Darolutamide Potentiates the Antitumor Efficacy of a PSMA-targeted Thorium-227 Conjugate by a Dual Mode of Action in Prostate Cancer Models

Abstract

Purpose: Androgen receptor (AR) inhibitors are well established in the treatment of castration-resistant prostate cancer and have recently shown efficacy also in castration-sensitive prostate cancer. Although most patients respond well to initial therapy, resistance eventually develops, and thus, more effective therapeutic approaches are needed. Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and presents an attractive target for radionuclide therapy. Here, we evaluated the efficacy and explored the mode of action of the PSMA-targeted thorium-227 conjugate (PSMA-TTC) BAY 2315497, an antibody-based targeted alpha-therapy, in combination with the AR inhibitor darolutamide.

Experimental design: The in vitro and in vivo antitumor efficacy and mode of action of the combination treatment were investigated in preclinical cell line-derived and patient-derived prostate cancer xenograft models with different levels of PSMA expression.

Results: Darolutamide induced the expression of PSMA in androgen-sensitive VCaP and LNCaP cells in vitro, and the efficacy of darolutamide in combination with PSMA-TTC was synergistic in these cells. In vivo, the combination treatment showed synergistic antitumor efficacy in the low PSMA-expressing VCaP and in the high PSMA-expressing ST1273 prostate cancer models, and enhanced efficacy in the enzalutamide-resistant KUCaP-1 model. The treatments were well tolerated. Mode-of-action studies revealed that darolutamide induced PSMA expression, resulting in higher tumor uptake of PSMA-TTC, and consequently, higher antitumor efficacy, and impaired PSMA-TTC-mediated induction of DNA damage repair genes, potentially contributing to increased DNA damage.

Conclusions: These results provide a strong rationale to investigate PSMA-TTC in combination with AR inhibitors in patients with prostate cancer.

©2021 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.

Darolutamide and PSMA-TTC combination shows synergistic antitumor efficacy in vitro. Isobolograms for the in vitro combination effect of PSMA-TTC and darolutamide on the proliferation of VCaP (A) and LNCaP (B) prostate cancer cells. PSMA (FOLH1) expression in VCaP (C) and LNCaP (D) cells treated with 5 kBq/mL PSMA-TTC and/or 2 μmol/L darolutamide as determined by qRT-PCR 48 hours after treatment (n = 2). The expression is presented as fold change compared with untreated cells. Cell surface PSMA expression as determined by flow cytometry in darolutamide or enzalutamide-treated VCaP (E) and LNCaP (F) cells. DMSO served as a baseline control and is depicted with a dashed line. CI, combination index; DMSO, dimethyl sulfoxide.

Figure 2.

Darolutamide induces PSMA expression and shows synergistic antitumor efficacy with PSMA-TTC in the VCaP prostate cancer model. Male CB17-Scid mice were treated with vehicle, a single dose of 150 or 300 kBq/kg PSMA-TTC (intravenous, total protein dose 0.14 mg/kg, treatment day indicated with a green arrow) and/or 100 mg/kg darolutamide [(twice daily)× 23, orally, treatment period indicated with a blue bar]. A, Growth curves of VCaP tumors (n = 11–12). B, VCaP tumor weight at the end of the study (n = 11–12). C,PSMA RNA expression in VCaP tumors as determined by qRT-PCR (n = 2–4). D, PSMA protein expression in VCaP tumors as determined by immunoblotting (n = 4–7). E, Thorium-227 accumulation in VCaP tumors 66 hours after dosing (n = 4). F, Expression of phosphorylated Chk2 (pChk2) in VCaP tumors as determined by immunoblotting (n = 3–6). Signals from pChk2 were normalized to total Chk2. Statistical analyses were performed using linear models followed by Dunnett or Sidak method. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in comparison with vehicle. ##, P < 0.01; ###, P < 0.001 in comparison with the corresponding darolutamide monotherapy. †, P < 0.05; ††, P < 0.01; †††, P < 0.001 in comparison with the corresponding PSMA-TTC monotherapy.

