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Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in… Lallous, Nada; Volik, Stanislav V; Awrey, Shannon; Leblanc, Eric; Tse, Ronnie; Murillo, Josef; Singh, Kriti; Azad, Arun A; Wyatt, Alexander W; LeBihan, Stephane; Chi, Kim N; Gleave, Martin E; Rennie, Paul S; Collins, Colin C; Cherkasov, Artem Jan 26, 2016

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RESEARCH Open AccessFunctional analysis of androgen receptormutations that confer anti-androgenresistance identified in circulating cell-freeDNA from prostate cancer patientsNada Lallous1, Stanislav V. Volik1,3, Shannon Awrey1, Eric Leblanc1, Ronnie Tse1, Josef Murillo1, Kriti Singh1,Arun A. Azad2, Alexander W. Wyatt1, Stephane LeBihan1,3, Kim N. Chi2, Martin E. Gleave1, Paul S. Rennie1,Colin C. Collins1,3 and Artem Cherkasov1*AbstractBackground: The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethalcastration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, andenzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. SeveralAR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostatecancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations onthe functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.Results: We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene forthe presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identifyfour additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conductexperimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing toreveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting thebinding function 3 (BF3) site on the activity of CRPC-associated AR mutants.Conclusions: This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the directidentification of AR mutants from patients’ serum, and the functional characterization of these mutants in order toprovide personalized recommendations regarding the best future therapy.Keywords: Androgen receptor, Castration-resistant prostate cancer, Cell-free circulating DNA, Mutations, Drugresistance, Anti-androgens and steroidsBackgroundAdvances in prostate cancer (PCa) research have led tothe development of novel therapies for the metastaticcastration-resistance (CRPC) form of the disease, suchas two recent drugs abiraterone [1, 2] and enzalutamide[3, 4], which target the androgen receptor (AR) pathway.Abiraterone inhibits cytochrome P450 17A1 (CYP17A1),an enzyme responsible for the synthesis of testosteronethat, after conversion to dihydrotestosterone (DHT),binds to the androgen binding site (ABS) of the AR andactivates the AR signaling axis. Enzalutamide is a potentanti-androgen that competes with DHT and binds to theABS, preventing AR transcriptional activation. Unfortu-nately, patients with advanced PCa either do not re-spond to anti-androgen therapy due to pre-existingaberrations of CYP17, or AR or relapse to CRPC due toadaptive responses, or Darwinian selection of rare* Correspondence: artc@interchange.ubc.caNada Lallous and Stanislav V. Volik are co-first authors who equallycontributed to the work.Paul S. Rennie, Colin C. Collins and Artem Cherkasov are co-senior authors orlabs that contributed equally to this work.1Vancouver Prostate Centre, University of British Columbia, 2660 Oak St.,Vancouver, BC V6H 3Z6, CanadaFull list of author information is available at the end of the article© 2016 Lallous et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Lallous et al. Genome Biology  (2016) 17:10 DOI 10.1186/s13059-015-0864-1aberrations. Thus, while these therapies improve diseasemanagement and extend life for most patients, ultim-ately they are only palliative.In the majority of cases, CRPC is accompanied by re-activation of the AR signaling axis so that the receptorregulates its numerous target genes including PSA. Notsurprisingly, an impressive repertoire of mechanisms hasbeen identified that reactivate the AR signaling axis.These include upregulation of CYP17 [5], amplificationof the AR gene [6], expression of constitutive AR splicevariants [7, 8], or mutation of the AR itself [9–12]. It hasbeen demonstrated that mutations in the ABS of the ARcan lead to its activation by weak adrenal androgens,steroidal and non-steroidal ligands, and by mutation-driven conversion of AR inhibitors into agonists [13].For example, the AR substitution T878A, identified inthe LNCaP cell line, confers resistance to the anti-androgen hydroxyflutamide [14] and is promiscuouslyactivated by progesterone and 17β-estradiol [15]. Othermutations such as W742C/L and F877L are associatedwith resistance to the anti-androgens bicalutamide[16–18] and enzalutamide [19–21], respectively. Thus,identification and characterization of resistance-associatedAR mutations, as biomarkers for primary treatment ofboth naïve PCa and CRPC patients, are critically im-portant for predicting, as well as monitoring, patient’sresponse to therapy. This process is essential for the de-velopment of evidence-based precision oncology.The detection of circulating cell-free DNA (cfDNA)has recently emerged as a non-invasive diagnostic toolfor a variety of cancers, including CRPC, and as a tech-nology maximizing the efficacy of anticancer therapies[22, 23]. It is estimated that up to 3 % of tumor DNA isreleased into the circulatory system daily from the pro-cesses such as secretion, necrosis, and primarily apop-tosis [24]. In our previous work [25], we reported thedevelopment of a sequencing platform that allowed de-tection of a repertoire of AR mutations in cfDNAisolated from CRPC patients. This method enabled ef-fective sequencing of AR exon 8 from plasma samples of47/62 metastatic CRPC patients who were progressingon systemic therapy, and thus resulted in the identifica-tion of numerous AR mutations, including three previ-ously unreported ones.By improving the sequencing and data analysis pro-cesses, we were able to sequence the cfDNA of the 15CRPC patients that were previously excluded due to lowyield of DNA in their plasma samples, to validate all ofthe mutations reported in our previous work [25] and toidentify new candidate mutations.Importantly, in the current work, we have also carriedout in vitro characterization of all AR mutations identi-fied in 62 CRPC patients together with seven ARmutants previously reported in the literature (L702H,W742L, W742C, V716M, V731M, T878S, and M896T),to ascertain the exact mechanisms of resistance to ARpathway inhibitors (Fig. 