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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: Mol Cancer Ther. 2017 Aug 3;16(10):2281–2291. doi: 10.1158/1535-7163.MCT-17-0259

Bypassing drug-resistance mechanisms of prostate cancer with small-molecules that target androgen receptor chromatin interactions

Kush Dalal 1, Meixia Che 2, Nanette S Que 3, Aishwariya Sharma 1, Rendong Yang 4, Nada Lallous 1, Hendrik Borgmann 1, Deniz Ozistanbullu 1, Ronnie Tse 1, Fuqiang Ban 1, Huifang Li 1, Kevin J Tam, Mani Roshan-Moniri 1, Eric LeBlanc 1, Martin E Gleave 1, Daniel T Gewirth 3, Scott M Dehm 2, Artem Cherkasov 1, Paul S Rennie 1
PMCID: PMC5628141  NIHMSID: NIHMS894380  PMID: 28775145

Abstract

Human androgen receptor (AR) is a hormone-activated transcription factor that is an important drug-target in the treatment of prostate cancer. Current small molecule AR-antagonists, such as enzalutamide, compete with androgens that bind to the steroid binding pocket of the AR ligand binding domain (LBD). In castration-resistant prostate cancer (CRPC), drug-resistance can manifest through AR-LBD mutations that convert AR-antagonists into agonists, or by expression of AR-variants lacking the LBD. Such treatment resistance underscores the importance of novel ways of targeting the AR. Previously, we reported the development a series of small molecules that were rationally designed to selectively target the AR DNA binding domain (DBD) and, hence, to directly interfere with AR-DNA interactions. In the current work we have confirmed that the lead AR DBD inhibitor indeed directly interacts with the AR-DBD and tested that substance across multiple clinically relevant CRPC cell lines. We have also performed a series of experiments that revealed that genome-wide chromatin binding of AR was dramatically impacted by the lead compound (although with lesser effect on AR variants). Collectively, these observations confirm the novel mechanism of anti-androgen action of the developed AR-DBD inhibitors, establishing proof-of-principle for targeting DNA-binding domains of nuclear receptors in endocrine cancers.

Introduction

In advanced prostate cancer (PCa), the main therapeutic target is the androgen receptor (AR), a transcription factor that regulates expression of genes required for tumor growth (1,2). The AR shares a domain organization with other members of the nuclear receptor (NR) family, consisting of an N-terminal domain (NTD), a central DNA-binding domain (DBD) and C-terminal ligand-binding domain (LBD) (3). Binding of endogenous androgens, such as testosterone or dihydrotestosterone (DHT), to the LBD promotes AR nuclear localization, dimerization via the DBD, and engagement with androgen response elements (AREs) located in enhancers of target genes (1,2). Antiandrogens in current clinical use, including enzalutamide, block AR activation by functioning as competitive antagonists of the ligand binding pocket (LBP) on the LBD (1,4-5). Drug-resistance can occur when mutations in the LBP render these molecules ineffective, when cells begin synthesizing constitutively active variant forms of the AR that lack the LBD such as AR-V7 (6-9), and/or when cells display AR gene amplification and overexpression of AR protein (10). This drug-resistant state, termed castration-resistant PCa (CRPC) is responsible for nearly all prostate cancer-specific deaths.

Although various cell signaling pathways and AR-associated co-factors also influence CRPC progression, their value as targets for PCa treatment is still unclear. On the other hand, it is clear that the AR remains a master regulator of genes required for tumor growth in CRPC. Accordingly, the AR represents the most direct target to explore inhibition with small molecules that attack molecular functions beyond steroid binding at the LBP. For example, the AR-NTD targeted inhibitor EPI-506 was derived by high throughput screening (11) and is now being tested in clinical trials. Despite the clinical potential of EPI-506, its drug-design is challenged by intrinsic disorder in the AR-NTD (2), which cannot be analyzed by X-ray crystallography. Hence, rational drug-design is only possible for the AR-DBD and -LBD domains where crystallographic information is available (1,12,13). In this regard, drug-development efforts at the AR-LBD have been focused on interfering with hormone binding (4, 14) or blocking co-factor recruitment sites such as the Binding-Function 3 (BF3) surface exposed pocket (15-17).

The AR-DBD is composed of two zinc fingers, the first of which contains the P-box recognition helix responsible for ARE specificity and the second which contains the D-box site responsible for dimerization (13). In previous studies, we analyzed the crystal structure of the AR-DBD:DNA complex to identify a surface exposed pocket near the protein-DNA interface that could be targeted with small molecules (18,19). The lead compound, VPC-14449, potently inhibited full-length AR in LNCaP cells, down-regulated PSA expression and suppressed growth of xenografts in mouse models (19). However, the mechanism of action of VPC-14449 has not been fully defined. To this end, the purpose of this study was to investigate VPC-14449 treatment in diverse CRPC cell lines exhibiting clinically-relevant drug-resistance mechanisms including expression of mutated AR or constitutively active AR variants lacking the LBD. VPC-14449 suppressed cell viability, AR transcriptional activity and target-gene expression in every model system tested. Mechanistically, VPC-14449 reduced the ability of full-length AR as well as AR variants to interact with chromatin, although the effects on full-length AR were more pronounced. Collectively, these findings highlight the potential for AR DBD-specific inhibition to overcome drug-resistance in CRPC.

