the effect of f877l and t878a mutations on androgen ... · the discovery of novel ... f877l...

Cancer Biology and Signal Transduction

The Effect of F877L and T878A Mutations onAndrogen Receptor Response to EnzalutamideStefan Prekovic1, Martin E.vanRoyen2, Arnout R.D.Voet3,4, Bart Geverts2, ReneHoutman5,Diana Melchers5, Kam Y.J. Zhang3,Thomas Van den Broeck1,6, Elien Smeets1, Lien Spans7,Adriaan B. Houtsmuller2,8, Steven Joniau6, Frank Claessens1, and Christine Helsen1

Abstract

Treatment-induced mutations in the ligand-binding domainof the androgen receptor (AR) are known to change antagonistsinto agonists. Recently, the F877L mutation has been describedto convert enzalutamide into an agonist. This mutation wasseen to co-occur in the endogenous AR allele of LNCaP cells,next to the T878A mutation. Here, we studied the effects ofenzalutamide on the F877L and T878A mutants, as well asthe double-mutant AR (F877L/T878A). Molecular modelingrevealed favorable structural changes in the double-mutant AR

that lead to a decrease in steric clashes for enzalutamide.Ligand-binding assays confirmed that the F877L mutationleads to an increase in relative binding affinity for enzaluta-mide, but only the combination with the T878A mutationresulted in a strong agonistic activity. This correlated withchanges in coregulator recruitment and chromatin interactions.Our data show that enzalutamide is only a very weak partialagonist of the AR F877L, and a strong partial agonist of thedouble-mutant AR. Mol Cancer Ther; 15(7); 1702–12. �2016 AACR.

IntroductionGlobally, prostate cancer is the fourth most common cancer

and the fifth leading cause of cancer-related death in men (1). Asthis cancer is hormone-dependent, the blockade of the androgenreceptor (AR) signaling is an effective therapeutic strategy formenwith advanced metastatic prostate cancer. The discovery of novelcompounds that inhibit the AR and their clinical application hasled to the improvement in survival time. One such compound isthe potent antiandrogen enzalutamide, which has been approvedunder the nameXtandi for use in postchemotherapy patients withmetastatic castration-resistant prostate cancer (mCRPC; refs. 2, 3).Enzalutamide acts as a direct competitor for dihydrotestosterone(DHT) binding to the ligand-binding pocket of the AR. In addi-tion, it reduces the binding of the AR to DNA, and inhibits therecruitment of AR coactivators (4). Importantly, enzalutamidesignificantly prolonged the survival of men with mCRPC after

chemotherapy by amedian of 4.8months in comparisonwith theplacebo group (5). On the other hand, enzalutamide has nobeneficial effect in a portion (�20%) of patients, while themajority of patients who initially respondwill eventually developresistance towards this therapy (6). It is imperative, therefore, tostudy the different mechanisms that can lead to enzalutamideresistance.

Prior studies of resistance against the earlier generation anti-androgens, bicalutamide (Bic) and hydroxyflutamide (Hof), ledto the discovery of mutations that changed the ligand-bindingcharacteristics of the AR and thus lead to the change from anantagonistic to an agonistic behavior (7, 8). Balbas and colleaguestransduced LNCaP cells (which carry the T878A mutation) withconstructs driving overexpression of a library of mutated AR andwere able to show that the F877Lmutation converts enzalutamideto an agonist (9). In addition, LNCaP xenograft tumors whichexpress the AR F877L mutant show sustained growth in castratedmice treated with enzalutamide (10). Another study in LNCaPcells pointed out that the F877L mutation had arisen in theendogenous AR T878A allele when cells were treated with enza-lutamide for a prolonged period of time (10). It should be notedthat the LNCaP cells are alsomutated in themismatch repair gene(MSH2), along with over 1,800 other mutations and have arelatively unstable genome (11). However, the detection of verylow levels of the same mutation in the circulating tumor DNA ofpatients who were treated with ARN-509 (ARN) spiked a highclinical interest in the F877L mutation (12).

Several studies reported the co-occurrence of the F877L andthe T878A mutations (F877L/T878A, hereinafter referred to asthe double-mutant AR) in cell line models (10, 12, 13).Interestingly, this double-mutant AR was described in a patientwho progressed on enzalutamide treatment (14). Here, wewanted to compare the agonistic activity of enzalutamide onthe AR F877L, as well as on the double-mutant AR. We dis-covered that the LNCaP-specific T878Amutation modulates theF877L responses to enzalutamide.

1Molecular Endocrinology Laboratory, Department of Cellular andMolecular Medicine, KU Leuven, Leuven, Belgium. 2Department ofPathology, Erasmus MC, Rotterdam, the Netherlands. 3Structural Bio-informatics Team, Division of Structural and Synthetic Biology, Centerfor Life Science Technologies, RIKEN,Yokohama, Japan. 4Laboratoryfor Biomolecular Modeling and Design, Department of Chemistry, KULeuven, Leuven, Belgium. 5Pamgene International, the Netherlands.6Department of Urology, University Hospitals Leuven, Leuven, Bel-gium. 7Laboratory forGenetics ofMalignant Disorders, Department ofHuman Genetics, KU Leuven, Leuven, Belgium. 8Erasmus OpticalImaging Center, Erasmus MC, Rotterdam, the Netherlands.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Frank Claessens, Molecular Endocrinology Laboratory,KU Leuven, Campus Gasthuisberg O&N1, Herestraat 49 Box 901, Leuven 3000,Belgium. Phone: 321-633-0253; Fax: 321-6343-0735; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0892

�2016 American Association for Cancer Research.