1). To accomplish this task, weengineered each one of 24 distinct AR mutants (con-taining single and multiple amino-acid substitutions),and determined in vitro effects of four current AR antago-nists (enzalutamide, hydroxyflutamide, bicalutamide, andARN509) on all mutants, as well as investigated their invitro responses to four different steroids including DHT,progesterone, estradiol, and hydrocortisone. As the result,we present evidence that all identified AR mutations pro-vide evolutionary escape routes from androgen blockade,thus highlighting the need for novel AR inhibitors thatbind to the AR outside of the ABS. Finally, we demon-strate that VPC-13566, one of our recently developed classof AR inhibitors bearing a quinolone scaffold [26] that dir-ectly interferes with AR recruitment of co-chaperones andactivating cofactors via binding to the BF3 surface [27,28], effectively inactivates the AR signaling axis for all 24CRPC-associated AR mutants.ResultsDeep sequencing reveals AR mutations in cfDNAIn the current study, we used data from a patient co-hort we previously reported [25]. We showed thatmutations in the AR ABS contributed to treatment re-sistance in a subset of patients and presented the possi-bility of detecting these mutations in cfDNA at thepoint of progression [25]. Due to low DNA yield(<30 ng), 15 patients were not amenable to sequencing.In order to overcome this limitation, we have WGA2-amplified and sequenced cfDNA from these patientsand modified the pipeline we developed previously [25]to enable detection of mutations in WGA2 cfDNA (seethe ‘Methods’ section for more details). We have alsoperformed experimental validation of the redesignedpipeline using direct comparison of WGA2 and non-amplified data for subset of cfDNA samples as well asalternative sequencing platforms (see Additional file 1:Supplementary data, Table S1).In total, mutations were detected at 13 nucleotide po-sitions in the coding region of exon 8 in 14/62 (23 %) ofpatients (Table 1). The frequency of these mutations inpatients’ cfDNA ranged from 0.11 % to 23 %. Mutationsat two positions were silent, while mutations in theremaining 11 resulted in 12 distinct amino-acid substitu-tions (no nonsense mutations were detected). Two mis-sense mutations were detected in multiple patients:H875Y (n = 7) and T878A (n = 4). By including theWGA2 sequencing, we were able to report four newmutations (H875Q, D891H, E898G, and T919S) thatwere neither identified in our previous study [25] nordescribed in the literature.Lallous et al. Genome Biology  (2016) 17:10 Page 2 of 15We previously discussed the validation of sequencingresults using MiSeq resequencing of AR exon 8 ampli-cons and additional DNA samples from VC-012 andVC-041 patients. Inclusion of WGA2 sequencing dataallowed us to extend the validation. For example, wehave identified M896V and S889G mutations in theWGA2 sequence of the patient VC-012 at the first time-point; both mutants were supported by the unamplifiedsequence data from the first and/or the second time-points. We reported that for VC-012, four additionalmutations were identified at the second (post-enzaluta-mide) time-point, including F877L/T878A and T878A/S889G (Fig. 2).The cfDNA sample from the first time-point of VC-001patient was collected at progression on abiraterone priorto commencing enzalutamide, but the patient’s mutationstatus was not reported due to low cfDNA yield. The sec-ond sample was collected approximately 3.5 months laterat the point of progression on Enzalutamide. After WGA2sequencing, two single (H875Y and T878A) and onecombined (H875Y/T878A) mutation were detected atthe first time-point, in addition to a silent mutationL874L (Table 1). Two additional substitutions, D891Hand T878A/D891H, were detected at enzalutamide pro-gression. Similar to patient VC-012, we detected mul-tiple AR haplotypes at both time-points for VC-001,none of which contained more than two missensemutations.AR transcriptional activation by steroidsThe response of AR mutants to increasing concentra-tions of DHT has been measured using a luciferase-reporter transcription assay in PC3 cells transientlytransfected with either wild-type or mutated AR. The ex-pression level of all of the mutants was evaluated bywestern blotting (Additional file 2: Figure S1). Only twopoint mutations, T878S (EC50 = 0.019 nM) and T919S(EC50 = 0.030 nM), made the receptor slightly moreFig. 1 AR mutations identified in CRPC patients. a AR gene organization showing the AR-LBD mutants. b AR mutants mapped on the X-ray structure(PDB: 2 AM9) of the LBD (cartoon representation, in gray) in complex with testosterone (TES, ball-and-stick representation, in cyan). AR mutants encodedby exon 8 are shown in magenta ball-and-stick representation. The rest of the mutants are shown in blueLallous et al. Genome Biology  (2016) 17:10 Page 3 of 15sensitive to DHT compared to the wild type (EC50 = 0.047nM) (Table 2). Some mutants such as H875Q/T919S andW742L/C appeared to be over-stimulated by higher con-centrations of DHT (Fig. 3a). A noteworthy mutant wasW742L that showed an approximately two-fold higherlevel of transcriptional activity than the wild-type AR athigh concentrations of DHT (approximately 500 nM)(Table 2, Fig. 3a).Taken together, these data illustrate the heterogeneousresponses of AR mutants towards activation by DHT;ranging from mutation-driven enhancement of AR lig-and sensitivity, to creation of ‘super-active’ variants ofthe receptor. It is noteworthy that these CRPC mutantsmay also present higher affinities toward other steroidsin order to overcome the effect of androgen deprivation[29–31]. Therefore, we tested the response of wild-typeand mutated AR to activation by three other steroids: es-tradiol, progesterone, and hydrocortisone. The wild-typeAR was only mildly stimulated by progesterone concen-trations higher than 100 nM and was not activated withestradiol or hydrocortisone at concentrations as high as500 nM (Additional file 3: Figure S2). Regarding activa-tion with estradiol, many mutants demonstrated a stron-ger response to this steroid compared to the wild-typereceptor (Fig. 3b). It has been reported that H875Y,T878A, and T878S could be activated by estrogensTable 1 AR mutations detected in CRPC patientsPatient Amino acid change Mutant read count Wild-type read count Total read count Percent mutantVC-001-t1* T878A 39 13,231 13,270 0.29H875Y 36 13,351 13,387 0.27H875Y/T878A 251 