An Autophagy-Targeting Chimera Induces Degradation of Androgen Receptor Mutants and AR-v7 in Castration-Resistant Prostate Cancer

Abstract

Genetic alterations play a pivotal role in various human diseases, particularly cancer. The androgen receptor (AR) is a crucial transcription factor driving prostate cancer progression across all stages. Current AR-targeting therapies utilize competitive AR antagonists or pathway suppressors. However, therapy resistance often emerges due to AR mutations and AR splice variants, such as AR-v7. To overcome this, we developed ATC-324, an AR degrader using the innovative protein degradation technology platform AUTOphagy-TArgeting Chimera (AUTOTAC). ATC-324 was designed to comprise enzalutamide, an AR inhibitor, as a target-binding ligand and YT 6-2, a ligand of the autophagy receptor p62/SQSTM1, as an autophagy-targeting ligand. ATC-324 induces the formation of the AR/p62 complex, leading to autophagy-lysosomal degradation of AR. Importantly, ATC-324 effectively degrades AR mutants frequently detected in prostate cancer and codegrades AR-v7 as a heterodimer with full-length AR. ATC-324 reduces nuclear AR levels and downregulates the target gene expression of AR and AR-v7, leading to cytotoxicity in AR-positive prostate cancer cells. We also provide evidence of the therapeutic potential of ATC-324 in vivo as well as ex vivo bone organ culture. Moreover, ATC-324 remains potent in enzalutamide-resistant prostate cancer cells. These results demonstrate the potential of the AUTOTAC platform to target previously considered undruggable proteins and overcome certain drug resistance mechanisms. Significance: The characterization of an AUTOTAC-based degrader capable of inducing autophagic degradation of wild-type and mutated androgen receptors demonstrates the potential of this approach for targeting castration-resistant prostate cancer and overcoming drug resistance.

Graphical abstract

Figure 1.

AR-targeting AUTOTACs activate p62-mediated autophagy. A, Chemical structures of AR-targeting AUTOTACs, ATC-323, ATC-324, and ATC-325. The p62-binding and AR-binding moieties are labeled with pink and blue boxes, respectively. B, Immunoblot analysis in WT and p62^−/− MEF cells treated with DMSO (vehicle), YT 6-2 (5 µmol/L), enzalutamide (10 µmol/L), and ATC-323, -324, -325 (5 µmol/L) for 24 hours. The levels of β-actin were examined as a loading control. C, Cell-based p62 oligomerization assay in HEK293T cells treated with ATC-324 and 325 (5 µmol/L, 24 hours). D, Immunofluorescence images of p62 and LC3 colocalization in HeLa cells treated with ATC-324 (2 µmol/L, 24 hours) in the presence or absence of bafilomycin A1 (100 nmol/L, 18 hours) under confocal microscopy. E–G, Quantification of the number of p62 (E) or LC3 puncta (F) and p62⁺LC3⁺puncta (G) in each cell. n = 50 cells. H and I, Immunoblot analysis of LC3 level in LNCaP cells treated with ATC-324 at indicated concentrations for 24 hours (H) and densitometry of H (I). The protein levels were normalized to β-actin. J and K, Immunoblot analysis of LC3 level in LNCaP cells treated with ATC-324 at indicated duration at 5 µmol/L (J) and densitometry of J (K). The protein levels were normalized to β-actin. Data are shown as means ± SEM (n = 3 independent experiments), and statistical significance was analyzed using two-way ANOVA, followed by Tukey post hoc procedures.

Figure 2.