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Materials and MethodsPlasmid constructs

Expression vectors, pSG5 and pEGFP-C1, containing cDNA ofwild-type flag- or EGFP-tagged human AR, respectively, (15) wereused to generate themutations in the ligand-bindingdomain (LBD)by two-step PCR site-directed mutagenesis. The resulting PCRfragments were ligated into pGEM-T Easy (Promega) and reclonedas AsuII/BglII fragments into the pSG5-flag-WT AR construct or thepEGFP-C1-WT AR construct. The WT DBD/LBD fragment of ARinserted into pSG5 backbone (16) was used to introducemutationsvia two-step PCR site–directed mutagenesis. The same cloningstrategy as described for the full-length AR constructs was used.The construct containing double-tagged (N-ter YFP and C-ter CFP)WT AR (17) has been used to generate mutations in the LBD usingQuikChange II kit (Agilent Technologies). A plasmid containingpCMV-b-Gal was acquired from Stratagene, the luciferase reporterconstruct driven by 4 copies of a classical ARE and the VP16 AD-NTD(WT) expression vector have been described elsewhere(16, 18). All expression vectors were sequence verified.

Cell cultureHep3B and LAPC4 were provided by the A.B. Houtsmuller and

colleagues (Erasmus Medical Center, Rotterdam, the Nether-lands) and the Laboratory for Experimental Medicine and Endo-crinology, respectively, whereas PC-3 and COS-7 cells wereobtained from the ATCC andwere authenticated by short tandemrepeat DNA profiling by Genetica. HEK293T cell line was a kindgift from the Laboratory of Biosignaling and Therapeutics (KULeuven) in 2004. HEK293T, PC-3, and COS-7 cells were culturedin DMEM supplemented with 10% FCS, while Hep3B cells werecultured in MEMa with GlutaMAX supplement and withoutnucleosides (Gibco). LAPC4 cells were cultured in Iscove's Mod-ified Dulbecco's Medium (Gibco) supplemented with 10% FCS.

Androgen receptor transactivation assayHEK293T or PC-3 cells were seeded at a density of 15,000 cells per

well or 10,000 cells perwell, respectively, in 96-well plates. Cellsweretransfected with 10 ng of expression vector of the receptor (wild-typeor mutant), 100 ng of reporter construct, and 10 ng of the pCMV-b-Gal expression vector per well. On the following day,mediumwasaspirated and fresh medium without or with compounds wasadded. Cells were stimulated with DHT (0.1 nmol/L and 1 nmol/L)obtained from Sigma-Aldrich; enzalutamide (Sequoia Research Pro-ducts Ltd, Selleckchem and Medivation, Inc.; kindly provided byDr. Ganesh Raj, UT Southwestern Medical Center, Urology Clinic,Dallas, TX), ARN509 (ARN;MedChemExpress), Bicalutamide (Astra-Zeneca), hydroxyflutamide (Schering-Plough), abiraterone acetate(Sequoia Research Products Ltd), Galaterone (Sequoia ResearchProducts Ltd), AZD3514 (AZD; MedChem Express), ASC-J9 (Med-Chem Express), EPI-001 (Epi; a kind gift from Dr. Marianne Sadar,Department of Pathology, LaboratoryMedicine, University of BritishColumbia, Vancouver, British Columbia, Canada), and ARAN com-pounds [Asinex (http://www.asinex.com) and Specs] were used atconcentration of 10 mmol/L. All the compounds were dissolved inDMSO (Acros Organics). Cells were harvested in Passive Lysis buffer(Promega) and the luciferase and b-galactosidase measurementswere done according to the protocol described previously (19).

Modeling of the androgen receptor ligand interactionModeling was done using the Molecular Operating Environ-

ment (MOE, The Chemical Computing Group; ref. 20). The AR

structure was taken from PDB entry 4OJB, and resistancemutations were introduced. Subsequently, the structure wasoptimized using Protonate_3D and the Amber99 force fieldembedded in MOE (21). Conformations of the AR ligands(DHT, enzalutamide, ARN, and RD162) were calculated usingthe MMFF94x force field. As a reference for the correct torsionalangle between the 3-fluoro-cyano-benzyl group and the thiox-oimidazolidin, PDB entry 2NW4 was utilized (22). The top fivebinding modes for all the receptor–ligand pairs were visuallyinspected and exhibited nearly identical binding propertieswith the exception of enzalutamide fitted in the WT and theT878A AR LBD. As a positive control DHT and Hof were dockedinto the agonistic conformation of AR WT or T878A LBD,respectively. The top docking scores exhibited a similar dockingmode within 0.4 Å RMSD deviation of the crystallographicallydetermined structure.

The ligands were docked into the ligand binding pocket of thereceptor followed by a short energyminimization allowingminor"inducedfit" changes to the protein and ligand conformation. Thebinding mode was scored using the London Free Energy scoringfunction for prediction of affinity and rescored using the DrugS-coreX scoring function for analysis of the complementarity andclashes between the ligand and the receptor atoms (23).