Materials and Methods

Compounds

VPC-14449 (4-(4-(2,4-bromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine) was synthesized by Life Chemicals (Niagra, ON). A correction to the published structure of VPC-14449 was made in 2017 (20,21). Pyrvinium pamoate was purchased from Enamine (Monmouth Jct., NJ). Enzalutamide was obtained from Haoyuan Chemexpress (Shanghai, China). R881 synthetic androgen was obtained from Sigma.

Plasmid and constructs

Full-length human AR (hARWT) was encoded on a pcDNA3.1 expression plasmid. Mutations in the AR-LBD or -DBD in hARWT of were generated using a QuikChange kit (Agilent). PIPE cloning (19) was used to replace the AR-DBD with the rat glucocorticoid receptor (GR) DBD, yielding ARw/GRDBD as outlined in Table S1. A plasmid encoding the GST-tagged rat C552A AR-DBD mutant was previously described (13).

Luciferase, cell viability and secreted PSA

All PCa cell-lines cells were obtained in 2013, authenticated by IDEXX Laboratories in August 2014 and tested weekly for mycoplasma contamination. LNCaP (ATCC, CRL-1740), C4-2 (Provided by Dr. C.J. Ong), and MR49F/MR49C (Provided by Dr. A. Zoubeidi, 22) were androgen starved for 2 days in RPMI 1640 media supplemented with 5% charcoal stripped serum (RPMI + CSS) prior to transfection. 22Rv1 cells (ATCC, CRL-2505) were maintained in RPMI + 10% CSS. Cells were seeded at 5000 cells/well (96-well, 24 hrs) and transfected with 50 ng/well ARR3tk-Luc plasmid, 2 ng/well SV40 Renilla-luc and 0.15 μL/well TransIT20/20 reagent (TT20, Mirus) for 48 hrs. Cells were treated with compounds (0.1% DMSO vehicle final) and 0.1 nM R1881 androgen (22Rv1 = no R1881) for 24 hrs, followed by cell lysis and luminescence measurement as described (19). PC-3 cells (ATCC, CRL-1435) were transfected with hARWT, mutant, or ARw/GRDBD chimera plasmids (50 ng), ARR3tk-Luc (50 ng) and TT20 (0.3 μL/well) as described above. Lysates (40 μL) from luciferase assays were separated by 10% SDS-PAGE and probed with polyclonal AR-N20 AR-NTD specific (Santa Cruz) antibody, polyclonal anti-actin antibody (Sigma), and polyclonal anti-PARP/anti-cleaved PARP antibody (Sigma). Cell viability tests were performed by culturing cells as described above (96 well plate, 5000 cell/well), treating with compounds/R1881 for 72 hrs and incubating with 30 μL/well Promega CellTiter 96® AQueous Cell Proliferation MTS Assay (1 hr) followed by ABS492nm measurement. Secreted PSA measurements from LNCaP cells were performed as described (19).

Protein Purification

Rat AR-DBD (550-637) C552A was expressed and purified as described (13) with the addition of a gel filtration step carried out on a Superdex S75 column in a modified PBS buffer (pH 7.0) containing 200 mM NaCl and 1 mM DTT (PBS200D). Eluted protein was concentrated using a spin filter (MWCO 3,000) and stored at -80°C. The C552A mutant is less prone to aggregation at high concentrations and is fully functional in binding DNA.

Isothermal Titration Calorimetry

Titrations were performed with a VP-ITC calorimeter (Microcal, Inc) at 25°C and analyzed with Origin 7 software. To maximize the weak signal resulting from VPC-14449-protein binding, several buffers were tested, with the optimal ITC buffer determined to be PBS (pH 7.0), 200 mM NaCl. Prior to titration, reducing agents were removed from the AR-DBD using a Superdex S-75 column equilibrated in ITC buffer. Titrations against VPC-14449 were carried out in ITC buffer supplemented with DMSO (2% v/v final) in both protein and ligand solutions. AR-DBD (1.0 mM) was loaded into the titration syringe and injected into VPC-14449 (0.1 mM) that was loaded into the cell. 10 μL aliquots (5 min intervals) were injected, discarding heat from the first injection during analysis. The measured heat of dilution with protein (1 mM) titrated into ITC buffer was subtracted from the titration of AR-DBD into VPC-I4449 to yield the ligand binding isotherm. Titration of the AR-DBD against a synthetic AR response element was performed (40 mM HEPES pH 7.4, 100 mM NaCl) using a DNA duplex containing the two AGAACA half-sites arranged as direct repeats (top strand: 5′-TTC AGAACA TCA AGAACC A, hexameric half-sites underlined).