AUTOTAC ATC-324 induces selective autophagic degradation of AR. A, Immunoblot analysis of AR level in LNCaP cells treated with YT 6-2 (5 µmol/L, 24 hours), enzalutamide (10 µmol/L, 24 hours), and ATC-323, 324, 325 (5 µmol/L, 24 hours). The levels of β-actin were examined as a loading control. B and C, Immunoblot analysis of AR level in LNCaP cells treated with ATC-324 at indicated concentrations for 24 hours (B) and densitometry of B (C). The protein levels were normalized to β-actin. D and E, Immunoblot analysis of AR level in LNCaP cells treated with ATC-324 at indicated duration at 5 µmol/L (D) and densitometry of D (E). The protein levels were normalized to β-actin. F and G, Immunofluorescence images of p62 and AR in LNCaP cells treated with enzalutamide (10 µmol/L, 12 hours) and ATC-324 (5 µmol/L, 12 hours) under confocal microscopy (G) and the colocalization was analyzed using the Pearson correlation coefficient (total n = 25 cells). H, Immunoblot analysis of AR in control and p62 knockdown LNCaP cells treated with ATC-324 (5 µmol/L, 6 hours). I, Immunoblot analysis of AR in LNCaP cells treated with ATC-324 (5 µmol/L, 24 hours) in the presence or absence of E64D and pepstatin A. The levels of β-actin were examined as a loading control. Data are shown as means ± SEM (n = 3 independent experiments), and statistical significance was analyzed using one-way ANOVA, followed by Tukey post hoc procedures.

Figure 3.

ATC-324 inhibits proliferation and induces apoptosis in AR-positive prostate cancer cells. A, WST-1 assay of PC3, DU145, LNCaP, and 22Rv1 cells in four replicates, treated at the indicated concentrations of vehicle, YT 6-2, enzalutamide, and ATC-324 for 24 hours. n.s., not significant; PCa, prostate cancer. B and C, Clonogenic cell survival assay of 22Rv1 cells in triplicates, exposed to vehicle, YT 6-2, enzalutamide, and ATC-324 at indicated concentrations (B) and the quantification of B (C). D, Immunoblot analysis of cleaved caspase-3 in PC3, DU145, LNCaP, and 22Rv1 cells treated with ATC-324 at indicated concentrations for 24 hours. The levels of β-actin were examined as a loading control. Data are shown as means ± SEM (n = 3 independent experiments), and statistical significance was analyzed using one-way ANOVA, followed by Tukey post hoc procedures.

Figure 4.

ATC-324 degrades AR-LBD mutants and AR-v7/AR-FL heterodimer. A, Immunoblot analysis of AR with or without LBD mutation (L702H, F877L, T878A, M896V, and H874Y) in PC3 cells transfected with the indicated plasmid and treated with ATC-324 (5 µmol/L, 24 hours). The levels of β-actin were examined as a loading control. B and C, Immunoblot analysis of AR-FL and AR-v7 in 22Rv1 cells treated with ATC-324 at the indicated concentrations for 24 hours (B) and densitometry of B (C). The protein levels were normalized with β-actin. D and E, Fluorescence images of the PLA of FLAG and p62 in PC3 cells (D) and quantification of the number of red puncta (E). The cells were transfected with untagged AR-FL and FLAG-AR-v7 plasmids and treated with indicated chemicals (5 µmol/L, 12 hours). n >30 cells. F, Immunoblot analysis of FLAG in PC3 cells transfected with indicated plasmids and treated with or without ATC-324 (5 µmol/L, 24 hours). The protein levels were normalized to β-actin and the densitometry values of FLAG corresponding to AR-FL and AR-v7 were labeled below the panels. G, Immunoblot analysis of FLAG in PC3 cells transfected with indicated plasmids and treated with or without ATC-324 (5 µmol/L, 24 hours). The protein levels were normalized to β-actin and the densitometry values of FLAG corresponding to AR-FL and AR-v7 were labeled below the panels. Data are shown as means ± SEM (n = 3 independent experiments), and statistical significance was analyzed using one-way ANOVA, followed by Tukey post hoc procedures.

Figure 5.