Whole cell competition assayHEK293T cells were seeded at a density of 30,000 cells per well

in 48-well plates. Cells were transfected with 375 ng of ARexpression constructs, and 75 ng of pCMV-b-Gal expressionvector. On the day after the transfections, medium was refreshedand cells were incubated overnight. Subsequently, cells weretreated with a dilution series (0.1 nmol/L to 10 mmol/L) ofcompound with 1 nmol/L [3H]-labeled mibolerone (PerkinElmer). After an incubation period of 90 minutes at 37�C,medium was aspirated, cells were washed three times with ice-cold PBS, and lysed in 100 mL of Passive Lysis buffer (Promega).Cell lysates (75 mL)were transferred to scintillation vials and 2mLof LumaSafe Plus (PerkinElmer) was added. Radioactivity mea-surements were done in a liquid scintillation counter (LK Wallac1216 RackBeta, Wallace).

Western blot analysisTotal protein extracts (in Passive Lysis buffer) were run on a

precast NuPAGE novex 4%–12% Bis-Tris Gel (Invitrogen). Afterelectrophoresis, the proteins were blotted on a polyvinylidenedifluoride transfer membrane (GE Healthcare). Antibodiesagainst GAPDH (Santa Cruz Biotechnology) and AR (in-houseantibody against N-terminus of AR as described in ref. 15), wereused. The secondary antibodies anti-rabbit IgG (P0217 fromDako) and anti-Mouse IgG (P0260 from Dako) were conjugatedto horseradish peroxidase, and visualized by immunodetectioncombined with chemiluminescence (Western Lightning Plus-ECL; PerkinElmer).

Double-hybrid assayCOS-7 cells were seeded at a density of 30,000 cells per well in

48-well plates. On the following day, cells were transfected with25 ng of AR DBD/LBD expression vector with or without themutations, 250 ng of the VP16 AD-NTD(WT) construct, 250 ng ofthe reporter construct, and 50 ng of pCMV-b-Gal construct. Cellswere stimulated with either DHT (10 nmol/L) or enzalutamide(10 mmol/L), overnight. Cells were harvested in Passive Lysis

Molecular Analysis of Androgen Receptor Mutants

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buffer (Promega) and the luciferase and b-galactosidase measure-ments were done as indicated above.

Acceptor photobleaching fluorescence resonance energytransfer

Acceptor photobleaching fluorescence resonance energy trans-fer (FRET) was performed using a Zeiss LSM510 confocal laserscanningmicroscope equipped with a Plan-Neofluar 40�/1.3 NAoil objective (Carl Zeiss) at a lateral resolution of 100nm(17). Forexcitation of CFP and YFP, an Argon laser at 358 and 514 nmwasused. Both the CFP and YFP images were taken prior to photo-bleaching of the acceptor fluorophore (514 nm at high laserpower). Apparent FRET efficiency was estimated according to theinstructions described in ref. (17). The abFRET datasets weretested for normality using the Kolmogorov–Smirnov tests, andthe datasets were compared using the one-tailed Student t test.

MARCoNI assayTo investigate the modulation of coregulator interaction with

the WT AR and mutant receptors, we used the MARCoNI assay(Pamgene). HEK293T cells were seeded at a density of 20millioncells per T175 flask. On the following day, cells were transfectedwith 85 mg of ARWT, AR F877L, AR T878A, or AR double-mutantexpression constructs. After 24 hours, cells were incubated inserum-free mediumwithout or with compounds (DHT, Hof, andenzalutamide) for 30 minutes. Subsequently, cells were scrapedand pelleted. TheMARCoNI assay was performedwith cell lysatesas described previously (24). Each sample was processed on threeseparate arrays. Subsequently, the binding of AR to each individ-ual coregulator peptide was quantified. The FQNLF motif of theAR was discarded as the antibody to detect the AR was raisedagainst a peptide that partly covered this motif. For each peptide,Student t test was applied to compare the receptor binding level tothat of the nonstimulated control. All calculations and statisticswere performed using R (version 3.1.2; ref. 25).

Generation of stable cell linesStable Hep3B cells were generated to constitutively express the

EGFP-tagged AR with either the F877L mutation or the F877L/T878A double mutation. The expression of EGFP-fusions wasvisualized by fluorescence microscopy and functionality of thereceptor was confirmed by transfection of an ARE-driven lucifer-ase reporter construct. Clones with moderate induction factor(F877L clone: 13.3� 5.5; F877L/T878A clone: 20.4� 11.8) afterDHT stimulation were selected (Supplementary Fig. S12). Hep3Bcell lines expressing EGFP-fused ARWT, AR T878A, or AR A574Dwere reported previously (26).

Fluorescence recovery after photobleachingCells were seeded in 6-well plates on a cover glass, ligands

were added, and cells were incubated overnight. For eachexperiment, a cover glass with stably transfected Hep3B cellswas placed in a preheated ring. Cells were kept at 37�C and usedfor no longer than 90 minutes. A Zeiss LSM510 META confocallaser scanning microscope equipped with a 40�/1.3NA oil-immersion objective, an argon laser (30 mW) and an acousto-optic tunable filter was used. Fluorescence recovery after photo-bleaching (FRAP) analysis was performed according to theinstructions by van Royen and colleagues (27). For each treat-ment group, 30 cells were measured by FRAP in two indepen-dent experiments. All curves were normalized to fluorescent

intensity before bleaching. The FRAP data were quantitativelyanalyzed by comparing the experimental data to curves gener-ated using a previously described Monte Carlo approach (28).In short, a large set of computer-simulated FRAP curves with a3-population model was generated, containing a diffusingfraction (ranging 1 to 3 m2/s) and two bound (immobile)fractions (ranging from 0.65 to 0.8 s and 3 to 15 s, respectively).Ranges in the simulation are based on quantitative FRAPanalysis of AR curves in previous work (29). The top 10simulated curves that are fitting best to the experimental curve(by ordinary least squares) were averaged and provide theproperties of the experimental data.