RT-PCR experiments

LNCaP and C4-2 cells were androgen starved (48 hr, RMPI + CSS) before 24 h treatment with DMSO (0.1%), DMSO + 1 nM R1881, 5 μM enzalutamide or 5 μM VPC-14449. MR49F cells were cultured in RPMI 1640 media containing 5% FBS and treated with DMSO (0.1%), 5 μM enzalutamide or 5 μM VPC-14449. 22Rv1 cells were maintained in RPMI + 10% CSS and treated with 50 μM enzalutamide or VPC-14449 (48 hrs) or DMSO control. R1-AD1 and R1-D567 isogenic cell lines expressing full-length AR or ARv567es (23), respectively, were starved as described above, followed by stimulation with 1 nM R1881 (R1-AD1), or without androgen (R1-D567), and compound treatment (24 hrs R1-AD1; 48 hrs R1-D567). RNA was extracted from each treatment group using TRIzol (ThermoFisher) and used for cDNA synthesis (Superscript II, Invitrogen). RT-PCR (125 ng cDNA, 5 μM primers, Roche FastStart Sybr green) was performed on an ABI ViiA7 thermocycler (GAPDH control). RT-PCR primers are shown in Table S1.

Chromatin fractionation

Fraction of ∼1-2 × 107 of cells from 15 cm dishes were performed as previously described (24). Each cell line was cultured the same way as for RT-PCR experiments (androgen starvation) except that VPC-14449 was administered for 24 hrs at various concentrations followed by 4 hrs treatment with DHT (1 nM) prior to biochemical fractionation of cytosol, nuclear and chromatin fractions. Western blots for AR detection were performed with anti-AR-N20 (Santa Cruz, ARN20) or anti-ARV7 (Precison, AG10008) specific antibodies. Equal loading of samples and confirmation of successful subcellular fractionation was performed with a monoclonal antibody against cytosol α-Tubulin (Cell Signaling Technology, DM1A), a monoclonal antibody against nuclear Lamin A/C (Cell signaling Technology, 4C11), and a monoclonal antibody directed against histone-H3 protein (abcam, ab32356).

ChIP-PCR and ChIP-seq

Chromatin immunoprecipitation (ChIP) was performed for 3 independent biological replicate experiments as described (25) with the exception that: R1-AD1 and R1-D567 cells were seeded at 5 × 106 cells/10 cm plate in RPMI 1640, settled for 48 hrs, then treated with vehicle (0.1% DMSO) or VPC14449 (50 μM) in RPMI 1640 for 24 hrs. Cells were re-fed with RPMI 1640 + 10% CSS supplemented with 50 μM VPC-14449 (or DMSO control) and 1 nM DHT (or ethanol control) for an additional 4 hrs prior to cross-linking. Nuclear pellets were sonicated on ice for ten cycles at 45% amplitude (10 s on/10 s off pulse for 1 min, 2 min rest) using a 450 Sonifier (Branson). Lysates were immunoprecipitated with AR-N20 (Santa Cruz) and Protein A/G Plus agarose beads pre-blocked with Salmon sperm DNA (Millipore). DNA was purified by PCR purification (Qiagen). Samples for ChIP-seq were processed exactly as samples for ChIP-PCR with the exception that blocking of Protein A/G beads was performed with tRNA (Sigma). Independent biological replicate experiments were performed for ChIP-seq in R1-AD1 (n=2) and R1-D567 (n=3) cells. 2 ng of DNA (ChIP-enriched and input) was used for library creation with a Truplex ChIP sample Preprarion Kit (Rubicon Genomics). ChIP-seq libraries were sequenced at the U of M Genomics Center using an Illumina HiSeq2000 with 1×50 bp settings. Data sets were deposited into the GEO database (accession number = GSE96084).

Analysis of ChIP-seq data

ChIP-seq reads were mapped to human genome version hg19 using BWA v0.7.10 (26) with default parameters. Reads with mapping quality <1 were discarded. ChIP-seq peak calling was performed using MACS v2 (27) with merged BAM files from two biological replicates of DHT-treated R1-AD1 cells immunoprecipitated with AR-N20 and input DNA from R1-AD1 cells as negative control. Default parameters were used with FDR cutoff of 0.01, yielding 3,556 AR binding sites. ChIP-seq heatmaps representing peak-centered AR or AR-V binding at these 3,556 AR binding sites was generated using a python-based script on raw reads from merged duplicate files from R1-AD1 cells and merged triplicate files from R1-D567 cells and visualized +/- 3kbp. Histograms of tag density around 3,556 called AR binding sites called in DHT-treated R1-AD1 cells were centered by peak summit and logarithm normalized average coverage in units of number of reads per million sequenced reads was counted within 3kbp relative to the peak center. Visualization of mapped ChIP-seq reads in BAM format was performed at the gene-track level using Integrated Genomics Viewer (IGV 2.3, Broad Institute, 28) with data merged from biological replicates. Data were normalized as reads per million mapped reads.

Results

VPC-14449 inhibits AR activity in androgen insensitive and enzalutamide resistant cell lines

We previously characterized novel AR-DBD specific compounds (VPC-14228/VPC-14449) predicted to bind near the P-box recognition helix (18). We also demonstrated that the corresponding inhibitory activity by both compounds was selective for the AR over other NRs, and that specific point-mutations in the AR-DBD rendered the molecules less effective (18,19). While these findings provided evidence for an on-target interaction, the compound-effects were mostly explored with transiently expressed AR, and down-stream effects were only tested in the androgen-dependent LNCaP cell line (19).