Figure 3.

PSMA-TTC and darolutamide show synergistic antitumor efficacy in the ST1273 prostate cancer PDX model. A, Growth curves of ST1273 tumors in female NMRI nude mice (n = 9–10) treated with vehicle, 250 kBq/kg PSMA-TTC (total protein dose 0.14 mg/kg, intravenous, treatment day indicated with a green arrow) and/or 15, 30, or 100 mg/kg darolutamide [(twice daily)× 21, orally; treatment period indicated with a blue bar]. B, Tumor volumes of individual ST1273 tumors shown in A on day 33. C, Growth curves of ST1273 tumors in female NMRI nude mice (n = 10) treated with vehicle, 75, 125, or 250 kBq/kg PSMA-TTC (intravenous, total protein dose 0.14 mg/kg; treatment day indicated with a green arrow) and/or 100 mg/kg darolutamide [(twice daily)× 21, orally; treatment period indicated with a blue bar]. For the generation of the mean tumor volume curves in A and C, the last measured tumor volume values of mice which were euthanized due to large tumor size were included into the calculation of the mean value in the graph until n ≧ 8 (last study day shown in graph). D, Tumor volumes of individual ST1273 tumors shown in C on day 19. Statistical analyses were performed on day 33 or day 19 for the studies shown in A and C, respectively, using the estimated linear model followed by Sidak method. **, P < 0.01; ***, P < 0.001 in comparison with vehicle. #, P < 0.05; ##, P < 0.01; ###, P < 0.001 in comparison with the corresponding darolutamide monotherapy. †, P < 0.05; ††, P < 0.01; †††, P < 0.001 in comparison with the corresponding PSMA-TTC monotherapy.

Figure 4.

Darolutamide shows enhanced antitumor efficacy in combination with PSMA-TTC in the KUCaP-1 prostate cancer PDX model. Male CB17-Scid mice (n = 10 mice/group) were treated with vehicle, 150 kBq/kg PSMA-TTC every 2 weeks (Q2W) × 2, intravenous, total protein dose 0.43 mg/kg, treatment days indicated with green arrows), 200 mg/kg darolutamide (once daily, orally, treatment period indicated with a blue bar) or their combination. A, Growth curves of KUCaP-1 PDX tumors. B, KUCaP-1 tumor weights at the end of the study. Statistical analyses were performed using linear models followed by Sidak method. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in comparison with vehicle on day 33; ###, P < 0.001 in comparison with darolutamide monotherapy on day 33.

Figure 5.

Darolutamide impairs the PSMA-TTC–mediated induction of DNA repair genes in the ST1273 PDX prostate cancer model. A, Enrichment plot of androgen-dependent genes after treatment with 250 kBq/kg PSMA-TTC alone (FDR = 0.004) or in combination with 100 mg/kg (twice daily) darolutamide (FDR = 0.0014). B, Enrichment plot of DNA repair genes after treatment with 250 kBq/kg PSMA-TTC alone (FDR = 0.0026) or in combination with 100 mg/kg (twice daily) darolutamide (FDR = 0.29). RNA-seq analysis of tumor samples was performed 72 hours after treatment. Data were compared with the untreated group and hallmark data sets from the Molecular Signatures Database v7.1. C, Expression of the differentially expressed genes included in the DNA repair hallmark gene set used in A. Red and blue colors indicate higher or lower expression of the selected genes compared with mean expression, respectively, after treatment with 250 kBq/kg PSMA-TTC alone or in combination with 100 mg/kg darolutamide in comparison with untreated control. D, γH2AX expression as determined by IHC in untreated ST1273 tumors and in ST1273 tumors treated with 250 kBq/kg PSMA-TTC alone or in combination with 100 mg/kg darolutamide (n = 3). IHC analysis was performed 336 hours after treatment. Scale bars indicate 100 μm. E, γH2AX expression in tumor tissues shown in D, quantified with HSA software. *, P < 0.05; **, P < 0.01 in comparison with untreated control. FDR, false discovery rate.