cancersArticleEvaluation of Darolutamide (ODM201) Efficiency on AndrogenReceptor Mutants Reported to Date in Prostate Cancer PatientsNada Lallous † , Oliver Snow , Christophe Sanchez ‡, Ana Karla Parra Nuñez ‡ , Bei Sun ‡, Ahmed Hussain ,Joseph Lee , Helene Morin, Eric Leblanc , Martin E. Gleave and Artem Cherkasov *,†



Citation: Lallous, N.; Snow, O.;Sanchez, C.; Parra Nuñez, A.K.; Sun,B.; Hussain, A.; Lee, J.; Morin, H.;Leblanc, E.; Gleave, M.E.; et al.Evaluation of Darolutamide(ODM201) Efficiency on AndrogenReceptor Mutants Reported to Date inProstate Cancer Patients. Cancers2021, 13, 2939. https://doi.org/10.3390/cancers13122939Academic Editor: Vasiliki TzelepiReceived: 22 May 2021Accepted: 8 June 2021Published: 11 June 2021Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2021 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, 2660 Oak St.,Vancouver, BC V6H 3Z6, Canada; nlallous@prostatecentre.com (N.L.); oliver_snow@sfu.ca (O.S.);christophe.sanchez997@gmail.com (C.S.); anaparranunez@gmail.com (A.K.P.N.); bei.sun@outlook.com (B.S.);ahmedbhus@gmail.com (A.H.); jlee@prostatecentre.com (J.L.); hmorin@prostatecentre.com (H.M.);eleblanc@prostatecentre.com (E.L.); m.gleave@ubc.ca (M.E.G.)* Correspondence: acherkasov@prostatecentre.com; Tel.: +1-604-875-4818; Fax: +1-604-875-5654† Lallous and Cherkasov labs contributed equally to this work.‡ Authors equally contributed to the work.Simple Summary: Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer in menand one of the leading causes of cancer-related death. The driver of PCa proliferation and growth isthe androgen receptor (AR) and inhibiting this receptor is the standard of care for patients, followingsurgery or radiotherapy. Unfortunately, the effectiveness of current therapeutics is temporary,with the cancer eventually developing drug resistance. Among the mechanisms of resistance are thearising mutations in the AR that make the receptor promiscuously activated by drugs or non-specificligands, thus promoting cancer progression. The aim of this study is to characterize the responses of44 AR mutants, derived from PCa patients, to available steroids that activate the receptor as wellas to various treatments currently used in the clinic. This work will help create a tool to guide themedical team in selecting the best personalized treatment option for each patient.Abstract: Resistance to drug treatments is common in prostate cancer (PCa), and the gain-of-functionmutations in human androgen receptor (AR) represent one of the most dominant drivers of progres-sion to resistance to AR pathway inhibitors (ARPI). Previously, we evaluated the in vitro responseof 24 AR mutations, identified in men with castration-resistant PCa, to five AR antagonists. In thecurrent work, we evaluated 44 additional PCa-associated AR mutants, reported in the literature,and thus expanded the study of the effect of darolutamide to a total of 68 AR mutants. Unlikeother AR antagonists, we demonstrate that darolutamide exhibits consistent efficiency against allcharacterized gain-of-function mutations in a full-length AR. Additionally, the response of the ARmutants to clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids(DHT, estradiol, progesterone and hydrocortisone), was also investigated. As genomic profilingof PCa patients becomes increasingly feasible, the developed “AR functional encyclopedia” couldprovide decision-makers with a tool to guide the treatment choice for PCa patients based on their ARmutation status.Keywords: androgen receptor; antagonists; castration-resistant prostate cancer (CRPC);darolutamide; drug resistance; mutations1. IntroductionThe emergence of drug resistance in prostate cancer (PCa) is a prominent factor ofits progression to castration-resistant PCa (CRPC). Notably, human androgen receptor(AR) remains the main driver in both PCa and CRPC [1]. Therefore, the use of androgendeprivation therapy (ADT) has been the standard of care for PCa patients, relying on directinhibition of AR signalling axes [2]. The remarkable plasticity [3] of the AR in responseCancers 2021, 13, 2939. https://doi.org/10.3390/cancers13122939 https://www.mdpi.com/journal/cancersCancers 2021, 13, 2939 2 of 11to targeted therapies is now well recognized, and there is an impressive