To assess the activity of the lead inhibitor (VPC-14449) in models of clinical CRPC, AR transactivation and cell viability experiments were performed in LNCaP, C4-2, MR49F, and 22Rv1 cell lines, which were chosen to explore the ability of VPC-14449 to bypass drug-resistance conferred by LBP mutations (hydroxyflutamide-resistant T877A in LNCaP/C4-2; enzalutamide-resistant F876L in MR49F, 22) or via expression of AR variants lacking the LBD (AR-V7 in 22Rv1). Luciferase reporter assays showed that endogenous AR transactivation was inhibited by VPC-14449 in LNCaP, C4-2 and MR49F cells stimulated with the synthetic androgen R1881 (sub μM IC50, Fig. 1A), with less potency against AR-V activity in 22Rv1 (no R1881, low μM IC50, Fig. 1A). In addition, high levels of enzalutamide increased reporter activity in MR49F cells, but had no effect in 22Rv1 cells (Fig. 1A, grey). Western blot analysis confirmed that AR protein levels were not affected by the compound (Fig. 1A, bottom) and toxic effects of VPC-14449 over 24 hours treatment were ruled out by the lack of PARP cleavage in all cell types, in contrast to a strong signal for PARP cleavage in cells treated with an alternative AR-DBD inhibitor, pyrvinium pamoate (Fig. 1A, bottom, 29). Cell viability assays demonstrated that VPC-14449 suppressed growth in every tested cell line while enzalutamide resistance was apparent in both MR49F and 22Rv1 cells (Fig. 1B). Similarly, VPC-14449 inhibited AR transactivation and cell viability in a second enzalutamide resistant cell line (MR49C, F876L mutation, 30) (Fig. S1). Importantly, the viability of AR-negative PC-3 cells was not affected by any of the compounds, indicating the specific action of enzalutamide or VPC-14449 on the AR signalling pathway (Fig. S1). This data demonstrates that VPC-14449 can inhibit AR signalling across five CRPC cell lines each reflecting resistance to therapies targeting the AR-LBD.

Figure 1. VPC-14449 inhibits AR-transcriptional activity and cell viability in drug-resistant PCa cell lines.

Figure 1

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(A) Top - Cells (RPMI+CSS) were co-transfected with ARR3tk-Luc and Renilla-Luc plasmids (48 hrs). With the exception of 22Rv1, 0.1 nM R1881 was used to stimulate the AR, followed by compound treatment (24 hrs). Error bars = mean ± SEM of 4 replicates normalized to constitutive Renilla expression. 100% refers to normalized luminescence without compound (DMSO vehicle). Curves were fitted to a sigmoid dose-response with variable slope equation (GraphPad). Bottom - Western blots of cell lysates (AR-N20/Actin antibodies) following 24 hrs compound-treatment or DMSO (Pyr = pyrvinium pamoate). Lysates were probed with anti-PARP/cleaved PARP antibodies as a measure of apoptosis. (B) Cell viability was measured in cells cultured as in A following 72 hrs compound-treatment with 1 nM R1881 (except 22Rv1 = no R1881). Error bars = mean ± SEM of 4 replicates per point. 100% = viability (cell-growth) over 72 hrs stimulated by R1881 without compound (DMSO vehicle: LNCaP, C4-2, MR49F), or with DMSO alone (22rv1). 0% = viability without R1881 treatment (LNCaP, C4-2, MR49F), or at t=0 hrs (22rv1).

Binding of VPC-14449 analyzed by mutagenesis and direct interaction with the AR-DBD

We originally proposed that the VPC-14449-DBD interaction depends on Q592 and Y594 side-chains (19), but did not explore proximal residues predicted to also facilitate binding. Thus, to identify other key residues, we mutated K591-L595 (KQKYL) of the AR and performed reporter assays in PC-3 cells. Three point mutants - Q592D, K593D and Y594D - weakened inhibition of the full-length AR (1 μM VPC-14449), revealing the central lysine as another critical side-chain for ligand recruitment (Fig. 2A). In addition, simultaneous mutations to the binding site were introduced with a chimera construct in which the AR-DBD was replaced with the glucocorticoid receptor DBD, where the KQKYL sequence is replaced by QHNYL. Reporter assays with over-expressed AR showed that the chimera remained R1881-inducible and sensitive to enzalutamide inhibition, but resisted VPC-14449 inhibition compared to the wild type AR (Fig. 2B).

Figure 2. Specificity of VPC-14449 for the AR-DBD.