13,019 13,270 1.89VC-001-t2 T878A 30 12,626 12,656 0.24H875Y 0 13,307 13,307 0.00H875Y/T878A 230 12,426 12,656 1.82T878A/D891H 158 10,383 10,541 1.50D891H 8 10,533 10,541 0.08VC-005 E894K 170 10,745 10,915 1.56VC-012-t1 M896V 1,270 5,985 7,255 17.51VC-012-t1* S889G 307 4,769 5,076 6.05M896V 273 5,838 6,111 4.47VC-012-t2 S889G 103 8,202 8,305 1.24M896V 31 8,934 8,965 0.35H875Y 49 10,355 10,404 0.47T878A 300 9,760 10,060 2.98F877L/T878A 141 9,919 10,060 1.40T878A/S889G 35 8,270 8,305 0.42VC-014* E898G 237 11,919 12,156 1.95VC-015 T878A 218 9,502 9,720 2.24VC-017 T878A 99 12,626 12,725 0.78VC-018* H875Y 223 10,382 10,605 2.10VC-021* H875Q 251 9,846 10,097 2.49T919S 238 9,004 9,242 2.58VC-022 D880E 12 10,902 10,914 0.11VC-040 H875Y 479 7,560 8,039 5.96VC-041-t1 H875Y 1,521 7,260 8,781 17.32VC-041-t2 H875Y 4,665 15,662 20,327 22.95VC-053 H875Y 136 9,064 9,200 1.48VC-063 H875Y 270 14,874 15,144 1.78VC-064 L882I 17 15,670 15,687 0.11Newly reported samples sequenced from WGA2 DNA are marked with a *. Each horizontal line in the table represents a particular haplotype, hence multiple linesfor some data points. Only patients with mutations detected in cfDNA or in WGA2 cfDNA are shownLallous et al. Genome Biology  (2016) 17:10 Page 4 of 15(reviewed in [13] and validated in our assay). In thecurrent work, we show that some of the newly reportedmutants such as D891H, T878A/D891H, and T878A/S889G were stimulated up to eight-fold higher than thewild-type receptor in the presence of 500 nM estradiol(Fig. 3b, Table 2).Nine AR mutants could be effectively stimulated bymuch lower concentrations of progesterone comparedwith the wild-type AR (EC50 = 104 nM). For example,mutant T878S revealed an EC50 value of 0.53 nM (pro-gesterone), which is 200 times higher affinity than thewild-type AR. Similarly, mutants T878A/S889G, T878A,T878A/D891H, F877L/T878A, H875Y, H875Y/T878A,S889G, and D891H exhibited EC50 in the range ofnanomolar progesterone concentrations (Fig. 3c, Table 2).Some of these mutants were previously reported to beactivated by progesterone (such as T878A/S and H875Y)[9, 13, 31, 32].The wild-type AR (and most of the mutants) did notexhibit any significant transcriptional activation withhydrocortisone at a concentration up to 500 nM, withthe exception of the L702H mutant (EC50 = 25 nM)known to be activated by hydrocortisone [15] andH875Y/T878A (EC50 = 105 nM) (Fig. 3d, Table 2).AR transcriptional inhibition by AR antagonistsWe have tested four current non-steroidal AR antago-nists: hydroxyflutamide [14], bicalutamide [16, 17], enza-lutamide [33–35], and ARN509 [36] (Table 3, Additionalfile 4: Table S2) for their effects on the transcriptionalFig. 2 Characterization of AR mutants identified in patient VC-012 after progression on bicalutamide and enzalutamide. a Two AR mutants wereidentified in the cfDNA isolated after patient progression on bicalutamide. Both mutants show agonist response to bicalutamide in an in vitrotranscription assay. b Four additional mutants were identified in the same patient VC-012 after progression on enzalutamide, all with various agonisteffects toward enzalutamide in vitro. The percentage in the charts only reflects the mutated form of the androgen receptor. Each concentration wasassayed in quadruplicate n = 4, with a biological replicate of n = 3. Results were averaged and normalized by expressing them as a percentage of thewild-type AR activity ± SEMLallous et al. Genome Biology  (2016) 17:10 Page 5 of 15activity of 24 AR mutants. Using the same luciferasetranscription assay described above, cells were stimu-lated with the non-metabolizable androgen R1881 andthen treated with the increasing concentrations of thedrugs. This experiment allowed us to identify specificAR mutations that decrease the sensitivity of the recep-tor to inhibition by these antagonists as well as thecharacterization of the mutations that transform currentanti-androgens into AR agonists. Thus, in addition tothe already well-documented F877L mutation that con-verts enzalutamide to a partial agonist, we showed thatthe compound mutation (F877L/T878A), that is alsopresent in the enzalutamide resistant cell line MR49F[37], converted this drug into a full agonist. We havealso observed that canonical hydroxyflutamide-resistantT878A and H875Y AR variants also confer a partialagonist effect to enzalutamide when the drug was ad-ministered at higher concentrations (Table 3, Fig. 2).When T878A is combined with other LBD mutations,such as T878A/D891H or T878A/S889G, the activationeffect of enzalutamide on the mutant is retained, andseemingly enhanced. Of note, in the panel of 24 studiedAR variants, the experimental drug ARN509 behavedvery similarly to enzalutamide (Table 3 and Additionalfile 4: Table S2), which was not surprising, consideringthat a very high degree of structural resemblance existsbetween the two chemicals. Moreover, our results indi-cate that two mutants - H875Y and F877L - appear tobe more resistant to ARN509 than to enzalutamide(Additional file 4: Table S2).In accordance with a previous study [20], we foundthat the well-documented enzalutamide- and ARN509-resistant mutant, F877L, can effectively respond to theolder anti-androgens, bicalutamide and hydroxyfluta-mide (Fig. 4, Additional file 4: Table S2). A similar ob-servation was also made for the double mutant F877L/T878A that provides a profound agonist function tohydroxyflutamide, enzalutamide, and ARN509, but canTable 2 The inhibition of AR mutants by VPC-13566 and their activation by various steroidsAR construct IC50 of VPC-13566Inhibition (μM)EC50 of DHTactivation (nM)EC50 of estradiolactivation (nM)EC50 of progesteroneactivation (nM)EC50 of hydrocortisoneactivation (nM)WT 1.73 0.05 >500 104.0 >500L702H 6.13 8.00 >500 172.0 25.0V716M 1.06 0.14 >500 329 .0 >500V731M 0.99 0.09 >500 115.0 >500W742L 2.29 33.60 >500 >500 >500W742C 3.43 4.74 >500 293.0 >500H875Y 1.34 0.14 68.0 10.20 >500H875Q 0.79 0.43 >500 >500 >500F877L 0.37 0.08 >500 >500 >500T878A 2.56 0.06 144.0 0.57 >500T878S 0.43 0.02 100.0 0.53 >500D880E 1.14 0.11 >500 177.0 >500L882I 0.84 0.20 >500 >500 >500S889G 10.48 0.37 230.0 17.20 >500D891H 2.35 0.12 173.0 31.0 >500E894K 1.20 0.25 >500 143.0 >500M896V 0.59 4.50 >500 >500 >500M896T 0.10 >500 >500 >500 >500E898G 1.24 0.45 >500 >500 >500T919S 1.29 0.03 >500 123.0 >500H875Q/T919S 0.63 0.18 >500 >500 >500T878A/S889G 13.20 0.12 94.0 0.36 >500T878A/D891H 13.40 0.40 100.0 0.49 >500H875Y/T878A 