Androgen receptor degraders overcome common resistance mechanisms developed during prostate cancer treatment

Abstract

Androgen receptor (AR) antagonists, such as enzalutamide, have had a major impact on the treatment of metastatic castration-resistant prostate cancer (CRPC). However, even with the advent of AR antagonist therapies, patients continue to develop resistance, and new strategies to combat continued AR signalling are needed. Here, we develop AR degraders using PROteolysis TArgeting Chimeric (PROTAC) technology in order to determine whether depletion of AR protein can overcome mechanisms of resistance commonly associated with current AR-targeting therapies. ARD-61 is the most potent of the AR degraders and effectively induces on-target AR degradation with a mechanism consistent with the PROTAC design. Compared to clinically-approved AR antagonists, administration of ARD-61 in vitro and in vivo results in more potent anti-proliferative, pro-apoptotic effects and attenuation of downstream AR target gene expression in prostate cancer cells. Importantly, we demonstrate that ARD-61 functions in enzalutamide-resistant model systems, characterized by diverse proposed mechanisms of resistance that include AR amplification/overexpression, AR mutation, and expression of AR splice variants, such as AR-V7. While AR degraders are unable to bind and degrade AR-V7, they continue to inhibit tumor cell growth in models overexpressing AR-V7. To further explore this, we developed several isogenic prostate cell line models in which AR-V7 is highly expressed, which also failed to influence the cell inhibitory effects of AR degraders, suggesting that AR-V7 is not a functional resistance mechanism for AR antagonism. These data provide compelling evidence that full-length AR remains a prominent oncogenic driver of prostate cancers which have developed resistance to AR antagonists and highlight the clinical potential of AR degraders for treatment of CRPC.

Fig. 1

ARD-61 is a potent PROTAC AR degrader that reduces full-length AR levels in diverse prostate cancer cell lines. a) Structures of AR antagonists 1–4, VHL ligands 5–6, and synthesized PROTAC AR degraders 7–10. b) Western blot analysis of AR degraders 7–10. Degradation of AR protein can be blocked by AR antagonist ARI-16 (4), VHL ligand (5), NEDD8 activating E1 enzyme inhibitor MLN4924, and proteasome inhibitor MG132 in LNCaP and VCaP cells with a 2 hour (hr) pretreatment of these drugs and 6 hr treatment of degrader. c) Representative western blots of protein collected from whole cell lysates from a variety of AR-positive prostate cancer cell lines, with different AR mutation statuses (CWR-R1, LNCaP) and AR variant expression (CWR-R1, 22Rv1), treated with increasing concentrations of the lead compound ARD-61 (7) for 24 hours. PARP cleavage (cPARP) was utilized as a readout of apoptosis. AR target genes PSA (KLK3) and NKX3.1 shown for LNCaP. AR-FL, AR full-length; AR-V7, AR variant 7. VCaP cells treated with increasing concentrations of ARD-61 for 24 hours had total AR levels quantified using the ProteinSimple® assay (n = 3). d) Quantitative proteomic analysis of LNCaP and VCaP cells after 6 hr treatment of 100 nM ARD-61 compared to control (DMSO). AR is highlighted as the most downregulated protein in both cell lines; red dashes indicate proteins that met significance (>0.5 change compared to control; >0.05 p-value, n = 3). Tandem mass spectroscopy was performed on tandem mass tagged whole cell lysates.

Fig. 2

ARD-61 decreases AR signaling and inhibits prostate cancer cell growth in vitro and in vivo. a) Growth curves of VCaP cells assayed by Cell Titer Glo ATP assay® treated with increasing concentrations of ARD-61 and enzalutamide for 5 days (n = 6). b) RNA-sequencing was performed on VCaP cells treated with 100 nM ARD-61 for 6 hr compared to control (DMSO, n = 3). AR target genes, in red with a few representative genes labeled, were significantly downregulated, as illustrated by gene set enrichment analysis (GSEA – inset). c) Western blot of whole tumor lysates from pharmacodynamics studies to confirm ARD-61 induces degradation of AR protein in LNCaP prostate xenograft tumor tissues in intact mice at 6, 24, and 48 hr. Efficacy analysis showed that ARD-61 at 50 and 25 mg/kg, IP, qD, 5 times a week can inhibit tumor growth (n = 10) in both LNCaP (d) and VCaP (e) models (mice intact), even after cessation of dosing (indicated by black dashed line on the graph).