Figure 2

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(A) PC-3 cells were transfected with the wild type/mutant AR and ARR3tk-luc as in Fig. 1A. Error bars = mean ± SEM of 6 replicates per point. *=p-value < 0.05; **=p-value < 0.01. (B) PC-3 cells were co-transfected with wild-type or ARw/GRDBD receptor plasmids and ARR3tk-Luc as in Fig. 1A. 100% = luminescence recorded without compounds (DMSO vehicle). (C) Isothermal titration calorimetry of AR-DBD against VPC-14449 (dotted lines) is overlaid with the titration of the protein into buffer (heavy lines). (Top) The raw ITC data expressed as the change in thermal power with respect to time over the titration period. (Bottom) The lower panel shows the integrated heats of AR-DBD against VPC-14449 (open squares) and protein into buffer (filled circles). The AR-DBD C552A protein (1 mM) was in the syringe and the inhibitor VPC-14449 (100 μM) was in the cell.

To directly assess VPC-14449 binding to the AR DBD, we employed Isothermal Titration Calorimetry (ITC) with the recombinant rat AR DBD. Due to the high protein concentration used in the titrations, heats of dilution from the protein was significant, therefore, this value was subtracted from the binding isotherm in subsequent analyses. Titrating 1 mM AR-DBD into 0.1 mM VPC-14449 indicated a modest interaction between protein and compound, with a calculated binding constant (KD) of ∼64 μM and N-value of 0.53 (Fig. 2C). Control experiments measuring the affinity between the AR-DBD and an oligonucleotide harboring an ARE revealed a strong interaction (56.8 nM), demonstrating that the protein was properly folded and competent for DNA binding (Fig. S2).

VPC-14449 circumvents clinically relevant mutations and can be co-administered with enzalutamide

Given that VPC-14449 inhibited T877A and F876L AR mutations (Fig. 1A), we hypothesized that VPC-14449 would bypass other clinically-relevant mutations in the AR-LBD. To test this, we considered a set of mutations (L701H, W741C, W741L, H874Y) that are induced by anti-androgen treatment based on their frequent detection in tissues and circulation of CRPC patients, but are absent in patients with localized treatment-naïve PCa (31-33). As patient derived cell lines harboring these mutations were not available, we performed luciferase reporter assays with transient over-expression of wild-type or mutant AR. Consistent with the DBD as the site of action, the transcriptional activity of every tested AR-LBD mutant was inhibited, to various degree, by VPC-14449 (Fig. 3A).

Figure 3. VPC-14449 bypasses AR-LBD mutations and exhibits additive inhibition with enzalutamide.

Figure 3

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(A) Transfection of PC-3 cells with the wild type or AR-LBD mutants and ARR3tk-luc, and luciferase reporter assay, were performed as in Fig. 2A. (B) Cell viability experiments (Left) with LNCaP cells were performed as in Fig. 1B but with IC25 (75% Activation) levels for enzalutamide (130 nM) and VPC-14449 (665 nM) either individually or in combination. Similarly, secreted PSA (Right) from LNCaP were measured following treatment with IC25 values of enzalutamide (40 nM) and VPC-14449 (140 nM). Error bars = mean ± SD of 4 replicates per point.

Because VPC-14449 and enzalutamide target distinct sites on the AR, we next tested for possible combinatorial effects. Cell viability and secreted PSA measurements in LNCaP cells simultaneously treated with doses of enzalutamide and VPC-14449 corresponding with their respective IC25 values produced ∼50% inhibition in each assay (Fig. 3B), indicating an additive effect of co-administration.

Down-regulation of target genes of full length AR and AR variants in response to VPC-14449

Given that VPC-14449 inhibited transcriptional activity of full-length AR and AR variants, we next analyzed the consequences of VPC-14449 on expression of AR gene targets in the panel of CRPC cell lines. R1881 dependent expression of KLK3 (PSA), TMPRSS2 and FKBP5 in LNCaP and C4-2 cells was suppressed by 5 μM VPC-14449 or enzalutamide (Figs. 4A and 4B). R1-AD1 cells, which were derived from heterogeneous CWR-R1 cell line and express full-length AR (23), displayed a similar VPC14449-mediated inhibition of FKPB5 and FASN, two genes under AR regulation in that cell line (23,25) (Fig. 4C). The GR-regulated gene, FKPB52, served as a negative control in all RT-PCR experiments and was unaffected by treatments with androgens or AR inhibitors.

Figure 4. Down-regulation of full length and variant target-genes by VPC-14449 treatment.

Figure 4

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In all RT-PCR experiments, extracted mRNA was analyzed for relative amounts of AR or AR variant target-genes in each cell line. (A) LNCaP, (B) C4-2 or (C) R1-AD1 cells (RPMI+CSS) were cultured with or without 1 nM R1881 and 5 μM of the indicated compounds (24 h). (D) MR49F cells were cultured in RPMI + 5% FBS and compounds. (E) 22Rv1 were maintained in RPMI + 10% CSS before adding compound (50 μM, 48 hrs, no R1881). (F) R1-D567 cells were maintained in RPMI + 5% CSS and tested as in E. *=p-value ≤ 0.05, **=p-value ≤ 0.01 comparing expression between DMSO+R1881 and compound+1881 (LNCaP, C4-2, R1-AD1), between FBS/DMSO and FBS/compounds (MR49F) and between CSS/DMSO and CSS/compounds (22Rv1, R1-D567). Error bars = the mean ± SD with 3 replicates per bar. Experiments were repeated in at least two independent biological replicates.