10.80 1.26 63.0 0.66 105.0F877L/ T878A 11.70 0.81 >500 5.70 >500The IC50 values of the inhibition by VPC-13566 and the EC50 values of the activation by DHT, estradiol, progesterone, and hydrocortisone are reported for thewild-type AR and the 24 studied mutants. For steroid activation, we tested a concentration range up to 500 nM, therefore mutants showing no activation or veryweak activation in the studied range are presented with EC50 values >500 nMLallous et al. Genome Biology  (2016) 17:10 Page 6 of 15be effectively suppressed by bicalutamide (Table 3,Additional file 4: Table S2). These important cases illus-trate that the use of cfDNA sequencing technologycould revive older treatment options for some CRPCpatients that have developed resistance to enzaluta-mide. These results once again emphasize the import-ance of an evidence-based approach to precisiononcology for prostate cancer patients.We recently reported that H875Y and T878A AR mu-tations were identified in patients progressing on abira-terone or had previously received it [25]. Romanel et al.also showed the emergence of T878A and L702H mu-tants in 13 % of patients progressing on abiraterone [38].As none of the tested mutants were activated with abira-terone in our assay (Additional file 4: Table S2), the se-lection for these mutants after abiraterone treatmentseems important for the AR promiscuous activation byother steroids, especially progesterone. This suggestioncould be supported by the increase in progesteronelevels after abiraterone therapy [39].Inhibition of AR mutants via BF3 siteOne promising strategy for combating mutation-drivendrug resistance could be to develop drugs that act onthe AR outside of the ABS region. This paradigm is ex-emplified by the recently described Binding Function-3(BF3) - a protein-protein interaction site that is essen-tial for AR transcriptional activity and is involved inrecruiting AR co-regulators such as FKBP52 and Bag-1 L [27, 40]. Previously, we reported on the develop-ment of quinolone derivatives that selectively inhibitthe AR through its BF3 functionality at clinically relevantconcentrations [26, 41–44]. One such AR inhibitor - 2-(7-methyl-1H-indol-3-yl) quinolone (called VPC-13566),demonstrated an IC50 of 1.73 μM in PC3 cells transfectedwith WT-AR plasmid (Fig. 5a). Importantly, using a TR-FRET assay, we showed that VPC-13566 was able to dis-place a FITC labeled BAG1L peptide (residues 1-20) fromthe BF3 pocket of AR, proving its binding to the suggestedsite. However, the compound VPC-14449 [45], which tar-gets the DNA binding domain of AR, was not able to dis-place BAG1L from its pocket (Fig. 5b). In the currentstudy, we have employed the described luciferase-basedassay to assess the transcriptional activity of 24 AR mu-tants in response to varying concentrations of VPC-13566 (Table 2, Additional file 4: Table S2). Under thesame conditions, using an MTS cell proliferation assay,we assessed the toxicity of this compound in non-Fig. 3 Steroid activation of AR mutants in comparison with the wild-type receptor in luciferase reporter assay. While most of the AR mutantsshowed similar or lower affinity to the activation by DHT (a), when compared to wild-type, several variants presented better activation by estradiol (b),progesterone (c), or hydrocortisone (d) than the wild-type. PC3 cells were transfected with both wild-type or mutated AR and a reporter plasmidpARR3-tk-luciferase. After 48 h post transfection, cells were treated with increasing concentrations of steroids. The graphs represent the average ± SEMof three independent experiments with four replicates each. The activity of each mutant in the presence of a steroid was normalized to the wild-typestimulated by 500 nM of the same steroidLallous et al. Genome Biology  (2016) 17:10 Page 7 of 15transfected PC3 cells and only noticed a mild toxicityat 50 μM (Fig. 5c). We hypothesized that the AR BF3inhibitor should not be activated by any of the muta-tions, because they mainly cluster in the AR ABS, a re-gion spatially distant from the BF3 site, and therefore,should not affect the interaction of VPC-13566 withthe protein. Indeed, VPC-13566 effectively suppressedthe transcriptional activity of all 24 AR mutants withthe corresponding IC50 values in the range of 0.12 to13.4 μM (Table 2, Additional file 4: Table S2).DiscussionRepertoire and functionality of AR mutationsThe AR is a multi-domain, ligand-inducible transcriptionfactor composed of an N-terminal part, followed by aDNA binding (DBD) domain – the functional site of thereceptor, a hinge region, and finally, a C-terminal LBDportion that is encoded by exons 5-8 and is known to beprone to mutations (Fig. 1) [46]. The incidence of ARmutations is rare in untreated prostate cancer, and is es-timated to be in the range of 15 % in CRPC patients[47]. It has been reported that certain AR mutations cancause treatment failure of conventional AR antagonists,and can promote progression of PCa to its lethal CRPCstate [13, 48, 49]. The accumulating evidence suggeststhat temporal monitoring of PCa patients being treatedwith AR pathway inhibitors can help detect the emer-gence of resistant AR mutants that drive CRPC.Several of these CRPC-associated mutations are welldocumented in the literature, including hydroxyfluta-mide- and bicalutamide-resistant AR variants T878Aand W742L/C, respectively. Other substitutions, includ-ing L702H [10], V716M [50], V731M [51], T878S [11],and H875Y [9], have been associated with receptorpromiscuity – that is, an increased AR sensitivity toother steroids (progesterone, hydrocortisone, estradiol,and so on) or to AR antagonists. We have also recentlyreported four additional CRPC-associated variants,S889G, D880E, L882I, and E894K located on exon 8 ofthe AR gene [25].In the current work, we employed a modifiedmutation-identification pipeline that allowed us to estab-lish a detailed cfDNA-based mutation status of all 62CRPC patients reported in [25], thus including 15 pa-tients that were previously excluded from the analysisdue to low DNA yield in their blood samples. The appli-cation of an improved sequence analysis approachallowed for the identification of four additional muta-tions in the AR (H875Q D891H, E898G, and T919S).We have also identified cases where two mutationsoccur on the same haplotype: T878A/D891H, T878A/S889G, F877L/T878A, H875Y/T878A, and H875Q/T919S. We hypothesized that understanding the poten-tial clinical