repertoire ofAR-genomic and non-genomic mechanisms of treatment escape, including gain-of-functionmutations in the androgen-binding site (ABS) of the receptor [4–8]. It used to be widelyaccepted that such resistance occurs as the result of treatment selection of pre-existing drug-resistant sub-clones of AR. However, a recent study [9] demonstrated that downregulationof DNA mismatch repair (MMR) and homologous recombination (HR) play a significantrole in adaptive mutability in colorectal cancers, occurring as a response to therapeuticpressure. Such adaptive mutability has also been reported for epidermal growth factorreceptor (EGFR) in non-small cell lung cancers [10]. Similarly, in PCa there is a 4-folddifference in mutation rates in metastases compared to primary tumours [11], and a subsetof metastatic PCa presents a hypermutated MMR leading to oncogene activation andtumour heterogeneity [12]. Therefore, adaptive mutability could rapidly contribute to theemergence of acquired drug-resistant sub-clones in advanced PCa.Our group and others have previously demonstrated that the treatment pressuredirected at the AR by clinically used AR antagonists leads to drug-induced mutationsin the AR androgen-binding site (ABS), changing the pocket characteristics and induc-ing receptor activation by adrenal/prostate androgens, by steroidal and non-steroidalligands and, notably, by the AR antagonists themselves [13–16]. Recently, Ledt et al.analyzed the circulating tumour cell free DNA (cfDNA) of 892 patients with advancedPCa, and demonstrated that 32% of patients with AR alterations present nonsynonymousmutations (SNVs or indels) [17]. Similarly, by analyzing the cBio cancer genomics portaldata base [18,19], we found that the frequency of AR mutants can vary between patientcohorts and can reach up to 15% in metastatic CRPC [4,20]. We also reported the resultsof functional characterization of 24 AR mutants identified in liquid biopsies from CRPCpatients or reported in the literature, and demonstrated that all these mutants exhibitedresistance to at least one of four available AR antagonists, including hydroxyflutamide,bicalutamide, enzalutamide and apalutamide [13].The remarkable plasticity of the AR under selective pressure of AR pathway inhibition(ARPI), coupled with the marked heterogeneity and negative prognostic significance ofits cfDNA mutants, indicates that there is no “one size fits all” treatment for PCa patients.Furthermore, the results of our initial functional characterization of clinically observed ARmutants clearly indicate the need for novel AR antagonist(s) capable of inhibiting all formsof AR mutants.Darolutamide, a structurally distinct AR antagonist compared to ABS antagonists hy-droxyflutamide, bicalutamide, enzalutamide and apalutamide (Figure 1), showed completeinhibition of several documented AR-resistant mutants [21] and might provide broaderantagonist activity with emergent AR mutants. Hence, we evaluated the inhibition of 44PCa-associated AR mutants identified in the literature and public databases by darolu-tamide. Additionally, the response of the AR mutants to most clinically used bicalutamideand enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesteroneand hydrocortisone), was investigated.Cancers 2021, 13, 2939 3 of 11Cancers 2021, 13, x 3 of 11    Figure 1. Chemical structures of clinically used AR antagonists. 2. Materials and Methods 2.1. Constructs Full-length human AR (WT-AR) was encoded on a pcDNA3.1 expression plasmid (Life Technologies, Carlsbad, CA, USA). The AR point mutations were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) as per manufacturer’s instructions using WT-AR as the template. The mutagenic oligonucleotide primers were designed individually with the desired mutation in the mid-dle of the primer with ~10–15 bases of correct sequence on both sides (the sequences of the used primers are presented in Table S1). 