MR49F cells contain ∼45% of AR protein bearing the F876L mutation (23). To avoid excessive stimulation of wild-type AR, MR49F cells were cultured in FBS-supplemented media containing naturally-occurring androgens. VPC-14449 suppressed target-gene expression, with either no effect (KLK3, TMPRSS2) or agonist effect (FKBP5) resulting from enzalutamide treatment (Fig. 4D). To determine if the AR and AR-V7 responsive M-phase mitotic gene UBE2C (34) was affected by VPC-14449, we performed RT-PCR analysis in 22Rv1 cells cultured under castrate conditions to prevent hormone-stimulation of the endogenous AR. Significant reduction of UBE2C expression was observed with 50 μM VPC-14449 (Fig. 4E) without any apparent effect of enzalutamide. Similar results were observed with R1-D567 cells that express only the truncated ARv567es variant (23), with FKBP5 and FASN expression moderately suppressed by VPC-14449 (Fig. 4F). Taken together, these findings demonstrate that VPC-14449 blocks the transcriptional program driven by mutant forms of the AR, or truncated AR variants.

AR-chromatin interactions are diminished in the presence of VPC-14449

22Rv1 cells, which express full-length AR as well as several truncated AR variants (AR-Vs) including AR-V7 (35), were used to analyze the effects of VPC-14449 on AR sub-cellular localization dynamics. Analysis of whole cell, cytosolic, and nuclear extracts revealed nuclear translocation of full-length AR in cells stimulated with DHT, whereas AR-V7 was constitutively nuclear (Fig. 5A). VPC-14449 did not alter expression or nuclear localization of AR or AR variants in these experiments (Fig. 5A and Ref. 19). Separation of nuclear extracts into soluble and insoluble (chromatin bound) fractions revealed a reduction in association of full-length AR with chromatin in cells treated with >1 μM VPC-14449. The effect of VPC-14449 on chromatin binding of AR-V7 was less apparent in 22Rv1 cells, even at a concentration of 50 μM (Fig. 5A).

Figure 5. AR/ARV7 interactions with chromatin are diminished after VPC-14449 treatment.

Figure 5

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(A) Western blots of indicated subcellular fractions of AR and AR variants as revealed by AR-NTD antibody (and indicated antibodies for loading controls) in 22Rv1 cells treated with VPC-14449 (24 hrs) in the absence or presence of 1 nM DHT (4 hrs). (B) Western blots for full-length AR using chromatin bound fractions from LNCaP and R1-AD1 cells treated with VPC-14449 (24 hrs) and DHT (4 hrs). (C) Effect of VPC-14449 on association of ARv567es with chromatin (treated for 24 hrs, no DHT) in R1-D567 cells. (D) Chromatin fractions from CWR-R1 cells treated with VPC-14449 in the absence (left panel) and presence (right panel) of DHT and blotting for AR-V7. (E) Western blot to detect the full-length AR in chromatin fractions from C4-2 (androgen insensitive) and MR49F (enzalutamide resistant) cells treated with VPC-14449 and DHT. (F) ChIP analysis of inhibitory effect of VPC14449 on androgen-mediated recruitment of AR to FASN AR binding site (ARBS), an ARBS in the FKBP5 enhancer, and the TSC2 exon 37 ARBS in R1-AD1 cells. Cells were treated with 50 μM VPC14449 or vehicle (DMSO) in the presence of 1 nM DHT or vehicle (ethanol, ETH). Data represent fold enrichment of PCR signal in ChIP DNA following immunoprecipitation with an AR-specific antibody vs. non-specific IgG control (arbitrarily set to 1). Data represent mean ± S.D. of 3 independent experiments each performed in triplicate. (G) The effect of VPC14449 on constitutive ARv567es occupancy of FASN, FKBP5, and TSC2 ARBS in R1-D567 cells was assessed as in F by ChIP-qPCR.

To test this mechanism more broadly, we performed chromatin fractionation in an expanded panel of AR-positive cells treated with VPC-14449. In LNCaP and R1-AD1 cells, VPC-14449 inhibited androgen-induced chromatin binding of AR at concentrations > 1 μM (Fig. 5B). Similar to 22Rv1 cells, higher concentrations of VPC-14449 (>10 μM) were required to inhibit ARv567es chromatin binding in the R1-D567 cell line (Fig. 5C) and AR-V7 binding in CWR-R1 cells (Fig. 5D). In C4-2 (androgen insensitive) or MR49F (enzalutamide resistant) cell lines, VPC-14449 at higher concentrations (>10 μM) was also required to inhibit binding of AR to chromatin (Fig. 5E).