significance of these mutations would requirein vitro functionalization to determine whether and howthe mutations modulate the activity of the AR.The measured responses of 24 single and double ARmutants to four AR antagonists, hydroxyflutamide,bicalutamide, enzalutamide, and ARN509, revealed thatall these drugs can behave as AR agonists in the con-text of certain mutations (Table 3). In particular, thefirst generation antagonist hydroxyflutamide demon-strated an activating behavior towards the vast majorityof the AR variants (with a notable exception of F877L),ranging from weak to strong agonist for mutationsT878A/S, H875Y, F877L/T878A, T878A/D891H, andT878A/S889G.Resistance to bicalutamide was also observed for themajority of the AR mutants, with the notable examplesof W742L/C, T878A/S, S889G, D891H, and M896V/Tmutations conferring strong bicalutamide activating phe-notypes (Additional file 5: Figure S3). A partial agonistTable 3 The response of CRPC-associated AR mutations to anti-androgen treatmentsAR mutants Agonist response to treatmentHydroxyflutamide Bicalutamide Enzalutamide ARN509L702H Partial Partial No NoV716M Partial Partial No NoV731M Partial Partial No NoW742L Partial Yes No NoW742C Partial Yes No NoH875Y Yes Partial Partial PartialH875Q Partial Partial No NoF877L Partial No Partial partialT878A Partial Yes Partial partialT878S Partial Yes Partial partialD880E Partial Partial No NoL882I Partial Partial No NoS889G Yes Yes No NoD891H Yes Yes No NoE894K Partial Partial No NoM896V Partial Yes No NoM896T Partial Yes No NoE898G Partial No No NoT919S Partial Partial No NoH875Q/T919S Partial Partial No NoT878A/S889G Yes Yes Partial PartialT878A/D891H Yes Yes Partial NoF877L/ T878A No Yes Yes YesH875Y/T878A Yes Yes Partial PartialResults presented in Additional file 4: Table S2 are summarized here. Weconsidered as partial agonist a drug that inhibited a mutant at lowconcentrations and stimulated its activity at high concentrationsLallous et al. Genome Biology  (2016) 17:10 Page 8 of 15response to bicalutamide was observed for the majorityof the studied mutants, especially when bicalutamide ispresent at high concentrations. In particular, whenbicalutamide is administered above 16 μM, a profoundactivation was detected for single mutations V716M,V731M, H875Y, and for the mutation combinationsT878A/D891H and T878A/S889G. Knowing that thesteady state concentration of bicalutamide in the serumof prostate cancer patients is 8.9 μg/mL or approxi-mately 20 μM [52], we were surprised to find that inour assay this drug activated the wild-type AR in thesame concentrations range (approximately 16 μM)(Additional file 4: Table S2).In addition to the documented case of enzalutamide-resistant mutation F877L, we identified that the combin-ation F877L/T878A behaved as full agonist and did notshow any inhibition in presence of enzalutamide. Athigher doses of enzalutamide, we observed a stimulationof such mutants as T878A, H875Y, T878A/D891H, andT878A/S889G.Fig. 4 AR mutants associated with enzalutamide resistance in CRPC patients. a Molecular dynamics (MD) model of AR LBD (cartoon representation, ingray) in complex with enzalutamide (ball-and-stick representation, in blue). The residues presented as gray sticks are found to be mutated in patientsprogressing on enzalutamide treatment. b The F877L mutant showed an agonist response to enzalutamide in an in vitro cell-based assaybut was inhibited by the first generation anti-androgens hydroxyflutamide and bicalutamide. Each concentration was assayed in quadruplicaten = 4, with a biological replicate of n = 3. Results were averaged and normalized by expressing them as a percentage of WT AR activity ± SEMLallous et al. Genome Biology  (2016) 17:10 Page 9 of 15The functionalization of these 24 CRPC-associated ARmutants revealed that ARN509 generally exhibits dose-response profiles similar to those of enzalutamide, in-cluding agonist stimulation of mutants F877L, H875Y,and T878A.These experiments indicate that any of the 24 AR mu-tants could drive resistance to at least one of the investi-gated drugs. These observations point to a complex anddynamic repertoire of AR mutations, driving therapeuticresistance in CRPC. This is underscored by the observa-tion that the AR LBD haplotypes and haplotype ratios incfDNA of individual CRPC patients can rapidly changein response to treatment regimens.An interesting example is a patient VC-012 (Fig. 2).This individual underwent bicalutamide treatmentthat resulted in the development of CRPC. At thistime-point, it was established that the patient’s ARharbored mutations M896V and S889G in the ratio of47 % to 53 %, respectively. In our assay both of thesemutations demonstrated profound resistance to bica-lutamide (Fig. 2) and likely underlie the patient’s pro-gression on bicalutamide. Importantly, both of thesemutations were sensitive to enzalutamide and couldbe fully inhibited in vitro by 5 μM of the drug. Not-ably, patient VC-012 was switched to enzalutamideand after approximately 4 months of treatment, hiscfDNA was collected and sequenced. At this time-point,the percentage of M896V and S889G had decreased to4.7 % and 15.6 %, respectively. Remarkably, cfDNA se-quencing revealed four new AR LBD mutations thatemerged in response to enzalutamide administration.These new mutations included T878A (45.5 %), H875Y(7.4 %), and two double mutants F877L/T878A (21.4 %)and T878A/S889G (5.3 %). All four AR mutants demon-strated varying degrees of activation by enzalutamide inour assay (Fig. 2, Additional file 4: Table S2).Structural basis for agonistic conversion of bicalutamideand enzalutamideThe distinction between the agonistic and antagonistic ac-tions of an AR ligand is conventionally attributed to theinduced motion of helix 12 – one of the receptor’s foldsforming the ABS cavity of the AR LBD. In a simplifiedview, AR inhibitors are believed to push helix 12 outward,preventing the formation of a ligand-locked, functional(agonist) configuration of the protein [53, 54]. Usingmethods of in silico modeling, we have investigated thepossible structural basis for the agonist conversion of bica-lutamide toward M896V and S889G mutants (Additionalfile 5: Figure S3), as well as the agonist interactions ofFig. 