2.2. Steroid Activation Assay PC3 cells lacking the AR and authenticated by Genetica using STR profiling were maintained in RPMI 1640 media (Life Technologies) and 5% FBS (Hyclone Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO2. Cultures were routinely monitored for mycoplasma contamination. For the steroid activation assay, cells were seeded in 96-well plates (5000 cells/well) in RPMI 1640 medium with 5% charcoal-stripped serum (CSS) (Hyclone). After 24 h, cells were co-transfected with 25 ng of wild-type or mutated AR and 25 ng of the reporter plasmid pARR3-tk-luciferase using TransIT20/20 transfection reagent (3 μL/μg of DNA) (Mirus Bio LLC, Madison, WI, USA) in Opti-MEM serum-free media (Life Technologies) for 48 h according to manufacturer’s suggested protocol. Cells were stimulated with increasing concentrations of DHT, estradiol, progesterone or hydro-cortisone in 100% ethanol (0 to 500 nM). Control cells were treated with 100% ethanol Figure 1. Chemical structures of clinically used AR antagonists.2. Materials and Methods2.1. ConstructsFull-length human AR (WT-AR) was encoded on a pcDNA3.1 expression plasmid(Life Tech ologies, Carlsbad, CA, USA). The AR p i t mutations were generated usingthe QuikChang II Site-Directed Mutagenesis Kit (Agilent Tech ologies, Sant Clara, CA,USA) as per manufacturer’s instr ctio s u ing WT-AR as the template. The mutagenicoligonucl otide primers were designed individually with the desired mutation in themiddle of the primer with ~10–15 bases of correct sequence on both sides (the sequences ofthe used ri ers are presented in Table S1).2.2. Steroid Activation AssayPC3 cells lacking the AR and authenticated by Genetica using STR profiling weremaintained in RPMI 1640 media (Life Technologies) and 5% FBS (Hyclone Thermo FisherScientific, Waltham, MA, USA) at 37 ◦C and 5% CO2. Cultures were routinely monitored formycoplasma contamination. For the steroid activation assay, cells were seeded in 96-wellplates (5000 cells/well) in RPMI 1640 medium with 5% charcoal-stripped serum (CSS)(Hyclone). After 24 h, cells were co-transfected with 25 ng of wild-type or mutated ARand 25 ng of the reporter plasmid pARR3-tk-luciferase using TransIT20/20 transfectionreagent (3 µL/µg of DNA) (Mirus Bio LLC, Madison, WI, USA) in Opti-MEM serum-free media (Life Technologies) for 48 h according to manufacturer’s suggested protocol.Cells were stimulated with increasing concentrations of DHT, estradiol, progesterone orhydrocortisone in 100% ethanol (0 to 500 nM). Control cells were treated with 100% ethanolalone. At 24 h after treatment, the medium was aspirated off and the cells were lysed byCancers 2021, 13, 2939 4 of 11adding 60 µL of 1× passive lysis buffer (Promega, Madison, WI, USA) followed by shakingat room temperature for 15 min and two freeze/thaw cycles at −80 ◦C. Twenty microlitresof lysate from each well was transferred onto a 96-well white flat-bottom plate (Corning,NY, USA) and the luminescence signal was measured after adding 50 µL of luciferase assayreagent (Promega). The chemical oxidation of luciferin into oxyluciferin by the luciferaseis accompanied by light production that can be quantified as luminescence by a TECANM200Pro instrument. Each concentration was assayed in quadruplicate, n = 4, with atleast 3 biological replicates. For each steroid, results were averaged and normalized byexpressing them as a percentage of WT AR activity.2.3. AR Inhibition AssayPC3 cells were seeded and transfected as described above. At 48 h after transfection,medium was aspirated and replaced with medium containing 0.1 nM R1881 (PerkinElmer,Waltham, MA, USA) and either 0.1% DMSO (control) or serial dilutions of increasingconcentrations of AR inhibitors ranging from 0 to 25 µM. After 24 h, cells were lysedand AR-dependent luciferase activity was quantified. Each concentration was assayedin quadruplicate, n = 4, with at least 3 biological replicates. Results were averaged andnormalized by expressing them as a percentage of WT AR activity. Darolutamide waspurchased from MedKoo Biosciences (Cat#206514; Morrisville, NC, USA).2.4. Western BlottingTwenty microlitres of each of 8 replicates of DMSO/control-treated lysate from theluciferase assay were pooled and the total amount of protein was assayed by bicinchoninicacid assay (BCA) (Pierce™, Appleton, WI, USA). Equal amounts of protein samples wereloaded on a 10% SDS-PAGE gel and electrophoresed at 120 V for 90 min. Proteins weretransferred to PVDF membrane (Millipore, Burlington, MA, USA) at 25 V for 15 min usinga TransBlot® Turbo™ Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). Themembrane