ResultsT878A enhances the partial agonistic effects of enzalutamide onAR F877L

To unravel possible interferences between the F877L and theT878Amutations, we first compared the response of the AR WT,AR F877L, AR T878A, and the AR double-mutant F877L/T878Ato several compounds on a transactivation assay. Reporterassays were performed in HEK293T and PC-3 cells (Fig. 1 andSupplementary Fig. S1). The mutations had some effect on themaximal DHT induction: 1 nmol/L DHT resulted in inductionfactors of approximately 82, 71, 28, and 22 respectively for ARWT, AR T878A, AR F877L, and the double mutant (Supple-mentary Fig. S2). To allow comparisons, the luciferase responseto 1 nmol/L DHT was put at 100. As expected, the DHT-inducedtransactivation of AR WT was inhibited by the antagonists,enzalutamide, ARN, bicalutamide, and Hof, to at least 25%.These effects were independent of the receptor levels (Supple-mentary Fig. S3). As expected, none of the antagonists hadrobust agonistic activity on WT AR. The induction of WT AR by10 mmol/L Hof rose to �6% of the 1 nmol/L DHT response;(Fig. 1A) which was comparable to the signal observed forcastrate levels of DHT (0.1 nmol/L). Thus, for AR WT, enzalu-tamide, ARN, and bicalutamide are full antagonists, whilehydroxyflutamide is a weak partial agonist.

For the LNCaP AR T878A mutant, hydroxyflutamide gainedfull agonistic activity (�98% of DHT 1 nmol/L signal) and lostits antagonistic properties (Fig. 1B), as reported in earlierstudies (30). The other compounds (enzalutamide, ARN, andbicalutamide) efficiently antagonize AR T878A. When it comesto the AR F877L mutant, enzalutamide and ARN led to anincrease in luciferase signal to approximately 10% and 14% ofthe signal induced by 1 nmol/L DHT, respectively (Fig. 1C andSupplementary Fig. S4). It should be noted that the level oftransactivation induced by these compounds was also similarto that of castrate levels of DHT (0.1 nmol/L). Importantly,both enzalutamide and ARN retain their antagonistic effects onDHT-activated AR F877L. In our assays, enzalutamide and ARNare only weak partial agonists on AR F877L (Fig. 1C andSupplementary Fig. S4), comparable with the report by Josephand colleagues (12). In case of the double mutant, however,enzalutamide and ARN were able to activate the receptor to asubstantial extent (33% and 34% of the response to 1 nmol/LDHT, respectively) and exhibited weaker antagonistic proper-ties than on AR WT, AR F877L, and AR T878A (Fig. 1D andSupplementary Fig. S4). In other words, enzalutamide and ARNare considerably stronger agonists of the double-mutant ARthan of the AR F877L. Surprisingly, the combination of the

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Mol Cancer Ther; 15(7) July 2016 Molecular Cancer Therapeutics1704




F877L mutation with the T878A mutation impaired the fullagonist activity of hydroxyflutamide (compare Fig. 1D with B).These effects are independent of AR levels and of the cell lineused (Fig. 1E and Supplementary Fig. S1).

Modeling the effect of the F877L and T878A mutations on theAR-enzalutamide interactions

To get an insight into the mechanisms how the T878A and theF877Lmutationswould convert the antagonist enzalutamide into

Figure 1.

T878A increases the partial agonistic effects of enzalutamide (Enza) on AR F877L. Transient transfection of AR WT or AR mutant together with an androgen-regulated luciferase reporter gene in HEK293Twas performed to investigate the effect ofmutations on the transactivational response to selected compounds (conc.10 mmol/L). DHT was used in two different concentrations (1 nmol/L and 0.1 nmol/L) and all the values were normalized to DHT 1 nmol/L (100%). The meanand SEM of five independent experiments in triplicates are shown. AR WT (A), AR T878A (B), AR F877L (C), AR F877L/T878A (D), and Western blot analysis (E)showing equal expression levels of all receptor constructs with or without 1 nmol/L DHT. Hof, hydroxyflutamide; Bic, bicalutamide.






an agonist, wemodeled the latter into the agonistic conformationof the AR ligand-binding pocket (Fig. 2A). In this agonisticconformation, the interactions of testosterone (T) or DHT withthe R753 side chain are crucial (31, 32). However, if a similarinteraction between R753 and enzalutamide is preserved, thelatter cannot be accommodated in the agonistic conformationof AR WT or AR T878A. The top scoring orientation for enzalu-tamide in theWT structure significantly deviated from the bindingmode observed in the mutant or protein in the modeled antag-onistic conformation (33)with an8ÅRMSDdeviation andanon-negative predicted binding energy of 54 kcal/mol. Similarly,oriented and bad scoring binding modes were observed in thetop solutions for both ARN and RD162.

Indeed, there is a strong steric hindrance of enzalutamide withthe phenylalanine at position 877, as well as with the threonineresidue at position 878 (Fig. 2B). The F877L mutation resolvesmost of this, as leucine has a more flexible side chain comparedwith phenylalanine. This leucine can adopt a rotamer positionwithout steric hindrance for enzalutamide, thus allowing the ARLBD to assume an agonistic conformation (Fig. 2C). This mech-anism clearly differs from the mechanisms of agonism of theflexible bicalutamide, which fits as an agonist into a nearby cavityof the AR W741C/L mutants, thus stabilizing the agonistic con-formation of the AR LBD in an alternative way (34). Moreover,modeling enzalutamide into the ligand-binding pocket of thedouble-mutant AR predicted even fewer steric clashes, as well asa moderate improvement in docking scores compared with thesingle F877L mutant (Table 1; Supplementary Fig. S5). Similarresults were obtained for ARN and RD162 (Table 1).

