Published OnlineFirst May 16, 2016; DOI: 10.1158/1535-7163.MCT-15-0892 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide Stefan Prekovic1, Martin E.van Royen2, Arnout R.D.Voet3,4, Bart Geverts2, Rene Houtman5, 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 domain that lead to a decrease in steric clashes for enzalutamide. of the androgen receptor (AR) are known to change antagonists Ligand-binding assays confirmed that the F877L mutation into agonists. Recently, the F877L mutation has been described leads to an increase in relative binding affinity for enzaluta- to convert enzalutamide into an agonist. This mutation was mide, but only the combination with the T878A mutation seen to co-occur in the endogenous AR allele of LNCaP cells, resulted in a strong agonistic activity. This correlated with next to the T878A mutation. Here, we studied the effects of changes in coregulator recruitment and chromatin interactions. enzalutamide on the F877L and T878A mutants, as well as Our data show that enzalutamide is only a very weak partial the double-mutant AR (F877L/T878A). Molecular modeling agonist of the AR F877L, and a strong partial agonist of the revealed favorable structural changes in the double-mutant AR double-mutant AR. Mol Cancer Ther; 15(7); 1702–12. Ó2016 AACR. Introduction chemotherapy by a median of 4.8 months in comparison with the placebo group (5). On the other hand, enzalutamide has no Globally, prostate cancer is the fourth most common cancer beneficial effect in a portion (20%) of patients, while the and the fifth leading cause of cancer-related death in men (1). As majority of patients who initially respond will eventually develop this cancer is hormone-dependent, the blockade of the androgen resistance towards this therapy (6). It is imperative, therefore, to receptor (AR) signaling is an effective therapeutic strategy for men study the different mechanisms that can lead to enzalutamide with advanced metastatic prostate cancer. The discovery of novel resistance. compounds that inhibit the AR and their clinical application has Prior studies of resistance against the earlier generation anti- led to the improvement in survival time. One such compound is androgens, bicalutamide (Bic) and hydroxyflutamide (Hof), led the potent antiandrogen enzalutamide, which has been approved to the discovery of mutations that changed the ligand-binding under the name Xtandi for use in postchemotherapy patients with characteristics of the AR and thus lead to the change from an metastatic castration-resistant prostate cancer (mCRPC; refs. 2, 3). antagonistic to an agonistic behavior (7, 8). Balbas and colleagues Enzalutamide acts as a direct competitor for dihydrotestosterone transduced LNCaP cells (which carry the T878A mutation) with (DHT) binding to the ligand-binding pocket of the AR. In addi- constructs driving overexpression of a library of mutated AR and tion, it reduces the binding of the AR to DNA, and inhibits the were able to show that the F877L mutation converts enzalutamide recruitment of AR coactivators (4). Importantly, enzalutamide to an agonist (9). In addition, LNCaP xenograft tumors which significantly prolonged the survival of men with mCRPC after express the AR F877L mutant show sustained growth in castrated mice treated with enzalutamide (10). Another study in LNCaP cells pointed out that the F877L mutation had arisen in the 1Molecular Endocrinology Laboratory, Department of Cellular and endogenous AR T878A allele when cells were treated with enza- Molecular Medicine, KU Leuven, Leuven, Belgium. 2Department of lutamide for a prolonged period of time (10). It should be noted Pathology, Erasmus MC, Rotterdam, the Netherlands. 3Structural Bio- informatics Team, Division of Structural and Synthetic Biology, Center that the LNCaP cells are also mutated in the mismatch repair gene for Life Science Technologies, RIKEN, Yokohama, Japan. 4Laboratory (MSH2), along with over 1,800 other mutations and have a for Biomolecular Modeling and Design, Department of Chemistry, KU relatively unstable genome (11). However, the detection of very 5 Leuven, Leuven, Belgium. Pamgene International, the Netherlands. low levels of the same mutation in the circulating tumor DNA of 6Department of Urology, University Hospitals Leuven, Leuven, Bel- gium. 7Laboratory for Genetics of Malignant Disorders, Department of patients who were treated with ARN-509 (ARN) spiked a high Human Genetics, KU Leuven, Leuven, Belgium. 