was then blocked for 30 min at room temperature with 5% non-fat skim milk inTBS, followed by incubation with 1/1000 dilution of AR (441) mouse monoclonal antibody(sc-7305, Santa Cruz Biotechnologies, Dallas, TX, USA) and GAPDH Antibody (G-9) HRPmouse monoclonal antibody (sc-365062, Santa Cruz Biotechnologies) overnight at 4 ◦C.Membranes were then washed and incubated with 1/1000 dilution of Donkey Anti-MouseIgG Polyclonal Antibody (IRDye® 680RD; 925-68072; LI-COR Biosciences, Lincoln, NE,USA) for 1 h at room temperature, washed 3 times with TBS 0.1% Tween 20 (Sigma-Aldrich,St. Louis, MO, USA) and bands visualized using Odyssey Li-Cor Scanner.3. ResultsCharacterization of resistance-associated AR mutants is critically important for pre-dicting and monitoring patients’ response to therapy and ultimately, for the developmentof evidence-based precision oncology practices [22,23]. In this work, we have expandedthe list of functionally characterized AR mutants with an additional 44 variants reported in“The Androgen Receptor Gene Mutations Database World Wide Web Server” of McGillUniversity [24]. These were associated with PCa and mainly localized in either the ligand-binding domain (LBD) or the DNA-binding domain (DBD) of the receptor (Table S1). Weevaluated the response of these 44 AR mutants to increasing concentrations of four steroids(dihydrotestosterone (DHT), progesterone, hydrocortisone and estradiol) as well as threeclinically used AR antagonists, including the first-generation bicalutamide, the second-generation enzalutamide and the most recently approved darolutamide [25].3.1. AR Transcriptional Activation by SteroidsThe response of AR mutants to increasing concentrations of DHT was measured usinga luciferase reporter transcription assay in PC3 cells transiently transfected with eitherwild-type (WT) or mutated AR. The expression level of all of the mutants was evaluatedby Western blotting (Figure S1). Some mutants were activated by lower concentrations ofCancers 2021, 13, 2939 5 of 11DHT than WT (EC50 = 0.14 nM), such as A587V (EC50 = 0.06 nM), K631T (EC50 = 0.05 nM),Q671R (EC50 = 0.05 nM), V756A/I (EC50 = 0.07 and 0.09 nM, respectively), S783N(EC50 = 0.09 nM), Q799E (EC50 = 0.05 nM) and D891N (EC50 = 0.07 nM) (Table 1 andTable S2). Twelve mutants (T576A, A587V, L595M, K721E, G751S, V758A/I, Y764C, S783N,Q799E, D891N and Q903R) with similar or higher affinities than WT were over-stimulatedand reached higher levels of transcriptional activation up to 2 times more than WT(Figure 2A and Table S2). Another set of mutants (L723F, G725D, L745F, N757D, S760P,V867M and L881Q) were stimulated by higher concentrations of DHT with EC50s rangingfrom three to ~40 nM; however, they reached higher levels of activity. One such example isL723F (EC50 = 43 nM), showing 2.5-fold increased activity compared to the wild-type AR(Figure 2B and Table S2). These new results illustrate the very heterogeneous response ofAR mutants to DHT, ranging from complete insensitivity to hyper-activation.Table 1. The activation of AR mutants by various steroids. The EC50 values of the activation by DHT, estradiol, progesteroneand hydrocortisone are reported for the wild-type AR and the 44 studied mutants. For steroid activation, we tested aconcentration range up to 500 nM; therefore, mutants showing no activation or very weak activation in the studied rangeare presented with EC50 values > 500 nM.AR Construct EC50 of DHT(nM)EC50 of Estradiol(nM)EC50 of Progesterone(nM)EC50 of Hydrocortisone(nM)WT 0.14 >500 144 >500T576A 0.12 >500 115 >500K581R weak >500 >500 >500A587V 0.06 >500 168 >500A588S 1.15 >500 >500 >500L595M 0.14 >500 167 >500C620Y >500 >500 >500 >500R630Q 0.10 >500 151 >500K631T 0.05 >500 118 >500E666D 0.11 >500 269 >500Q671R 0.05 >500 170 >500G684A 0.17 >500 148 >500K721E 0.27 >500 >500 >500A722T 0.67 >500 >500 >500L723F 42.66 >500 >500 >500G725D 31.12 >500 >500 >500L745F 23.27 >500 >500 >500A749T 17.56 >500 >500 >500A749V 140.3 >500 >500 >500M750I >500 >500 >500 >500G751S 0.11 >500 134 >500F755L 0.66 >500 >500 >500T756A 0.43 >500 >500 >500N757D 2.67 >500 >500 >500V758A 0.07 >500 >500 >500V758I 0.09 >500 >500 >500S760P 3.33 >500 >500 >500Y764C 0.40 >500 >500 >500S783N 0.09 >500 162 >500S792P >500 >500 >500 >500Q799E 0.05 >500 