Modeling of hydroxyflutamide into the agonistic conformationof the ligand-binding pocket of the double-mutant AR suggestedless favorable contacts between Leu877 and the dimethyl groupof hydroxyflutamide (Supplementary Fig. S6; SupplementaryTable S1), predicting a less efficient AR activation.

Modeling of DHT into the WT and mutant AR also indicatedslightly reduced docking scores for the F877L and the double-

mutant AR compared with the WT AR (Supplementary Fig. S7)and the T878A mutant (data not shown).

The F877L mutation leads to an increase in the affinity of thereceptor for enzalutamide

Whole cell competition assays with 1 nmol/L 3H-labeledmibolerone, were performed to investigate whether bindingaffinities of the mutated receptors for DHT and enzalutamidehave changed. A decrease was observed in relative binding affin-ities (RBA) of DHT for both mutant receptors tested when com-paredwith theWT receptor [RBA(DHT)wt¼ 100, RBA(DHT)877¼75.81, RBA(DHT)877/878 ¼ 77.51; Fig. 3A; Table 2]. On the otherhand, there was a drastic increase in RBA of enzalutamide for bothAR F877L and AR F877L/T878A when compared with the AR WT[RBA(Enza)wt¼1.38, RBA(Enza)877¼ 12.77, RBA(Enza)877/878¼19.28; Fig. 3A; Table 2].

Effect of mutations on the ligand-dependent N/C interactionsUpon binding of an agonist, the C-terminal LBD of the AR is

known to interact with the FQNLF motif in the N-terminaldomain (35). To measure these N/C interactions, FRET andmammalian double-hybrid assay were performed (Fig. 3B andSupplementary Fig. S8). DHT induced N/C interactions in boththe FRET and themammalian double-hybrid assays (35), whereasno AR N/C interactions were observed for enzalutamide-boundARWT.While there was only a trend towards N/C interactions for

Figure 2.

The double mutant enables an optimalagonistic conformation for binding ofenzalutamide (Enza). A, in silicomodels for enzalutamide interactionwith the LBD; enzalutamide(contacting the side chain of R752)was docked into the agonisticconformation of the ligand bindingpocket of WT (B), F877L (C), and thedouble mutant (D).

Table 1. Docking scores of enzalutamide (Enza), ARN-509, and RD162

Docking scores (kcal/mol)WT F877L F877L/T878A

Enza / �10.4 �15.1ARN / �10.8 �15.9RD / �10.1 �14.8

NOTE: Docking simulations were used to predict the affinity of the compounds(kcal/mol) according to the top solutions for the different ARmutants. Decreaseof the docking score is in relation increased stability of the receptor–ligand pair.

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the AR F877L single mutant, high signals for N/C interactionswere observed for enzalutamide-bound double-mutant AR (Fig.3B and Supplementary Fig. S8).

Comparison of enzalutamide-induced coregulator recruitmentby WT and mutant receptors

To explorewhetherDHT, enzalutamide, andhydroxyflutamidecould induce a modulation of the interaction of WT and mutantARwith coregulators, we performed aMARCoNI assay for the full-length receptors. The natural ligand (DHT) induced strong inter-actions of coactivator peptides with both WT and all mutantreceptors, while corepressor interactions were downregulated(Fig. 4A and Supplementary Fig. S9). Unsupervised clusteringshowed that a very similar binding pattern was observed for thehydroxyflutamide-bound AR T878A (Supplementary Figs. S10and S11) and the enzalutamide-bound double-mutant AR. Infact, out of all enzalutamide-stimulated ARs, the double mutantresembled most closely to the pattern observed for DHT-stimu-lated receptors (Fig. 4A and B and Supplementary Fig. S11). Incontrast, no strong effects on coregulator interactions could beobserved for enzalutamide-bound AR WT or AR T878A (Fig. 4Aand B and Supplementary Fig. S11).

Comparison of the intranuclear mobility of ARWT andmutantreceptors

Nuclear mobility of a transcription factor can be studied byFRAP. The extent of immobilization is strongly correlated withchromatin-binding characteristics (27). For all receptors tested in

our FRAP experiments, DHT induced nearly overlapping redis-tribution curves from which very similar immobile fractions canbe calculated (Fig. 5A and D).

Hydroxyflutamide-stimulated AR F877L reacted like the inac-tive DNA-binding defective mutant, while the curves of thehydroxyflutamide-bound AR T878A and the double mutant werenearly superimposable on that of the DHT-bound AR WT (Fig.5B). The calculated immobilized fraction size and free diffusiontime reflected these similarities in mobility behavior (Fig. 5D;Supplementary Table S2).

We also compared the effect of enzalutamide on themobility ofthe AR mutants. The FRAP curves of the WT AR, AR T878A weresuperimposable with that of the inactive DNA-binding defectiveARA574D. Thedouble-mutant AR showeda slower redistributionpattern after photobleaching when compared with AR WT, ART878A, and AR F877L (Fig. 5C). Clearly, the AR double mutantapproaches the FRAP profile of the DHT-stimulated ARWT, whilethe curve for the enzalutamide-bound AR F877L mutant is moresimilar to that of the enzalutamide-bound AR WT (Fig. 5C).Comparable results were observed for ARN-stimulated AR WTand mutant receptors (Supplementary Fig. S13).