5 Characterization of the in-house developed AR inhibitor VPC-13566. a Dose-response curve illustrating the inhibiting effect of theVPC-13566 and enzalutamide on the AR transcriptional activity in PC3 cells transfected with wild-type AR plasmid. Data points represent themean of three independent experiments performed in four replicates each. Error bars represent the standard error of the mean ± SEM for n = 12 values.b The specific binding to the BF3 site was confirmed by BAG1L peptide (1-20) displacement using a TR-FRET assay. c The effect of VPC-13566 on PC3cell viability. % cell viability is plotted in dose dependent manner. Data points represent the mean ± SEM of two independent experiments performedin quadruplicateLallous et al. Genome Biology  (2016) 17:10 Page 10 of 15T878A, H875Y, F877L/T878A, and T878A/S889G vari-ants with enzalutamide (Fig. 4).Our analysis of the distribution of the 15 mutated resi-dues on the AR 3D structure demonstrated that they aremainly clustered around the AR steroid binding site andadjacent to helix 12 (Fig. 1). Previous structural studiesinto AR mutants have associated the T878A mutationwith an increase in the size of the LBD cavity, the conse-quence of replacing a threonine residue with a morecompact alanine residue. This size increase was attrib-uted to the ability of the mutated AR to bind hydroxy-flutamide in an agonist-like conformation [14]. Similarly,the F877L mutation was shown to increase the size ofthe AR ABS, thus enabling the receptor to accommodatethe enzalutamide moiety [20].Residue M896 belongs to the above-mentioned helix12 of the AR, and is in close contact with the sulfonyloxygen O15 of the bicalutamide molecule (1 Å), whichmakes the agonist conformation improbable with theWT-LBD (Additional file 3: Figure S2). The introduc-tion of a less bulky non-polar valine residue into the896-position should significantly increase the surfacearea of the ABS pocket, therefore allowing bicaluta-mide to bind in the closed agonist-like configuration.In a similar way, one could consider the effect ofH875Y substitution: some preliminary modeling resultsshowed that the hydroxyl group of the Y875 side chaincan directly interact with -C(O)NHCH3 to further ac-commodate the ligand into the agonist configuration ofthe LBD.The resistant character of the S889G substitutioncould be attributed to the increased mobility of helix 12in the AR. In fact, the S889 residue is positioned right atthe hinge of the helix 12, and introduction of the mostflexible amino acid – glycine – into that position shouldsignificantly increase the mobility of the hinge, thus fa-cilitating the motion of helix 12 toward the inbound lig-and (Additional file 5: Figure S3).We speculate that the double mutants F877L/T878Aand T878A/S889G combine the agonist effects of theconstituent single mutations. The intriguing possibilityof synergism arising from the LBD compound mutationsremains to be investigated.Targeting new sites on the AR could overcome themutation-dependent drug resistanceThe use of AR inhibitors that target the AR beyond itsconventional and mutation-prone ABS could provide aneffective strategy for addressing the problem of resist-ance, either alone or in combination with ABS-targetedagents such as enzalutamide. VPC-13566 has effectivelyinhibited all AR variants, including those that confer re-sistance to enzalutamide and to emerging drug candi-dates such as ARN509 (Table 2 and Additional file 4:Table S2). Thus, VPC-13566 could represent a viabletreatment option for CRPC patients. Moreover, our ARBF3 inhibitor VPC-13566 exhibits a novel, distinctivemode of action against the AR, and therefore, could beconsidered for combinatorial therapy with conventionalAR antagonists with a view to reducing toxicity and un-favorable side effects as well as delaying resistance ofconventional single agent therapy.ConclusionsGenomic analysis of cfDNA is a minimally invasivemethod for interrogating mechanisms of therapeutic re-sistance in CRPC patients. In the present study, we dem-onstrate that cfDNA sequencing can identify mutationscausally linked to resistance to therapies targeting theAR in CRPC. In vitro functionalization revealed that all24 investigated AR mutations exhibited resistance to atleast one of four AR inhibitors used in clinical practice.Moreover, some of the newly identified double AR mu-tants exhibited enhanced activation in presence of enza-lutamide and ARN509 and/or demonstrated elevatedsensitivity to stimulation by DHT or others steroids.These results underscore the importance of developingtherapeutics that target the AR at sites outside the ABS.Thus, it is significant that the AR inhibitor VPC-13566,targeting the AR BF3 pocket, can effectively block theactivity of all 24 AR mutants identified in CRPC patients.It will now be important to determine if co-targeting theAR with VPC-13566 and, for example, enzalutamide, candelay/overcome the resistance. Finally, the results of thisstudy suggest that precision oncology for the improvedmanagement of CRPC patients may be a feasible option toimprove patient care.MethodsPatient cohort and sequencing of exon 8 of the AR geneThe cohort of 62 patients with metastatic CRPC re-cruited at the British Columbia Cancer Agency (BCCA) -Vancouver Prostate Centre (VPC, Vancouver, BC, Canada)between August 2013 and March 2014 was describedpreviously [25]. In total, 19/62 (30 %) of patients wereswitched onto enzalutamide after collection of cfDNA.Clinico-pathological characteristics including prior andsubsequent therapies were recorded for each patient.Blood collection, DNA isolation, and quantification wereperformed as described previously [25].For this paper we have sequenced blood samples from15 patients that were reported as not sequenced in theprevious manuscript due to either low DNA yield (lessthan 30 ng as determined by Qubit 2.0 measurementusing Qubit dsDNA HS Assay Kit) or repeatedly failedto produce useable sequencing data (VC-024, VC-028and VC-044). DNA was amplified with the SigmaWGA2 kit (Cat No WGA2-10rxn or WGA2-50rxn) asLallous et al. Genome Biology  (2016) 17:10 Page 11 of 15per manufacturer’s instructions. The amplified materialwas sequenced with the Roche 454 GS FLX+ system,software version 2.9 as described in [25]. The ampliconsfrom three WGA2 samples with detected mutationswere also sequenced using an Illumina MiSeq sequencer;in all cases the results were concordant.Mutation callingThe biggest challenge in analysis of WGA2 amplifiedDNA is the significantly higher noise levels introducedby the genomic amplification. Therefore, we modifiedour pipeline to assure tighter