Figure 2. AR F876L mutation converts enzalutamide into an agonist and rescues enzalutamide-induced growth inhibition.
(A) A representative FACS histogram shows the induction of AR-dependent EGFP expression by enzalutamide in LNCaP-Pb.PSE.EGFP cells ectopically expressing AR F876L. The magnitude of induction by enzalutamide (10 μM) is comparable to that conferred by the endogenous androgen DHT (1 nM). Enzalutamide treatment of LNCaP-Pb.PSE.EGFP cells ectopically expressing AR WT effectively suppressed EGFP expression. Geometric-mean fluorescence intensity for WT treated cells: vehicle (348), enzalutamide (66.4), DHT (1554); for F876L cells: vehicle (345), enzalutamide (1051), DHT (1699). (B) Cotransduction of CV1 cells with an AR-regulated firefly luciferase construct, a constitutive Renilla luciferase construct, and one of the indicated AR constructs, recapitulates the pharmacology observed in the EGFP reporter system. These cells were treated with vehicle (DMSO), antiandrogens (1 μM), or the synthetic androgen R1881 (1 nM). A dual luciferase assay was conducted on cell lysates, the firefly signal was normalized to the constitutive Renilla activity, and the data are reported as relative light units (RLUs). Notably, the bisaryl-thiohydantoin antiandrogens (enzalutamide and ARN-509) effectively induce AR F876L transcriptional activity, while structurally discrete antiandrogens (hydroxyflutamide and bicalutamide) do not impact AR F876L activity in this assay. As expected, the transcriptional activity of AR W741C or AR T877A was induced by bicalutamide or hydroxyflutamide, respectively. (C) Quantitative reverse transcription–polymerase chain reaction analysis of LNCaP/AR F876L cells shows that enzalutamide (1 μM) can induce the expression of canonical AR-regulated gene products (i.e., PSA, TMPRSS2, SGK1, and FKBP5). Relative gene expression post therapy for LNCaP/AR WT cells is included as positive controls. FL = AR F876L, data are normalized to GAPDH and represented as mean ± SD, n = 3. (D) Cell proliferation data shows that overexpression of AR F876L in a human prostate cancer cell line sensitive to enzalutamide therapy can rescue cell growth. VCaP cells overexpressing either AR WT (solid lines) or AR F876L (dashed lines) were cultured in media containing full serum, treated with either vehicle (DMSO) or 10 μM enzalutamide, and the viable cell fraction was determined at the indicated time points (data is represented as mean ± SD, n = 3). (E) Cellular proliferation data shows that enzalutamide also rescues the growth of VCaP cells expressing AR F876L in androgen-depleted media. VCaP cells overexpressing either AR WT (solid lines) or AR F876L (dashed lines) were treated with vehicle (DMSO), 1 nM DHT, or 10 μM enzalutamide, and the viable cell fraction was determined at the indicated time points (mean ± SD, n = 3). (F) A time to progression study for mice bearing subcutaneous LNCaP/AR-WT (solid lines) or LNCaP/AR-F876L (dashed lines) xenografts further highlights the genotype-dependent pharmacology of enzalutamide. Inoculated animals were treated once daily through oral gavage with either vehicle or enzalutamide (30 mg/kg), and tumor size was monitored weekly (11–16 tumors per treatment group). While enzalutamide potently suppressed the growth of LNCaP/AR-WT tumors, LNCaP/AR-F876L tumors exposed to enzalutamide grew with kinetics roughly equivalent to either vehicle treatment arm. AR: androgen receptor; WT: wild-type.
Figure 2—figure supplement 1. Dose-dependent induction of EGFP expression by enzalutamide in LNCaP-Pb.PSE.EGFP cells expressing AR F876L.
Figure 2—figure supplement 2. Enzalutamide induces AR F876L nuclear translocation and DNA binding to AR enhancer elements.
Figure 2—figure supplement 3. Enzalutamide binds to AR F876L with higher affinity than to AR WT.
Figure 2—figure supplement 4. AR F876L mutation converts ARN-509 into an AR agonist.
Figure 2—figure supplement 5. Dose-dependent agonism in AR F876L expressing cells treated with enzalutamide or ARN-509.
Figure 2—figure supplement 6. Ectopic expression of AR F876L in CWR22Pc cells confers resistance to enzalutamide and rescues growth in androgen-depleted media.
Figure 2—figure supplement 7. Other amino acid substitutions at Phe876 modify the pharmacology of second-generation antiandrogens.
Figure 2—figure supplement 8. Bicalutamide is a modest inhibitor of AR F876L transcriptional activity.