Computational calculations of the fraction sizes of receptorsthat have an apparent long immobilization time revealed that theAR F877L/T878A double mutant, when stimulated with enzalu-tamide, behaved similarly to any of the AR variants tested withDHT, while the single mutant AR F877L behaved more like theantagonist-bound AR WT (Supplementary Table S2).

The double AR mutant is targetable by other AR antagonistsWe exploredwhether the double-mutant ARwould be resistant

to other antagonists nonrelated to enzalutamide or ARN. For this,we used the well-known AR axis inhibitors abiraterone acetate(primarily discovered as CYP17 inhibitors, however, it can alsodirectly inhibit the AR; refs. 36, 37), galaterone, Epi (this com-pound targets theNTDof theAR; ref. 38), AZD, andASC, aswell assome experimental ARAN compounds which we described earlierfor their full antagonistic activity on AR W742C and AR T878A(33). All of the tested compounds exhibited no agonistic and

Figure 3.

Ligand-binding affinities and N/C interactions. A, experimental binding affinities as obtained in a whole cell competition assay with 3H-Mibolerone. Bindingaffinity curves for DHT and enzalutamide (Enza) on AR WT and mutant receptors. Both WT and the mutant receptors show high affinity for DHT, while forenzalutamide there is an increase in affinity for the F877L and F877L/T878A mutants. B, acceptor photobleaching FRET for AR WT and the mutant ARs stimulatedwith either DHT or enzalutamide. Binding of DHT-induced intramolecular N/C interactions for WT, F877L, and F877L/T878A receptors. Induction of N/Cinteraction by enzalutamide was only observed for the double-mutant AR.

Table 2. Relative binding affinities for DHT and enzalutamide (Enza) of WT ARand the mutant ARs

RBAsWT F877L F877L/T878A

DHT 100 75.81 77.51Enza 1.38 12.77 19.28

NOTE: The binding affinities of DHT and Enza are presented as relative to theaffinity of WT AR for DHT (set at 100), as determined in whole cell competitionassay.






strong tomoderate antagonistic activity on the ARWT, AR T878A,AR F877L, and AR F877L/T878A (Fig. 6 and Supplementary Fig.S14) when tested on an androgen-responsive luciferase reporter.The only exception being ARAN-4, which slightly activated thedouble-mutant AR, although not to the same extent as enzaluta-mide (Supplementary Fig. S14).

Proliferation studies in LAPC4 cells (Supplementary Methods)also show that cells transfected with the double-mutant AR wererescued from the growth-suppressing effect of enzalutamidewhencompared with WT AR–transfected cells (Supplementary Fig.S15A). Furthermore, the same rescue was observed for T878A-transfected cells treated with hydroxyflutamide (SupplementaryFig. S15B). Abiraterone acetate, however, had a similar effect onproliferation of all the cells regardless of the receptor transfected(Supplementary Fig. S15C).

DiscussionIt is well documented that mutations in the AR can lead to

clinical resistance against antiandrogens, and can even turn thelatter into agonists. The AR T878Amutation of the AR for instanceconverts hydroxyflutamide to an agonist in both preclinical andclinical settings (8, 30). Recently, it has been proposed that the ARF877L mutation leads to enzalutamide resistance, as it mightconvert enzalutamide into an agonist (9, 10, 12, 39). Here, we

compared the effect of these mutations and their combination onthe biochemical properties of the AR.

Transactivation propertiesEnzalutamide is only a weak partial agonist for the AR F877L

mutation, while when this mutation co-occurs with T878A, thereis a dramatic increase in agonistic effect of enzalutamide. A similarobservation was made for the combination of L701H and T878Amutations, which resulted in greater responsiveness to cortisolcompared with the single L701H mutant (40). Possibly, in bothcases, the T878Amutation leads to a stabilization of the agonisticconformation of the receptor. This might be attained by thereduction in steric hindrance (discussed below) or by reposition-ing of helix (H) 11 and H12 towards the ligand-binding pocket,facilitating the transition into the agonistic mode of the AR asargued in refs. 30, 41.

In silico modelingThe antagonists that are currently used in the clinic all act via the

ligand-binding pocket, preventing the correct realignment of H12which is recognized by coactivators. However, the details of theirmechanismsof action are different (reviewed in refs. 41, 42). It hasbeen proposed that bicalutamide binds an additional bindingpocket (B-site) in the proximity of the androgen-binding pocket

Figure 4.

Coregulator recruitment for AR WT and mutants. A, MARCoNI assay heatmap representing AR-coregulator peptide interactions for DHT and enzalutamide(Enza)-stimulated WT AR and mutant ARs. Stimulation of receptors with DHT led to increased binding to peptides from coactivators (red) and loss ofbinding to corepressor peptides (blue); similar pattern was observed only for AR F877L/T878A stimulated with enzalutamide. B, data on selectedcoactivator peptides from the MARCoNI assay. Interaction of AR with NCOA3_609_631 and PRGC_134_154 peptides is evident upon agonistic stimulationof AR and absent in presence of antagonists. Hydroxyflutamide (Hof) and enzalutamide act as agonists on T878A and the double-mutant AR and are ableto induce interactions in a similar fashion as DHT.

Prekovic et al.