8Erasmus Optical clinical interest in the F877L mutation (12). Imaging Center, Erasmus MC, Rotterdam, the Netherlands. Several studies reported the co-occurrence of the F877L and Note: Supplementary data for this article are available at Molecular Cancer the T878A mutations (F877L/T878A, hereinafter referred to as Therapeutics Online (http://mct.aacrjournals.org/). the double-mutant AR) in cell line models (10, 12, 13). Corresponding Author: Frank Claessens, Molecular Endocrinology Laboratory, Interestingly, this double-mutant AR was described in a patient KU Leuven, Campus Gasthuisberg O&N1, Herestraat 49 Box 901, Leuven 3000, who progressed on enzalutamide treatment (14). Here, we Belgium. Phone: 321-633-0253; Fax: 321-6343-0735; E-mail: wanted to compare the agonistic activity of enzalutamide on [email protected] the AR F877L, as well as on the double-mutant AR. We dis- doi: 10.1158/1535-7163.MCT-15-0892 covered that the LNCaP-specific T878A mutation modulates the Ó2016 American Association for Cancer Research. F877L responses to enzalutamide. 1702 Mol Cancer Ther; 15(7) July 2016 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst May 16, 2016; DOI: 10.1158/1535-7163.MCT-15-0892 Molecular Analysis of Androgen Receptor Mutants Materials and Methods structure was taken from PDB entry 4OJB, and resistance mutations were introduced. Subsequently, the structure was Plasmid constructs optimized using Protonate_3D and the Amber99 force field Expression vectors, pSG5 and pEGFP-C1, containing cDNA of embedded in MOE (21). Conformations of the AR ligands wild-type flag- or EGFP-tagged human AR, respectively, (15) were (DHT, enzalutamide, ARN, and RD162) were calculated using used to generate the mutations in the ligand-binding domain (LBD) the MMFF94x force field. As a reference for the correct torsional by two-step PCR site-directed mutagenesis. The resulting PCR angle between the 3-fluoro-cyano-benzyl group and the thiox- fragments were ligated into pGEM-T Easy (Promega) and recloned oimidazolidin, PDB entry 2NW4 was utilized (22). The top five as AsuII/BglII fragments into the pSG5-flag-WT AR construct or the binding modes for all the receptor–ligand pairs were visually pEGFP-C1-WT AR construct. The WT DBD/LBD fragment of AR inspected and exhibited nearly identical binding properties inserted into pSG5 backbone (16) was used to introduce mutations with the exception of enzalutamide fitted in the WT and the via two-step PCR site–directed mutagenesis. The same cloning T878A AR LBD. As a positive control DHT and Hof were docked strategy as described for the full-length AR constructs was used. into the agonistic conformation of AR WT or T878A LBD, The construct containing double-tagged (N-ter YFP and C-ter CFP) respectively. The top docking scores exhibited a similar docking WT AR (17) has been used to generate mutations in the LBD using mode within 0.4 Å RMSD deviation of the crystallographically QuikChange II kit (Agilent Technologies). A plasmid containing determined structure. pCMV-b-Gal was acquired from Stratagene, the luciferase reporter The ligands were docked into the ligand binding pocket of the construct driven by 4 copies of a classical ARE and the VP16 AD- receptor followed by a short energy minimization allowing minor NTD(WT) expression vector have been described elsewhere "induced fit" changes to the protein and ligand conformation. The (16, 18). All expression vectors were sequence verified. binding mode was scored using the London Free Energy scoring fi Cell culture function for prediction of af nity and rescored using the DrugS- Hep3B and LAPC4 were provided by the A.B. Houtsmuller and coreX scoring function for analysis of the complementarity and colleagues (Erasmus Medical Center, Rotterdam, the Nether- clashes between the ligand and the receptor atoms (23). lands) and the Laboratory for Experimental Medicine and Endo- crinology, respectively, whereas PC-3 and COS-7 cells were Whole cell competition assay obtained from the ATCC and were authenticated by short tandem HEK293T cells were seeded at a density of 30,000 cells per well repeat DNA profiling by Genetica. HEK293T cell line was a kind in 48-well plates. Cells were transfected with 375 ng of AR b gift from the Laboratory of Biosignaling and Therapeutics (KU expression constructs, and 75 ng of pCMV- -Gal expression Leuven) in 2004. HEK293T, PC-3, and COS-7 cells were cultured vector. On the day after the transfections, medium was refreshed in DMEM supplemented with 10% FCS, while Hep3B cells were and cells were incubated overnight. Subsequently, cells were m cultured in MEMa with GlutaMAX supplement and without treated with a dilution series (0.1 nmol/L to 10 mol/L) of compound with 1 nmol/L [3H]-labeled mibolerone (Perkin nucleosides (Gibco). LAPC4 cells were cultured in Iscove's Mod- ified Dulbecco's Medium (Gibco) supplemented with 10% FCS. Elmer). After an incubation period of 90 minutes at 37 C, medium was aspirated, cells were washed three times with ice- Androgen receptor transactivation assay cold PBS, and lysed in 100 mL of Passive Lysis buffer (Promega). HEK293T or PC-3 cells were seeded at a density of 15,000 cells per Cell lysates (75 mL) were transferred to scintillation vials and 2 mL well or 10,000 cells per well, respectively, in 96-well plates.