147 >500I800T 0.55 >500 >500 >500R847G 0.17 >500 267 >500S866P >500 >500 >500 >500V867M 4.62 >500 >500 >500E873Q weak >500 >500 >500D880G 0.85 >500 >500 >500L881Q 40.58 >500 >500 >500Cancers 2021, 13, 2939 6 of 11Table 1. Cont.AR Construct EC50 of DHT(nM)EC50 of Estradiol(nM)EC50 of Progesterone(nM)EC50 of Hydrocortisone(nM)M887I 0.17 >500 253 >500D891N 0.07 134.2 135 >500A897T 0.15 >500 154 >500Q903R 0.74 >500 >500 >500G910E 0.25 >500 >500 >500K911R 0.11 >500 295 >500Q920R 0.15 >500 320 >500Cancers 2021, 13, x 5 of 11   wild-type (WT) or mutated AR. The expression level of all of the mutants was evaluated by Western blotting (Figure S1). Some mutants were activated by lower concentrations of DHT than WT (EC50 = 0.14 nM), such as A587V (EC50 = 0.06 nM), K631T (EC50 = 0.05 nM), Q671R (EC50 = 0.05 nM), V756A/I (EC50 = 0.07 and 0.09 nM, respectively), S783N (EC50 = 0.09 nM), Q799E (EC50 = 0.05 nM) and D891N (EC50 = 0.07 nM) (Table 1 and Table S2). Twelve mutants (T576A, A587V, L595M, K721E, G751S, V758A/I, Y764C, S783N, Q799E, D891N and Q903R) with similar or higher affinities than WT were over-stimulated and reached higher levels of transcriptional activation up to 2 times more than WT (Figure 2A and Table S2). Another set of mutants (L723F, G725D, L745F, N757D, S760P, V867M and L881Q) were stimulated by higher concentrations of DHT with EC50s ranging from three to ~40 nM; however, they reached higher levels of activity. One such example is L723F (EC50= 43 nM), showing 2.5-fold increased activity compared to the wild-type AR (Figure 2B and Table S2). These new results illustrate the very heterogeneous response of AR mutants to DHT, ranging from complete insensitivity to hyper-activation.  Figure 2. Steroid activation of AR mutants in comparison with the wild-type receptor in luciferase reporter assay. AR mutants that showed similar (A) or lower (B) affinities to DHT than WT but reached higher activation levels. Mutants that were better activated by estradiol and progesterone, compared to wild-type, are shown in (C,D), respectively. None of the mutants were activated by hydrocortisone. The graphs represent the average ± SE of three independent experiments with four replicates each. The activity of each mutant in the presence of a steroid was normalized to the wild type stimulated by 500 nM of DHT. Table 1. The activation of AR mutants by various steroids. The EC50 values of the activation by DHT, estradiol, proges-terone and hydrocortisone are reported for the wild-type AR and the 44 studied mutants. For steroid activation, we tested a concentration range up to 500 nM; therefore, mutants showing no activation or very weak activation in the studied range are presented with EC50 values > 500 nM. AR Construct EC50 of DHT (nM) EC50 of Estradiol (nM) EC50 of Progesterone (nM) EC50 of Hydrocortisone (nM) WT 0.14 >500 144 >500 T576A 0.12 >500 115 >500 K581R weak >500 >500 >500 A587V 0.06 >500 168 >500 A588S 1.15 >500 >500 >500 L595M 0.14 >500 167 >500 Figure 2. Steroid activation of AR mutants in comparison with the wild-type receptor in luciferasereporter assay. utants that sho ed si ilar (A) or lower (B) affinities to DHT than WT butreached higher activation levels. Mutants that were better activated by estradiol and progesterone,compared to wild-type, are shown in (C,D), respectively. None of the mutants were activated byhydrocortisone. The graphs represent the average ± SE of three independent experiments with fourreplicates each. The activity of each mutant in the presence of a steroid was normalized to the wildtype stimulated by 500 nM of DHT.We further evaluated the response of 44 studied AR variants to activation by estra-diol, progesterone and hydrocortisone. The wild-type AR was only mildly stimulated byprogesterone with EC50 in the range of 150 nM and was not activated with estradiol orhydrocortisone at concentrations as high as 500 nM (Table 1). Notably, D891N was the onlymutant in the cohort that demonstrated detectable transcriptional activity in the presenceof 100 nM estradiol, and at 500 nM it peaked with 5-fold increased transactivation (com-pared to the wild type). Three other mutants—A587V, G751S and Q799E—demonstratedmodestly enhanced activation by estradiol (Figure 2C and Table S2).Of the 44 studied