Fig. 3

Enzalutamide-resistant prostate cancer models remain sensitive to ARD-61 treatment. a) Half-maximum inhibitory concentration (IC:50) values of ARD-61 and enzalutamide on the growth of AR-positive and -negative cancer cell lines, including both prostate cancer and one AR-positive breast cancer cell line, BT474 (n = 6). ARD-61 shows an effect in both enzalutamide-sensitive and -resistant cells that are AR-positive, while there is no effect in the AR-negative prostate cancer and normal cell lines. b) RNA-sequencing was performed on parental LNCaP cells treated with 100 nM ARD-61 for 24 hours compared to control. AR target genes (red), with a few labeled representative genes, are significantly downregulated, as illustrated by gene set enrichment analysis (GSEA) plot (n = 3). c) RNA-sequencing was performed on enzalutamide-resistant (Enz^R) LNCaP cells with 100 nM ARD-61 for 24 hours compared to control. AR target genes (red) are downregulated from a low baseline (n = 3). GSEA of RNA-sequencing shows significant decreases of AR signaling targets normally upregulated by AR. d) Total RNA extracted from parental LNCaP and LNCaP Enz^R cells treated with ARD-61 for 24 hours at concentrations ranging from 1 nM to 10 µM and analyzed by Q-RT-PCR for the mRNA transcripts of the AR target genes PSA (KLK3), TMPRSS2, and NKX3-1 (n = 3). e) Growth curves of tumor volume illustrating the effects of ARD-61 and enzalutamide on castration-resistant VCaP xenografts. Mice were injected with VCaP prostate cancer cells subcutaneously, and when the tumors reached 200 mm³ in size, the mice were castrated. Once the tumor grew back to the pre-castration size, the animals were treated with control (n = 10) or enzalutamide (10 mg/kg). After three weeks (21 days marked with dashed red line), half of the mice (n = 8) treated with enzalutamide were switched to ARD-61 (50 mg/kg).

Fig. 4

The growth inhibitory effects of ARD-61 occur through degradation of full-length AR and are independent of AR-V7 expression. a) Schematic of the creation of CRISPR-induced overexpression clones in LNCaP cells. b) Representative western blots of protein collected from whole cell lysates from four representative cell lines derived from individual single cell clones with CRISPR-induced AR-V7 expression treated with increasing concentrations of ARD-61 for 24 hours (short and long western blot exposures). c) Growth curves of LNCaP-V7 clones vs parental cells treated with ARD-61 (top panel), enzalutamide (middle panel), or bicalutamide (bottom panel) (n = 6). d) Growth curves of AR-V7 expressing 22Rv1 cells treated with ARD-61 compared to enzalutamide, assayed by Cell Titer Glo ATP® (n = 6). e) RNA-sequencing was performed on LNCaP AR-V7 overexpressing cells treated with 100 nM ARD-61 for 24 hours compared to control. AR target genes, in red with a few representative genes labeled, are significantly downregulated, and illustrated by gene set enrichment analysis (GSEA) plot (inset). f) With similar analysis to (e), RNA-sequencing of LNCaP parental cells compared to LNCaP AR-V7 overexpressing clones shows an increase in AR pathway gene expression (GSEA plot; inset). g) Growth curves of tumor volume illustrating the effects of ARD-61 and enzalutamide on enzalutamide-resistant CWR-R1 Enz^Rin vivo. Castrated mice were injected subcutaneously with CWR-R1 Enz^R cells, and when the tumors reached 100 mm³ in size, the mice were treated with control (n = 10), enzalutamide (50 mg/kg, n = 10), or ARD-61 (50 mg/kg, n = 10) qd, 5 times a week throughout the duration of the experiment.