Given the ability of VPC-14449 to prevent large-scale interactions between the AR and chromatin across multiple cell lines, we explored the ability of VPC-14449 to block specific chromatin binding events of AR and AR variants using chromatin immunoprecipitation (ChIP) approaches in the isogenic R1-AD1 and R1-D567 cell lines. In R1-AD1 cells, recruitment of the full-length AR to AR binding sites (ARBS) within or near the FASN, FKBP5, and TSC2 genes (25), was stimulated by DHT treatment, but suppressed with 50 μM VPC-14449 (Fig. 5F) or 10 μM enzalutamide (Fig. S3A). However, in R1-D567 cells, VPC14449 inhibited constitutive binding of ARv567es to the FASN and FKBP5 ARBSs, but not the TSC2 exon 37 ARBS Fig. 5G). Enzalutamide, at a concentration of 10 μM, had no effect on ARV567es binding and is consistent with the lack of an LBD of this variant (Fig. S3B). Lowering the concentration of VPC-14449 (10 μM) still resulted in robust inhibition of full-length AR binding to ARBS (Fig. S3A), but resulted in less efficient inhibition of ARv567es binding (Fig. S3B). Control ChIP experiments that probed chromatin regions known to be unoccupied by AR or ARv567es showed no change in signal caused by treatment with DHT or VPC-14449 (Fig. S4). Collectively, these results demonstrate that moderate to high levels of VPC-14449 inhibits chromatin binding of diverse AR species, including mutant forms of full-length AR and truncated AR variants.

Mapping the AR or AR-v567es interactions with chromatin following VPC-14449 treatment

To test genome-wide changes in AR and ARv567es recruitment to ARBSs in response to VPC-14449, we performed ChIP-seq experiments in R1-AD1 and R1-D567 cells using an antibody targeted to the AR-NTD. Peak calling using data from R1-AD1 cells treated with DHT revealed 3,556 ARBSs that met a false discovery rate (FDR) cutoff of 0.01. These ARBSs displayed strong, peak-centered binding of DHT-activated AR when visualized as heatmaps (Fig. 6A). DHT-dependence of AR binding was evidenced by lower heatmap signal intensity in vehicle-treated cells. Consistent with chromatin fractionation data, the DHT stimulated recruitment of AR to these sites was inhibited by VPC-14449 treatment (Fig. 6A, left). Conversely, although visual inspection of data from R1-D567 cells provided strong evidence for peak-centered binding of ARv567es to these 3,556 ARBS, there did not appear to be any effect of VPC14449 at the 50 μM dose tested (Fig. 6A, right). To evaluate the effects of VPC-14449 more rigorously, we plotted signal intensities for AR and ARv567es binding at these 3,556 ARBS under the various treatment conditions. These plots confirmed that VPC-14449 reduced signal intensity of AR binding in R1-AD1 cells (Fig. 6B), but had a modest effect on ARv567es binding in R1-D567 cells (Fig. 6C).

Figure 6. Genome wide binding of AR and ARv567es to AREs are altered by VPC14449.

Figure 6

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(A) Heatmap of ChIP-seq signals ± 3kb around R1-AD1 AR peak midpoints from two biological replicate experiments for R1-AD1 cells (left panel). The same set of binding sites was also called from three biological replicate experiments for R1-D567 cells (right panel). Data are from cells treated with 50 μM VPC14449 or vehicle (DMSO) in the presence of 1 nM DHT or vehicle (ETH) as indicated. (B) Average ChIP-seq signals shown in mapped reads per base pair per peak normalized per 106 reads from 2 datasets at binding sites identified in R1-AD1 cells and (C) 3 datasets in R1-D567 cells. (D) Gene track view of ChIP-seq data at FASN locus in R1-AD1 and R1-d567 cells with indicated treatments. Common AR/ARv567es binding sites (ARBS) are indicated. (E) Gene track view of ChIP-seq data at FKBP5 locus in R1-AD1 and R1-D567 cells. Common AR/ARv567es binding site (ARBS) inside intron 5 is indicated. (F) Gene track view of ChIP-seq data at TSC2 locus. Common ARBS in TSC2 exon 37 is indicated.

Further evaluation of VPC-14449 effects on AR and ARv567es binding was performed at the gene-track level. As expected, VPC-14449 blocked DHT-dependent recruitment of AR to specific ARBS located near or within the FASN, FKBP5 and TSC2 genes in R1-AD1 cells (Figs. 6D-F). There also appeared to be only modest effects of VPC-14449 on ARv567es recruitment to FASN, FKBP5 and TSC2 ARBSs in R1-D567 cells (Figs. 6D-F). Collectively, our ChIP-seq data support the results obtained from fractionation and ChIP-PCR experiments, indicating that VPC-14449 has a more robust effect on genome-wide chromatin binding of AR compared with AR variants.

Discussion

Our earlier studies characterizing AR-DBD inhibitors provided the first evidence that selectively targeting the AR-DBD interactions could be rationally achieved (18,19). Here, we provide new evidence for the utility of DBD-specific inhibition in cell lines that reflect drug-resistance mechanisms in clinical CRPC, where VPC-14449 blocked reporter expression and down-regulated many known AR target-genes. In addition, ITC experiments provide evidence for direct binding between VPC-14449 and the AR-DBD. The weak binding affinity observed by ITC could arise from the absence of DNA, which might be required for the exact conformation of the AR-DBD where the binding pocket for VPC-14449 is correctly formed. Indeed, VPC-14449 was rationally designed to block DNA binding based on the X-ray structure of the AR-DBD bound to double-stranded DNA.