AR mutants, eight exhibited slightly higher levels of activationby progesterone compared to WT-AR (A587V, R630Q, G751S, R847G, M887I, D891N,K911R and Q920R) (Figure 2D and Table S2). Neither WT-AR nor any of the testedAR variants demonstrated any transcriptional activity when cells were stimulated by500 nM hydrocortisone. Three mutants in particular—A587V, G751S and D891N—madethe receptor promiscuous to activation by progesterone and estradiol while providing abetter affinity or higher activity in the presence of the DHT.Cancers 2021, 13, 2939 7 of 113.2. AR Transcriptional Inhibition by AR AntagonistsWe previously characterized CRPC-associated AR mutants that were mainly locatedin the vicinity of the receptor’s ligand-binding site [13,21]. All the previously studied ARmutants demonstrated activation by at least one clinically used AR antagonist. In thisstudy, we evaluated the response of an additional 44 PCa-associated AR mutants to darolu-tamide along with the drugs broadly used in the clinic, bicalutamide and enzalutamide(Figure 3 and Table S2). None of the mutants exhibited full activation with the tested drugs,with some of them demonstrating a partially agonistic response toward bicalutamide atconcentrations above 6 µM. Of those, A587V and L595M presented the most prominent par-tial agonist behavior corresponding to AR reactivation at concentrations as low as 3.25 µMof bicalutamide. All mutants were efficiently inhibited by the second-generation AR antag-onists enzalutamide and darolutamide, with no significant reactivation at concentrationsup to 25 µM (Figure 3 and Table S2).Cancers 2021, 13, x 7 of 11   demonstrated any transcriptional activity when cells were stimulated by 500 nM hydro-cortisone. Three mutants in particular—A587V, G751S and D891N—made the receptor promiscuous to activation by progesterone and estradiol while providing a better affinity or higher activity in the presence of the DHT. . .  r scri ti l I i iti    t ists  i l  t i  - i t   t t  t t  i l  l t          , .      t  t t  ti ti      l    a ta i t.   ,  l te  the response of an additional 4 PCa-a sociated AR mutants to daro-lutamide along with the drugs broadly used in the clinic, bicaluta ide    3 and Table S2). None of the mutants exhibited full ctivation with the tested drug , with some of them demo strating a partially agonistic response toward bicalutam-ide at conce tr ti ns above 6 μM. Of those, A587V and L595M pres nted the ost prom-inent partial agonist behavior corresponding to AR re ctivation t concentrations as low as 3.25 μM of bicalutamide. All mutants were efficientl  inhibited by th  second-ge era-tion AR antagonists e zalutamide and darolutamide, with no significant rea tivation at concentrations up to 25 μM (Figure 3 and Table S2).  Figure 3. AR mutants showing partial or full agonist response in presence of high concentrations of AR antagonists. (A) Previously characterized AR mutants activated by enzalutamide or bicalutamide treatment. (B) Newly characterized AR mutants showing signs of activation in presence of bicalutamide. Each concentration was assayed in quadruplicate, n = 4, with a biological replicate of n = 3. Results were averaged and normalized by expressing them as a percentage of WT-AR activity. Figure 3. AR mutants showing partial or full agonist response in presence of high concentrations of AR antagonists. (A)Previously characterized AR mutants activated by enzalutamide or bicalutamide treatment. (B) Newly characterizedAR mutants showing signs of activation in presence of bicalutamide. Each concentration was assayed in quadruplicate,n = 4, with a biological r plicate of n = 3. Results were averaged and normalized by expressing them as a percentage ofWT-AR activity.Cancers 2021, 13, 2939 8 of 114. DiscussionAcquired drug resistance represents a paramount danger for PCa patients. Thereare different mechanisms underlying the development of such resistance [3], yet ARremains one of the most dominant drivers in most scenarios. Thus, AR gain-of-functionmutations have been extensively reported in various clinical cohorts (Table S1) [4,13,17,18].Such mutations can promiscuously activate the receptor by non-specific steroids and/orby antiandrogen drugs. Thus, it has been previously observed that 24 