based on data from ref. 23 (GEO accession code GSM1145322), and show ETV1 recruitment to the MYC distal enhancer. b, ChIP-PCR validation of loss of ERG recruitment after JQ1 treatment in VCaP cells. Data show mean ± s.d. (n = 3) from one of two independent experiments. c, d, Knock-down of AR or ETS factor reduces MYC gene expression in VCaP and LNCaP cells. qRT–PCR for AR, ETS and MYC expression in siNT, siAR or siETS transfected cells. Data show mean ± s.d. (n = 3) from one of two independent experiments. e, A cartoon illustrating the mechanism of MYC loss by JQ1 in AR-positive VCaP and LNCaP cells. f, Anti-androgens but not JQ1 de-repress MYC expression in prostate cancer cells. Genome browser representation of AR and RNA Pol II binding events within the MYC gene locus. The y axis denotes reads per million per base pair (r.p.m. bp−1), the x axis denotes the genomic position. Note the AR recruitment to the same distal enhancer that is occupied by ERG (see Extended Data Fig. 8a), indicating that there is competition between the AR and ETS factors to bind to this enhancer region to regulate MYC gene expression. g, Heat map showing MYC expression values from VCaP microarray gene expression data. h, Anti-androgen restores DHT-repressed MYC expression in VCaP cells. qRT–PCR of MYC in VCaP cells treated with vehicle, DHT (10 nM), DHT plus JQ1 (500 nM), DHT plus MDV3100 (10 µM) or DHT plus bicalutamide (25 µM). Inability of JQ1 to de-repress MYC in this setting could be explained by the fact that both AR and ERG are de-recruited from the MYC distal enhancer, leading to net loss of MYC expression. i, MDV3100 and not JQ1 restores DHT-repressed MYC protein levels in VCaP cells. Immunoblot of MYC protein in VCaP cells pre-treated with vehicle, MDV3100 (10 µM) or JQ1 (500 nM) for 4 h followed by DHT (10 nM) for 20 h. Data show mean ± s.d. (n = 3) from one of two independent experiments. NS, not significant; *P ≤ 0.01; **P ≤ 0.001; **P ≤ 0.0001 by two-tailed Student’s t-test.

Extended Data Figure 9 JQ1 does not affect normal prostate growth and testosterone levels but reduces testis size in mice.

a, Comparison of JQ1 and MDV3100 treatment on VCaP cell viability in vitro. N = 8 wells of a 96-well plate per condition. VCaP cells were treated with MDV3100 or JQ1 for 8 days and assayed for viability with Cell-titerGLO. b, Gross images showing highly hormone-responsive seminal vesicles attached to prostate gland (red and black arrows, respectively) from male mice treated for 30 days with vehicle, JQ1(50 mg kg−1) or MDV3100 (10 mg kg−1). Vehicle or JQ1-treated mice show no change in the appearance of seminal vesicles. By contrast, MDV3100-treated animals show remarkable shrinkage of seminal vesicles. c, Mice treated with JQ1 do not show any adverse changes to anterior or ventral prostate morphology. The haematoxylin and eosin images show normal morphology of anterior and ventral prostate from vehicle- or JQ1-treated mice. MDV3100-treated mice show attenuated remnant glands of anterior or ventral prostate. d, Male mice (n = 3 per group) treated with vehicle or JQ1 for 30 days exhibit similar serum testosterone levels. Data represent the mean ± s.e.m. e, Gross analysis of testis from mice treated with vehicle or JQ1 for 30 days. f, Testis weight from vehicle control or JQ1-treated mice. Data represent the mean ± s.e.m. from n = 7 mice per group. NS, not significant; *P ≤ 0.0001 by two-tailed Student’s t-test.

Extended Data Figure 10 In vivo effects of BET bromodomain inhibition in VCaP xenograft model.

a, VCaP cells were implanted subcutaneously in mice and grown until tumours reached a size of approximately 100 mm3. Xenografted mice were randomized and then received vehicle, 50 mg kg−1 JQ1 or 10 mg kg−1 MDV3100 5 days a week as indicated. Calliper measurements were taken twice a week. Individual tumour volumes from different treatment groups at the end of the experiments with P values are shown. b, MDV3100 treatment leads to spontaneous metastasis. Mice bearing VCaP xenografts (subcutaneously engrafted) treated with vehicle (n = 6) or MDV3100 (n = 6) were assessed for spontaneous metastasis to the femur (bone marrow) and soft tissues such as liver and spleen. Genomic DNA isolated from these sites was analysed for metastasized cells by measuring human Alu sequence (by Alu-qPCR). MDV3100-treated mice showed spontaneous metastasis to femur and liver. Spleen did not show presence of human ALU sequences. c, As in a, for mice bearing VCaP xenografts treated with vehicle (n = 6), JQ1 (n = 6) or MDV3100 (n = 6). MDV3100-treated but not JQ1-treated mice showed metastasis to femur and liver. d, JQ1 or MDV3100 treatment does not affect animal weight. Mice from VCaP cell xenograft experiments treated with vehicle, 10 mg kg−1 MDV3100 or 50 mg kg−1 JQ1 were weighed at the time of calliper measurements. e, Individual tumour volume for vehicle- or JQ1-treated VCaP mouse xenograft (for data shown in Fig. 4c). Mean ± s.e.m. is plotted. Statistical significance was determined by two-tailed Student’s t-test.

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Asangani, I., Dommeti, V., Wang, X. et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 510, 278–282 (2014). https://doi.org/10.1038/nature13229

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