ATC-324 decreases the levels of both cytoplasmic and nuclear AR, including chromatin-bound AR, and suppresses the transcriptional activity of AR-FL and AR-v7. A, Immunoblot analysis of AR-FL and AR-v7 in subcellular fractions of 22Rv1 cells treated with ATC-324 for 24 hours at the indicated concentrations. B, Immunoblot analysis of AR-FL and AR-v7 in the chromatin-bound fraction of 22Rv1 cells treated with ATC-324 for 24 hours at the indicated concentrations. Subcellular fractionation was validated through the presence of HSP-90, transferrin receptor (TsfR), SP1, histone H1, and CK8. C, GSEA plots with the NES of the AR signature (29), NEPC signature (30), AR-v7-upregulated (31) and downregulated gene sets (32) in 22Rv1 cells treated with ATC-324 (5 µmol/L, 24 hours), compared with vehicle groups. D, qRT-PCR of AR-target genes (PSA, ATAD, KLK2, and TMPRRS2) and AR-v7–specific upregulated genes (NUP210, EDN2, UBE2C, and BUB1) in 22Rv1 cells treated with vehicle, YT 6-2 (5 µmol/L, 24 hours), enzalutamide (5 µmol/L, 24 hours), and ATC-324 (5 µmol/L, 24 hours). n = 3. E, Heatmap of the expression levels of indicated gene sets among vehicle-, YT 6-2 (5 µmol/L, 24 hours)-, enzalutamide (5 µmol/L, 24 hours)-, and ATC-324 (5 µmol/L, 24 hours)-treated 22Rv1 cells (n = 3). Expression levels of each gene were log₂-transformed, and the signature score was generated from GSEA using gene set variation analysis that is a nonparametric, unsupervised method for estimating variation of gene set enrichment through the samples of an expression data set. Data are shown as means ± SEM (n = 3), and statistical significance was analyzed using two-way ANOVA, followed by Tukey post hoc procedures. n.s., not significant.

Figure 6.

ATC-324 inhibits the growth of prostate cancer in xenograft models. A, Schematic of establishing LNCaP and 22Rv1 xenografts in NSG mice and the treatment schedule with ATC-324 (20 mg/kg). B and C, Body weight of LNCaP xenograft mice (B) and 22Rv1 xenograft mice (C) from the vehicle and ATC-324–injected groups measured at the end of the experiment. D, Tumor volume of LNCaP xenograft mice measured on the indicated day. E and F, Hematoxylin and eosin (H&E) staining (E) and IHC analysis of AR (F) of LNCaP xenograft tumor from vehicle and ATC-324–injected groups. G, Tumor volume of 22Rv1 xenograft mice measured on the indicated day. H–K, Hematoxylin and eosin staining (H) and IHC analysis of AR-FL and AR-v7 (I), Ki67 (J) of 22Rv1 xenograft tumor from vehicle and ATC-324-injected groups. K, Quantification of J. L and M Luminescence of bone pieces derived from luciferase-expressing 22Rv1 cells in the BICA assay (L) and the quantification of luminescence (M). The number of bone pieces ≥15. Data are shown as means ± SEM and statistical significance was analyzed using unpaired t tests and linear mixed-effects models, followed by multiple comparison correction. (LNCaP xenograft; Veh., ATC-324: n = 3/22Rv1 xenograft; Veh.: n = 3, ATC-324: n = 4). n.s., not significant. (A, Created with BioRender.com.)

Figure 7.

ATC-324 degrades AR and exhibits cytotoxicity in enzalutamide-resistant prostate cancer cells. A, WST-1 assay of control and enzalutamide-resistant LNCaP and LAPC4 cells in four replicates, treated with at indicated concentrations of vehicle, YT 6-2, enzalutamide, and ATC-324 for 5 days. B, Immunoblot analysis of AR in control and enzalutamide-resistant LAPC4 and LNCaP cells treated with increasing concentrations of YT 6-2, ATC-324, and enzalutamide. The protein levels were normalized to β-actin. Data are shown as means ± SEM and statistical significance was analyzed using two-way ANOVA, followed by Tukey post hoc procedures.