patient-derivedCRPC-associated AR mutants can be activated by at least one of the clinically used ARantagonists, including hydroxyflutamide, bicalutamide, enzalutamide and apalutamide.Only darolutamide later demonstrated complete suppression of all 24 AR mutants withsigns of reactivation only in presence of V716M [21] (Figure 3). In this work, we expand thestudy with 44 mutants located in both the LBD and DBD domains of the AR and describedin the McGill database [24], bringing the total number of mutants to 68.Ten of the studied mutants—C620Y, L723F, G725D, L745F, M750I, N757D, S760P,S792P, S866P and V867M—did not demonstrate any detectible transcriptional activity inthe presence of 0.1 nM of the synthetic active anabolic androgenic steroid, the standardAR agonist R1881, in our transcriptional inhibition assay. Out of those ten mutants, C620Y,M750I and S866P did not show any transcriptional activation also in the presence of500 nM DHT, while the other mutants (L723F, G725D, L745F, N757D, S760P, S792P andV867M) required higher concentrations of DHT to reach and surpass the wild-type level ofstimulation (Table 1, Table S2 and Figure 2). Only four mutants showed some activationin presence of estrogen and eight were stimulated by progesterone to similar or higherlevels as wild-type AR (Figure 2). Two mutants in particular, A587V and D891N, made thereceptor promiscuously activated by the DHT, estradiol and progesterone (Figure 2).In recent studies where patients were exposed to CYP17A1 inhibitor abirateroneand/or to first- and second-generation antiandrogens, the recurrent mutations were L702H,W742C/L, H875Y, F877L and T878A/S, that gained a significant spotlight and were char-acterized in our previous study [13]. Herein we report on documented but less noted ARmutants, yet most of them still demonstrated various degrees of activation by bicalutamide(Figure 3 and Table S2). Darolutamide and enzalutamide demonstrated generally verypotent inhibition of all the studied mutants.In summary, out of 68 experimentally evaluated AR mutants (24 reported in the previ-ous works and 44 presented in the current study), 25 demonstrated enhanced activationby DHT, 17 by progesterone, 12 by estradiol and 6 by hydrocortisone, compared to thewild-type receptor (Figure 4). The first-generation bicalutamide behaved as a partial orcomplete agonist for the majority, 43 out of 68, of studied AR mutants (63% of mutants).The second-generation antiandrogen, enzalutamide, demonstrated full or partial activa-tion of eight mutant variants, while the structurally distinct and most recently approveddarolutamide demonstrated significant activation in only one mutant at concentrationsup to 25 µM [21], which identifies a sequencing opportunity for this drug in men withprogressive CRPC with a gain-of-function mutation in the AR under selective pressure offirst-line ARPIs. Evaluating those mutants in mouse models is certainly needed to confirmthe in vitro data reported here and may be a direction for our future work.Cancers 2021, 13, 2939 9 of 11Cancers 2021, 13, x 9 of 11    Figure 4. Summary of functional characterization of AR mutants. We functionally characterized 68 AR mutants, mainly in the LBD, and few in the DBD and Hinge regions. Twenty-five of the stud-ied mutants were activated by DHT, 12 by estradiol, 17 by progesterone and 6 by hydrocortisone to higher levels than wild-type AR. AR mutants showed different response profiles in presence of first- (bicalutamide) and second (enzalutamide)-generation AR antagonists and the newly ap-proved darolutamide. Of the characterized mutants, 15% did not show any activity in our assay. 5. Conclusions Emergent AR mutations in men with advanced PCa treated with ARPI promote CRPC progression. The incidence of AR mutations was estimated to be around 15% for CRPC patients [4] and the availability of circulating tumour DNA assays now provide a sensitive method to serially detect (and treat) the emergence of resistant AR mutants. This current work expanded the list of experimentally evaluated AR mutants with 44 addi-tional examples (bringing the total to 68) and quantified their response to four major en-dogenous steroids and three clinically used AR antagonists, including darolutomide. Among these, only darolutamide demonstrated complete inhibition of 67 out of the 68 studied AR mutant variants, with no significant signs of partial of full activation at even higher concentrations. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: AR mutations identified in the ligand-binding domain (LBD) or the DNA-binding domain (DBD), Table S2: The response of PCa-associated mutants to increasing concentrations of steroids (dihydro-testosterone, progesterone, estradiol and hydrocortisone) and AR antagonists (bicalutamide, en-zalutamide and darolutamide), Figure S1: Western blot showing expression level of the PCa-associ-ated AR mutants. Author Contributions: Conceptualization: A.C., M.E.G. and N.L.; Methodology, validation and in-vestigation: N.L., A.K.P.N., B.S., C.S., A.H., J.L. and H.M.; Formal analysis: N.L., O.S., E.L. and A.C.; Resources: N.L. and A.C.; Writing—original draft preparation: N.L. and A.C.; Writing—review and editing: N.L., O.S., E.L., M.E.G. and A.C.; Visualization: N.L. and A.C.; Supervision, project admin-istration and funding acquisition: N.L. and A.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Figure 4. Su mary of functional characterizati n of AR mu ants. We functionally characterized68 AR mutants, mainly in the LBD, and few in the DBD and Hinge regio s. Tw nty-five of thestudied mutants were activated by DHT, 12 by estradiol, 17 by progesterone and 6 by hydrocortisoneto higher levels than wild-type AR. AR mutants showed different response profiles in presence offirst- (bicalutamide) and second (enzalutamide)-generation AR antagonists and the newly approveddarolutamide. Of the characterized mutants, 15% did not show any activity in our assay.5. ConclusionsEmergent AR mutations in men with advanced PCa treated with ARPI promoteCRPC progre sion. The incidence of AR mutations was estimated to be around 15% forCRPC patients [4] and the availab lity of circulating tumour DNA a says now provide asens tive method to serially detect (and treat) the emergence of resistant AR mutants. Thiscu rent work e list of experimentally evaluated AR mutants with 44 additionalexamples (bringing the total to 68) and quantified their respons to four maj r endogenoussteroids and three clinically used AR antagonists, i cludi g darolutomide. Among these,only darolutamide emonstrated complete inhibition of 67 out of the 68 studied AR mutantvariants, with no significant signs of partial of full activation at even higher concentrations.Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/cancers13122939/s1, Table S1: AR mutations identified in the ligand-binding domain (LBD) orthe DNA-binding domain (DBD), Table S2: The response of PCa-associated mutants to increasingconcentrations of steroids (dihydrotestosterone, progesterone, estradiol and hydrocortisone) andAR antagonists (bicalutamide, enzalutamide and darolutamide), Figure S1: Western blot showingexpression level of the PCa-associated AR muta ts.Author Contributions: Conceptualization: A.C., M.E.G. and N.L.; Methodology, validation andinvestigation: N.L., A.K.P.N., B.S., C.S., A.H., J.L. and H.M.; Formal analysis: N.L., O.S., E.L. a dA.C.; Resources: N.L and A C.; Writing—original draft prepar tio : N.L. and A.C.; Writing—reviewand editing: N.L., O S., E.L., M.E.G. and A.C.; Visualization: N.L. and A.C.; Supervision, projectadministration and funding acquisition: N.L. and A.C. All authors hav read and agreed to thepublished version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Cancers 2021, 13, 2939 10 of 11Data Availability Statement: Not applicable.Acknowledgments: The authors acknowledge Anna Kanyuka and Nicholas Pinette for their technicalsupport.Conflicts of Interest: The authors declare no conflict of interest.References1. Feng, Q.; He, B. Androgen Receptor Signaling in the Development of Castration-Resistant Prostate Cancer. Front. Oncol. 2019, 9,858. [CrossRef]2. Shore, N.D. Current and Future Management of Locally Advanced and Metastatic Prostate Cancer. Rev. Urol. 2020, 22, 110–123.[PubMed]3. Snow, O.; Lallous, N.; Singh, K.; Lack, N.; Rennie, P.; Cherkasov, A. Androgen receptor plasticity and its implications for prostatecancer therapy. Cancer Treat. Rev. 2019, 81, 101871. [CrossRef] [PubMed]4. 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