American Association for Cancer Research
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Supplementary Tables S1-S2 and Figures S1-S15 from The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

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journal contribution
posted on 2023-04-03, 15:48 authored by Stefan Prekovic, Martin E. van Royen, Arnout R.D. Voet, Bart Geverts, Rene Houtman, Diana Melchers, Kam Y.J. Zhang, Thomas Van den Broeck, Elien Smeets, Lien Spans, Adriaan B. Houtsmuller, Steven Joniau, Frank Claessens, Christine Helsen

Table S1: Docking scores for the ligands and the AR mutants. Table S2: FRAP simulation data. Figure S1: Luciferase reporter assay for AR negative prostate cancer cell line (PC3 cells). We used 100 ng of reporter construct; and 10 ng of androgen receptor constructs and B-Gal construct, each. Cells were stimulated overnight. Figure S2: Luciferase reporter assay: DHT stimulation of the WT and mutated receptors. Figure S3: Transactivation potential of AR WT or AR mutant in response to selected compounds was assessed by transient transfection of an androgen regulated luciferase reporter construct in HEK293T cells and increasing amounts of AR construct (5 ng/well, 50 ng/well and 100 ng/well). The mean and standard error of the mean of 3 independent experiments are shown. A) AR WT, B) AR T878A, C) AR F877L, D) AR F877L/T878A. Figure S4: Transactivation potential of AR WT or AR mutant in response to different batches of Enza. The mean and standard error of the mean of 3 independent experiments are shown. Figure S5: Visualization of the steric influence of T878A mutation on the binding of Enza. The modeled structure of human AR F877L/T878A can comfortably accommodate the binding of Enza (A). However the more bulky threonine 878 side chain in the single mutant AR F877L causes steric hindrance for the binding of the Enza (B). Figure S6: Influence of the double mutant AR on binding of Hof. In the single AR mutant, phenylalanine (877) is able to hydrophobically interact with the dimethyl function of the Hof, which is contacting alanine (878), thereby stabilizing Hof in the agonistic conformation of the ligand (A). In the double mutant however, the leucin (877) which is smaller and more flexible than phenylalanine is unable to interact with Hof (B). Figure S7: The DHT bound agonistic conformation of the AR LBD is depicted as a cartoon with the bound DHT in white sticks (A). The interaction of the DHT with the mutated residues is further highlighted for the WT AR LBD (B) and double mutant (C). The interaction score as represented in panel D. Figure S8: Mammalian double hybrid assay was used to asses induction of N/C interactions by DHT, Enza and Hof. A) AR WT, B) AR T878A, C) AR F877L, and D) AR F877L/T878. Figure S9: MARCoNI assay: clustering for DHT- and Enza-stimulated receptors. Figure S10: MARCoNI assay: clustering for DHT- and Hof-stimulated receptors. Figure S11: MARCoNI assay: clustering for DHT-, Enza- and Hof-stimulated receptors. Figure S12: Induction factors for Hep3B stable cell lines with AR F877L and AR F877L/T878A. Figure S13: FRAP curves and computational simulation data for ARN-stimulated AR. Figure S14: The double AR mutant is targetable by other AR antagonists. HEK293T cells were used to investigate the activity of ARAN-compounds on transactivation of AR. Transcriptional activity of AR was not induced by ARAN-4, ARAN-51 and ARAN-53. However, these compounds successfully reduced the DHT-induced transactivation of AR for both the WT and the mutant receptors. The mean and standard error of the mean of 3 independent experiments are shown. A) AR WT; B) AR F877L; C) AR F877L/T878A. Figure S15: Cell viability assay of LAPC4 cells transfected with either the WT AR or the mutant receptors. Expression of the double mutant AR rescued the cells from the effect of Enza (A; n=3), while the cells were rescued from the effect of Hof when the T877A or the double mutant AR were transfected (B; n=3). Abi was able to inhibit proliferation of LAPC4 regardless of the construct transfected (C; n=2). The mean and standard error of the mean are shown.



KU Leuven




Treatment-induced mutations in the ligand-binding domain of the androgen receptor (AR) are known to change antagonists into agonists. Recently, the F877L mutation has been described to convert enzalutamide into an agonist. This mutation was seen to co-occur in the endogenous AR allele of LNCaP cells, next to the T878A mutation. Here, we studied the effects of enzalutamide on the F877L and T878A mutants, as well as the double-mutant AR (F877L/T878A). Molecular modeling revealed favorable structural changes in the double-mutant AR that lead to a decrease in steric clashes for enzalutamide. Ligand-binding assays confirmed that the F877L mutation leads to an increase in relative binding affinity for enzalutamide, but only the combination with the T878A mutation resulted in a strong agonistic activity. This correlated with changes in coregulator recruitment and chromatin interactions. Our data show that enzalutamide is only a very weak partial agonist of the AR F877L, and a strong partial agonist of the double-mutant AR. Mol Cancer Ther; 15(7); 1702–12. ©2016 AACR.