We gained insight into the predicted compound-binding pocket by revealing new point- and simultaneous- mutations in the AR-DBD (K593D, KQK→QHN) that reduce, but not abolish, VPC14449 inhibition. Despite the tolerance of these substitutions, most mutations including S579D, F583D, and R586D, inactivate the receptor (18), indicating the structural and functional conservation of the DBD domain. For this reason, and given the lack of AR-DBD mutations in patients receiving therapy (36), we speculate that resistance could take longer to emerge from prolonged exposure to an AR-DBD specific compound compared to enzalutamide. In addition, co-administration with enzalutamide revealed no incompatibility with VPC-14449, suggesting that dual inhibition might pre-emptively select against resistance causing mutations in both DBD and LBD AR domains. Accordingly, VPC-14449 retained at least partial activity toward all forms of the AR bearing clinically-relevant LBD mutations in reporter assays. A subset of these AR-LBD mutants, including L702H, W742C and W742L, required higher levels of VPC-14449 to inhibit their transcriptional activity compared to the wild-type receptor. AR proteins expressed with these specific mutations were also partially resistant to other AR inhibitors, including those directed to the BF3 site as previously reported (17), and could be due to different protein folding of each mutant or changes in the recruitment of co-factors.

VPC-14449 was less effective against transcriptional activity mediated by AR variants compared to wild-type AR as observed at the reporter assay, targeted ChIP and genome-wide chromatin binding levels. Importantly, VPC-14449 significantly slowed the growth of variant driven 22rv1 cells (35), although the compound only partially inhibited AR-V7 transcriptional activity most likely due to the strong AR-specific promoter of the ARR3tk-luc construct (19) used in the reporter assay. Differential effects of VPC-14449 upon AR-Vs could stem from the absence of the LBD, which might change the conformation of the AR NTD/DBD core in a way that affects DNA recognition or the ability to bind VPC-14449. Alternatively, chromatin fractionation studies revealed that the majority of endogenously expressed AR-V7 in 22Rv1 cells is tightly associated with DNA and not present in the soluble nuclear fraction. Newly synthesized AR-V7 proteins might rapidly translocate to the nucleus and tightly associate to chromatin before VPC-14449 can exert its effect. By contrast, significant amounts of full-length AR remain in the soluble nuclear extract after R1881 stimulation, suggesting an equilibrium of free/chromatin-bound forms of the AR that may be more sensitive to compound-inhibition. Given the entire set of data, we conclude that VPC-14449 does not completely separate the AR or AR variants from chromatin, but the weakening of this interaction may manifest as growth and viability defects in PCa cells, likely by blocking access to specific genes regulating those processes.

To our knowledge, VPC-14449 is the only AR-specific inhibitor that is proven to directly affect chromatin binding of full-length AR or AR-Vs through the DBD. It remains to be seen if pyrvinium, another DBD-interacting compound, operates by the same mechanism. Notably, reports have shown that pyrvinium potently inhibits the transcriptional activity of several AR variants (29). Alternatively, pyrrole-imidazole polyamides, small-molecule DNA mimics that bind specific DNA sequences (37), can block AR recruitment to certain AREs, preventing transcriptional activity and suppressing tumor growth in PCa models (38). In addition to directly targeting the AR-DBD or AREs, AR or AR-V interactions with chromatin can be reduced by treatment with the bromodomain inhibitor JQ1, which targets the bromodomain and extraterminal (BET) family of chromatin-readers BDR2, 3, 4, and BRDT (39). Inhibition of BRD4, which itself interacts with the AR and regulates AR expression (39), affected the chromatin structure in such a way that blocked AR and AR-Vs from interacting with ARBSs across many cell types (25). Given its combinatorial effects with enzalutamide, additive or synergistic inhibition of VPC-14449 and JQ1 are also conceivable.

In conclusion, our findings establish proof-of-principle for targeted inhibition of the AR DBD to overcome AR-dependent drug-resistance mechanisms, prompting further pre-clinical development of such agents to refine their novel mechanism of action.

Supplementary Material

1

Acknowledgments

We thank Dr. A. Zoubeidi for access to MR49F and MR49C cells, and Dr. C.J. Ong for C4-2 cells.

Grant support: This work was funded by the Canadian Institutes of Health Research (P.S. Rennie and A. Cherkasov). The work was also supported by a Prostate Cancer Canada - Translational Acceleration Grant (P.S. Rennie and A. Cherkasov). The authors acknowledge support from the National Institutes of Health (Grant# R01CA174777; S.M. Dehm). Financial support was further provided by a Synergistic Idea Development Award from the US Department of Defense (W81XWH-14-1-0518 to S.M. Dehm; W81XWH-14-1-0519 to P.S. Rennie; W81XWH-14-1-0520 to D.T. Gewirth), and additionally the Terry Fox New Frontiers Program Project grant (TFRI Project 1062; P.S. Rennie and A. Cherkasov).

Footnotes

Rennie, Cherkasov and Dehm Labs contributed equally to this article.

K. Dalal and M. Che contributed equally to this article.

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