control and more precisefiltering of low-quality sequencing data. Raw sequencereads were mapped to the human genome (hg19) usingBWA and visualized using the Integrative GenomicsViewer (IGV) [55]. All possible non-reference baseswith greater than 25 Phred score at all amplicon loca-tions were quantified using bam-readcount v. 0.7.4(https://github.com/genome/bam-readcount) that allowsfor direct filtering of reads at each base position based onboth sequence phred quality score (only bases with phredscore >25 were scored) and mapping score (only baseswith mapping score of >18 were scored). The raw basecounts for each qualified base were converted to percent-age relative to sequence coverage at corresponding pos-ition. We defined mutation candidates as non-referencebases with percentage value greater than 4 standard devia-tions distant to the mean and exceeding 0.1 % and 1 %level in non-amplified samples and amplified samples, re-spectively, due to increased background of base substitu-tions in WGA2 data. All calls detected in more than 75 %of sequenced samples were discarded as artifacts. We per-formed manual curation of all detected calls, in somecases adding calls that were present in other sequencedsamples from the same patient, provided that they couldbe unambiguously identified as outliers on scatter plots ofmatching samples. Finally, we performed haplotype fre-quency estimation through manual curation of alignedreads in IGV. Reads with >7 mismatches with the refer-ence sequence were considered uninformative for sampleswith multiple mutation calls.ConstructsFull-length human AR (WT-AR) was encoded on apcDNA3.1 expression plasmid (Life technologies). TheLBD point mutations (single and multiple) were gener-ated using the QuikChange II Site-Directed MutagenesisKit (Agilent Technologies) as per manufacturer’s instruc-tions using WT-AR as the template. The mutagenicoligonucleotide primers were designed individually withthe desired mutation in the middle of the primer withapproximately 10 to 15 bases of correct sequence onboth sides.Steroid activation assayPC3 cells lacking the AR and authenticated by IDEXX La-boratories (Maine, USA) were maintained in RPMI 1640media (Life Technologies) and 5 % FBS (Hyclone ThermoFisher Scientific) at 37 °C and 5 % CO2. Cultures wereroutinely monitored for mycoplasma contamination. Forthe steroid activation assay, cells were seeded in 96-wellplates (5,000 cells/well) in RPMI 1640 medium with 5 %charcoal-stripped serum (CSS) (Hyclone). After 24 h, cellswere co-transfected with 25 ng of wild-type or mutatedAR and 25 ng of the reporter plasmid pARR3-tk-luciferaseusing TransIT20/20 transfection reagent (3 μL/μg ofDNA) (Mirus Bio LLC, Madison, WI, USA) in Optimemserum-free media (Life Technologies) for 48 h accordingto manufacturer’s suggested protocol. Cells were stimu-lated with increasing concentrations of DHT, estradiol,progesterone, or hydrocortisone in 100 % ethanol (0 to500 nM). Control cells were treated with 100 % ethanolalone. At 24 h after treatment, the medium was aspiratedoff and the cells were lysed by adding 60 μL of 1× pas-sive lysis buffer (Promega) followed by shaking at roomtemperature for 15 min and two freeze/thaw cycles at-80 °C . Twenty microliters of lysate from each wellwere transferred onto a 96-well white flat bottom plate(Corning) and the luminescence signal was measuredafter adding 50 μL of luciferase assay reagent (Promega).The chemical oxidation of luciferin into oxyluciferin bythe luciferase is accompanied by light production that canbe quantified as luminescence by a TECAN M200Pro in-strument. Each concentration was assayed in quadrupli-cate n = 4, with a biological replicate of n = 3. For eachsteroid, results were averaged and normalized by express-ing them as a percentage of WTAR activity.AR inhibition assayPC3 cells were seeded and transfected as described above.At 48 h after transfection, medium was aspirated and re-placed with medium containing 0.1 nM R1881 and either0.1 % DMSO (control) or serial dilutions of increasingconcentrations of AR inhibitors ranging from 0 μM to50 μM (hydroxyflutamide, bicalutamide, ARN509, enzalu-tamide, and VPC-13566). A non-stimulated/no R1881control was used. After 24 h, cells were lysed and AR-dependent luciferase activity was quantified. Each con-centration was assayed in quadruplicate n = 4, with abiological replicate of n = 3. Results were averaged andnormalized by expressing them as a percentage of WTAR activity.Western blottingTwenty microliters of each of the four replicates ofDMSO/control-treated lysate from the luciferase assaywere pooled with 20 uL of 5X sample buffer, boiled for5 min, and 20 uL of the mixture was loaded on a 10 %Lallous et al. Genome Biology  (2016) 17:10 Page 12 of 15SDS-PAGE gel and electrophoresed at 120 V for90 min. Proteins were transferred to PVDF membrane(Millipore) at 100 V for 1.5 h at 4 °C, blocked for 1 h atroom temperature with 5 % non-fat skim milk in TBS,followed by incubation with 1/1,000 dilution of AR N20antibody (sc-816, Santa Cruz Biotechnologies) over-night at 4 °C. Membranes were incubated with 1/5,000dilution of goat anti-rabbit IgG HRP (sc-2030, SantaCruz Biotechnologies) for 1 h at room temperature,washed five times with TBS 0.1 % Tween 20 (Sigma),and bands visualized using Super Signal West Femto(Thermo Scientific) and a digital imager (Syngene G Box).Lanthascreen TR-FRET displacement assayThe displacement of a fluorescein isothiocyanate (FITC)labeled BAG1L peptide (FITC-MAQRGGARRPRGDRERLGSR) from the BF3 pocket of the LBD by the com-pounds VPC13566 and VPC14449 was assessed using aTime-Resolved Fluorescence Energy Transfer (TR-FRET).Compounds were tested in the range of 0.41 to 100 μM ina final concentration of 1.5 % DMSO. The protein AR-LBD, the FITC-BAG1L peptide, and the LanthaScreen®Elite Terbium-labeled anti-His-tag antibody (Life Tech-nologies, PV5863) were used at final concentrations of100 nM, 500 nM, and 5 nM, respectively. Briefly, the(His)6-tagged AR-LBD was prepared at 4X final concen-tration in the buffer (150 mM Li2SO4, 50 mM HEPESpH7.5, 10 % Glycerol, 20 μM of DHT and 0.5 mMtris(2-carboxyethyl)phosphine) in the presence of 4XTb-anti-His-antibody (Mix A). Mix B contained 4XFITC-BAG1L peptide in 2 % DMSO. A three-fold serialdilution of the compounds was prepared at 100X finalconcentration in DMSO. The compounds were then di-luted 50-fold in buffer to get a 2X final concentrationand 2 % DMSO (Mix C). In a black flat bottom 384-well plate 5 μL of Mix A, 5 μL of Mix B, and 10 μL ofMix C were added.The plate was incubated at room temperature for 2 hand FRET was analyzed on Synergy-4 multi-plate readerwith the following settings: excitation, 340 nm; emission,495 nm and 520 nm. The emission ratio (520:495) wasanalyzed, normalized to the buffer, and plotted.Cell viability assayPC3 cells were plated at 5,000 cells per well in RPMI1640 containing 5 % CSS in a 96-well plate and treated,after 72 h, with 0.1 nM R1881 and VPC-13566 (0-50 μM).After treatment for 24 h, cell density was measured usingthe 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay accordingto the manufacturer’s protocol (CellTiter 961 AqueousOne Solution Reagent, Promega). The percentage of cellsurvival was normalized to the cell density of control wellstreated by vehicle and 0.1 nM R1881.Availability of supporting dataAll sequence data supporting the results of this articleare available in the European Nucleotide Archive (ENA)repository, with the study accession number PRJEB12109and a direct URL: http://www.ebi.ac.uk/ena/data/view/PRJEB12109.EthicsThis study was approved by the University of BritishColumbia Clinical Research Ethics Board (CREB) withthe certificate approval number H09-01628 (renewed on7 December 2015) and by the Vancouver Coastal HealthResearch Institute (VCHRI) with the certificate numberV09-0320. All patients provided informed written con-sent. All the experimental methods comply with theHelsinki Declaration.Additional filesAdditional file 1: Table S1. The results of the validation of a subset ofdetected mutations on MiSeq, Illumina. The run was designed to test 23mutations in 11 cfDNA samples (both amplified and non-amplified). WGAsamples are marked with *, n/a – sample not sequenced on MiSeq. Onlytwo calls were not supported on MiSeq. S889G call in VC-012-t1 unamplifiedcfDNA was not detected on original 454 run, or on MiSeq resequencing.(DOCX 23 kb)Additional file 2: Figure S1. Western blot showing expression level ofthe CRPC-associated AR mutants in PC3 transfected cells. (TIF 351 kb)Additional file 3: Figure S2. Activation of wild-type AR by dihydrotes-tosterone (DHT), progesterone, estradiol, and hydrocortisone. The graphsrepresent the average ± SEM of three independent experiments with fourreplicates each. (TIF 252 kb)Additional file 4: Table S2. The response of the CRPC-associatedmutants to increasing concentrations of anti-androgens. Abiraterone,bicalutamide, hydroxyflutamide, enzalutamide, ARN509, and an in-housedeveloped AR inhibitor VPC-13566 were tested against the 24 CRPC-associated AR mutants. Each concentration was assayed in quadruplicaten = 4, with a biological replicate of n = 3. Results were averaged andnormalized by expressing them as a percentage of WT AR activity ± SEM.(PDF 345 kb)Additional file 5: Figure S3. AR mutants associated with bicalutamideresistance in CRPC patients. (a) The AR LBD (cartoon representation, ingray) in complex with bicalutamide (ball-and-stick representation, inblue). The residues presented as gray sticks presented an agonist effect inthe presence of bicalutamide in luciferase reporter transcription assay.The B ring of bicalutamide occupies the position that would normally befilled by the indole ring of tryptophan in the non-mutated W741 position(shown in transparent gray) in the LBD. (B) AR mutants showing agonistresponses to bicalutamide by in vitro functional characterization. Eachconcentration was assayed in quadruplicate n = 4, with a biological replicateof n = 3. Results were averaged and normalized by expressing them as apercentage of WT AR activity ± SEM. (TIF 1129 kb)AbbreviationsABS: Androgen binding site; AR: Androgen receptor; BF3: Binding function 3;cfDNA: Cell-free DNA; CRPC: Castration-resistant prostate cancer;DHT: Dihydrotestosterone; LBD: Ligand binding domain; PCa: Prostate cancer.Competing interestsKNC reports receiving commercial research grants and honoraria fromAstellas and Janssen. No potential conflicts of interest were disclosed by theother authors.Lallous et al. Genome Biology  (2016) 17:10 Page 13 of 15Authors’ contributionsConception and design: AC, PSR, CCC, KNC, MEG, AWW, NL, and SVV.Development of methodology and data acquisition: SVV, NL, SA, EL, RT, JM,AAA, SL, and KS. Data analysis: NL, SVV, SA, EL, AC, PSR, and CCC. Writing andrevision of the manuscript: AC, CCC, PSR, NL, SVV, EL, SA, RT, JM, AAA, AWW,SL, MEG, and KNC. Study supervision: AC, PSR, CCC, NL, and SVV. All authorsread and approved the final manuscript.AcknowledgementsWe would like to thank Jan Rennie for editing the manuscript. We thank alsoDr. Takeshi Yamazaki, Dr. Huifang Li, Dr. Yohann Loriot, and Ravi Munugantifor discussions regarding this study. We acknowledge Alisse Saunders andHelene Morin for their technical support.FundingThis work was supported by Prostate Cancer Canada with generous supportfrom Canada Safeway (Grant SP2013-02), Movember Team Grant T2013-1,CIHR/Terry Fox Foundation New Frontiers Program Grant #TFF-116129, anoperating grant (#272111) from the Canadian Institutes of Health Research, aMovember Discovery Program award, and funding from the Canadian CancerSociety Research Institute (grant F12-03271). The salary of Nada Lallous wassupported by Award Number P50CA097186 from the National CancerInstitute.Author details1Vancouver Prostate Centre, University of British Columbia, 2660 Oak St.,Vancouver, BC V6H 3Z6, Canada. 2Department of Medical Oncology, BCCancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada.3Laboratory for Advanced Genome Analysis (LAGA), Vancouver ProstateCentre, 2660 Oak St., Vancouver, BC V6H 3Z6, Canada.Received: 14 September 2015 Accepted: 29 December 2015References1. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al. Abirateroneand increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995–2005.2. Li Z, Bishop AC, Alyamani M, Garcia JA, Dreicer R, Bunch D, et al. Conversionof abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature.2015;523:347–51.3. Beer TM, Armstrong AJ, Rathkopf DE, Loriot Y, Sternberg CN, Higano CS, etal. Enzalutamide in metastatic prostate cancer before chemotherapy. 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BriefBioinform. 2013;14:178–92.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Lallous et al. Genome Biology  (2016) 17:10 Page 15 of 15


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