(34). In contrast, for enzalutamide, it has been suggested thatwhen it binds to the AR it contacts residues on H11, thusdisrupting H11–H12 interactions which prevents H12 fromadopting the correct agonistic alignment with the LBD (9). Ourin silico model indicates that the F877L mutation expands theligand-binding pocket, allowing for enzalutamide tofit inside andinteract with R753 like the agonists DHT or T. Similarly, themolecular dynamics simulation by Balbas and colleagues pro-posed that F877L allows repositioning of enzalutamide from anantagonistic binding mode in such a way that steric clashesresulting in dislocation of H12 in WT AR are eliminated (9). Themodeling further proposes that the additional exchange of thre-onine at site 878 to alanine leads to extra reduction of sterichindrance for enzalutamide. This is corroborated by crystallo-graphic data of the bicalutamide-bound T878A mutant (34).Furthermore, according to our modeling, the F877L mutationleads to a reduced hydrophobic contact and putative solventexposure for DHT. This might be the explanation why themutantAR is less responsive to DHT.

Ourmodelingmay provide some insight valuable for design ofnext generation compounds. Putative future antiandrogens couldfor instance harbor a group incompatible with the F877L due tosteric hindrance (rigid bulky modifications) or polar incompat-ibility with the hydrophobic contact surface of either the phenyl-alanine or the mutant leucine.

Ligand bindingIn concordancewith the computationalmodeling,weobserved

a decrease in affinity of DHT for the receptors containing theF877Lmutation,while the affinity of enzalutamidewas increased.Although co-occurrence of F877L and T878A led to a strongincrease in enzalutamide agonism, only amodest further increasein affinity for the double-mutant AR was observed. Therefore, wepropose that F877L is crucial for the increase in affinity of

enzalutamide and ARN, while the co-occurrence with T878A(through mechanisms discussed above) is pivotal in stronglyenhancing the agonistic activity of enzalutamide.

N/C interactions and binding to coregulator peptidesWhen the AR is activated by agonists it undergoes a confor-

mational change that leads to close positioning of the NTD andLBD and thus interaction between these two domains (27, 43).The enzalutamide-bound ARWT or F877L did not show any N/Cinteractions. However, strong interaction was observed for thedouble-mutant AR bound by enzalutamide, or anymutant or WTreceptor stimulated by DHT. As we detected transactivation andchromatin binding (discussed below) for the enzalutamide-bound F877L receptor, it might be that the absence of N/Cinteraction poises the F877L receptor only for specific promoters,as it was observed that deletions/mutations of FQNLF selectivelydiminish the activity of the AR at specific promoter elements (44).

To investigate the effect of DHT and antiandrogens on AR–coregulator interactions, we employed the MARCoNI assay (24).The modulation of interactions by DHT was similar, but clearlydistinct from theone recorded for the ER stimulatedwith estradiol(45). As expected, we observed strong interactions of the agonist-boundARwith the coactivator peptides that contain FxxLFmotifs.TheMARCoNI data for DHT on theWT andmutant receptors wasvery similar. The slight drop in coactivator peptide binding for themutant receptors again strongly correlates with the decreasedresponsiveness to DHT.

ARWT treatedwith hydroxyflutamide or enzalutamide showedvery little changes in coregulator binding, which is expected forantagonists. Hydroxyflutamide induces some binding of AR WT,which is in concordance with its reported partial agonist activity,and the agonist activity of hydroxyflutamide on AR T878A corre-lates with the similarity of the peptide interaction pattern withthat of DHT-bound WT AR. The pattern of modulation of the

Figure 5.

Agonistic effects of enzalutamide(Enza) are reflected in differences inintranuclear mobility of the ARmutants. Fitted curves of dataobtained in FRAP experiments (first30 seconds). The data represent themean of two independentexperiments (30 cells). The followingcompounds were used: A, DHT(10 nmol/L); B, Hof (10 mmol/L); and C,enzalutamide (10 mmol/L). D, pie-charts depicting calculated fractionsize of the long and short immobilefraction, as well as mobile fractionattributed to each receptor. The longimmobile fraction size ofenzalutamide-bound double mutant issimilar to that of agonisticallyactivated AR WT and AR T878A.






single mutant receptors by enzalutamide cluster more closely tothe antagonistic enzalutamide-treated WT AR. In contrast, theenzalutamide stimulated double-mutant AR clusters togetherwith the agonistic hydroxyflutamide-bound WT and double-mutant AR, further supporting the fact that enzalutamide acts asa potent partial agonist of the double-mutant AR.

Clearly, all these data validate this assay for the simultaneouscomparisonof relative effects of agonists and antagonists on the invitro binding to a series of coregulator-derived peptides. In con-clusion, the MARCoNI data show that ligands with differentactivity can be separated according to their modulation of ARinteractions with coregulator peptides into full agonists, partialagonists, and antagonists.

Ligand-induced intranuclear behavior of mutant receptorsIt is believed that the immobile fraction of AR measured in

FRAP assays corresponds to the chromatin-bound receptor duringthe transcription activation process, because of the overlap of ARlocations with the sites of RNA synthesis (17). There is also astrong correlation between AR activity and the immobile fraction,

as observed for AR WT and AR T878A stimulated with agonistsand antagonists (43). Antagonist-bound ARs behave differently,having smaller immobile fractions, resembling that of DNA-binding defective receptors (26).

Using FRAP,weobserved thatwhile the AR F877Lmutant had asmaller enzalutamide-induced immobile fraction, the chromatin-bound fraction of enzalutamide-stimulated double-mutant ARwas similar to the fraction of the DHT-bound AR WT.

Inhibiting the F877L and T878A mutant ARsBoth enzalutamide and ARN share a 4-cyano-3-(trifluoro-

methyl)phenyl group with Bic and hydroxyflutamide requiredfor specific binding to the AR. Because of the structural similaritiesbetween enzalutamide and ARN, the F877L mutation is likely tobe enzalutamide/ARN structure-specific. We were able to showthat structurally diverse AR inhibitors (abiraterone acetate, gala-terone, and Epi) can completely abolish theDHT-activation of theF877L and double-mutant AR (38, 46, 47). Furthermore, we haveshown that abiraterone acetate effectively inhibited proliferationof cells harboring transfected double-mutant AR, while presence

Figure 6.

The AR double-mutant is targetable by other AR antagonists. HEK293T cells were used to compare the activity of several antiandrogens on transactivationof AR. Transcriptional activity of none of the AR mutants was induced by abiraterone acetate (Abi), galaterone (Gal), Epi, AZD, or ASC. Moreover, thesecompounds successfully reduced the DHT-induced transactivation of AR for both the WT and the mutant receptors. The mean and SEM of three independentexperiments are shown. AR WT (A), AR T878A (B), F877L (C), and AR F877L/T878A (D).

Prekovic et al.





of the double mutant led to sustained survival under enzaluta-mide treatment. These results illustrate that targeting AR withcompounds that bind differently to the LBD or with compoundsthat target other domains rather than the LBD,might be favorableand potential candidates for future treatment of enzalutamide-resistant patients harboring F877L mutation.

Clinical implicationsMutations such as T878A have been found recurrently in

mCRPC patients, in hydroxyflutamide-treated patients, but alsoin patients that were never treated with hydroxyflutamide (14,48). Moreover, the antiandrogen withdrawal syndrome, which isknown to occur in patients after cessation of antiandrogens suchas hydroxyflutamide, bicalutamide, or nilutamide treatment isknown to be partly explained by the occurrence of mutations inthe AR that convert the antagonists into agonists (49). Similarmechanisms have been anticipated for enzalutamide.

For now, only a limited number of genomic studies on post-enzalutamide patients have been reported. However, none ofthese identified the single F877L mutation in this population.Only one patient was found to have the F877L mutation, but itwas co-occurring in the same allele with the T878A mutation(14, 39). In addition, withdrawal syndrome seems to be eitherextremely rare or nonexistent after enzalutamide progression(50, 51). All this suggests that in the clinic, the frequency of pointmutations that convert enzalutamide to an agonist is likely low.Possibly, other events like AR overexpression or activation ofother signaling pathways are more relevant (14, 48).

ConclusionIn conclusion, the molecular modeling and ligand-binding

data, the transactivation data, the N/C interactions, the MAR-CoNI data, and the FRAP data all converge to the same hypoth-esis: the F877L mutation enhances enzalutamide binding, buthas a limited effect on the activation potential of this antag-onist. Only when F877L is combined with T878A, the receptorbecomes strongly activated by enzalutamide. In addition, thesemutant ARs can be targeted with antiandrogens such as abir-aterone acetate or galaterone, opening the possibility forsequential inhibition of AR with structurally diverse com-pounds. Further genomic studies similar to the one by Azadand colleagues should be performed to determine the identityand frequency of the AR mutations in post-enzalutamidepatients (14). A recent article by the same group describes the

agonist and antagonist activity of enzalutamide, hydroxyfluta-mide, bicalutamide, and experimental BF3 binding antagonistson a series of AR single or combined mutations which theyfound in castration-resistant prostate cancer (39). The datapresented here focus on the effect of the F877L/T878A muta-tion on the (ant-)agonist binding, the intramolecular N/Cinteractions, the coregulator interactions and the intranuclearmobility. Together with Lallous and colleagues, we conclude afull molecular characterization of AR mutations that occur inCRPC could provide recommendations for future personalizedtherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Prekovic, R. Houtman, F. Claessens, C. HelsenDevelopment ofmethodology:M.E. vanRoyen,D.Melchers, A.B.Houtsmuller,F. Claessens, C. HelsenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Prekovic,M.E. vanRoyen, R.Houtman,D.Melchers,T. Van den Broeck, E. Smeets, L. SpansAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Prekovic, M.E. van Royen, A.R.D. Voet, B. Geverts,R. Houtman, K.Y.J. Zhang, F. ClaessensWriting, review, and/or revision of the manuscript: S. Prekovic, M.E. vanRoyen, A.R.D. Voet, R. Houtman, K.Y.J. Zhang, T. Van den Broeck, E. Smeets,S. Joniau, F. Claessens, C. HelsenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. GevertsStudy supervision: S. Joniau, F. Claessens, C. Helsen

AcknowledgmentsThe authors thankH.Debruyn, R. Bollen, andD. Schollaert for their excellent

technical assistance and the colleagues from the Molecular EndocrinologyLaboratory for the helpful discussions.

Grant SupportThis work supported by research grants from the FWO-Vlaanderen

(G.0830.13N), KU Leuven (GOA/15/017), and Kom op tegen Kanker. F.Claessens is the principal investigator for all the grants previously mentioned.A.R.D. Voet was supported by RIKEN FPR fellowship.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 3, 2015; revised April 18, 2016; accepted April 26, 2016;published OnlineFirst May 16, 2016.

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2016;15:1702-1712. Published OnlineFirst May 16, 2016.Mol Cancer Ther Stefan Prekovic, Martin E. van Royen, Arnout R.D. Voet, et al. Response to EnzalutamideThe Effect of F877L and T878A Mutations on Androgen Receptor

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