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Targeting androgen receptor phase separation to overcome antiandrogen resistance

Nature Chemical Biology volume 18, pages 1341–1350 (2022)Cite this article

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Abstract

Patients with castration-resistant prostate cancer inevitably acquire resistance to antiandrogen therapies in part because of androgen receptor (AR) mutations or splice variants enabling restored AR signaling. Here we show that ligand-activated AR can form transcriptionally active condensates. Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role. AR liquid–liquid phase separation behaviors faithfully report transcriptional activity and antiandrogen efficacy. Antiandrogens can promote phase separation and transcriptional activity of AR-resistant mutants in a ligand-independent manner. We conducted a phase-separation-based phenotypic screen and identified ET516 that specifically disrupts AR condensates, effectively suppresses AR transcriptional activity and inhibits the proliferation and tumor growth of prostate cancer cells expressing AR-resistant mutants. Our results demonstrate liquid–liquid phase separation as an emerging mechanism underlying drug resistance and show that targeting phase separation may provide a feasible approach for drug discovery.

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Fig. 1: Ligand-stimulated AR translocated to nucleus and formed transcriptionally active condensates.
Fig. 2: All AR domains contribute to effective condensate formation while NTD plays a predominant role.
Fig. 3: AR phase separation mediates the mutation-induced resistance to antiandrogens.
Fig. 4: Identification of ET516 as a potential AR inhibitor from LLPS-based screen.
Fig. 5: ET516 specifically reduce AR transcriptional activity and inhibits growth of CRPC.

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Data availability

Data supporting the findings of this study are presented within the article, the accompanying source data files and the Supplementary Information. RNA sequencing data have been submitted to the Gene Expression Omnibus database under accession GSE185223. Three-dimensional rendered graphics have been uploaded to Figshare (https://doi.org/10.6084/m9.figshare.20484396). Source data are provided with this paper.

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Acknowledgements

We thank J. Yuan for critical reading of the manuscript. We also thank Z. Wang and Y. Zhang for their efforts in crosslinking mass spectrometry. Thank D. Gao and Z. Liu for providing VCaP cells. We would like to acknowledge the staff members of the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Science, China for providing technical support and assistance in MST and nanoscale differentiation scanning fluorimetry data collection and analysis. This work was supported by the National Natural Science Foundation of China (21877123 to G.Z., 81803560 to L.S.).

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Author notes

  1. These authors contributed equally: Jingjing Xie, Hao He.

Authors and Affiliations

  1. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Jingjing Xie, Wenna Kong, Ziwen Li, Lin Sun & Guangya Zhu

  2. Etern Biopharma, Shanghai, China

    Jingjing Xie, Hao He, Wenna Kong, Zhenting Gao, Daoqing Xie, Lin Sun, Xiaofei Fan, Xiangqing Jiang, Qiangang Zheng, Guo Li & Jidong Zhu

  3. Lingang Laboratory, Shanghai, China

    Guangya Zhu

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Contributions

J.X., J.Z., and G.Z. conceived the project. J.X., H.H., and G.Z. performed most of the experiments and data analysis. H.H. performed the data analysis of image and RNA sequencing. W.K. assisted in plasmid construction and Z.L. helped in arranging compound characterization data. Z.G. and D.X. performed MD computer simulations. L.S. and G.L. helped with compound optimization. X.F. performed xenograft mouse model experiments. X.J. and Q.Z. designed and synthesized the compounds. J.X., H.H., J.Z., and G.Z. wrote the manuscript.

Corresponding authors

Correspondence to Jidong Zhu or Guangya Zhu.

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Competing interests

J.Z. is a co-founder of Etern Biopharma. J.X., H.H., W.K., L.S., X.F., X.J., Q.Z., D.X., Z.G. and G.L. are employees of Etern Biopharma. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 DHT-stimulated androgen receptor formed phase-separated condensates.

a. Immunofluorescence (IF) of AR in VCaP cells (AR amplification and some V7). Fluorescence signal is shown alone (green), merged with DAPI stain (blue) (Scale bar, 5 μm) and zoomed view of merged image (Scale bar, 350 nm). b. Live imaging of mEGFP tagged AR in VCaP cells. mEGFP signal is shown alone (green), mEGFP merged with Hoechst stain (blue). Scale bar, 5 μm. c. The immunoblot analysis of AR proteins in LNCaP and VCaP cells stably expressed with AR–mEGFP. d. Live imaging of mEGFP tagged AR in PC3 cells. mEGFP signal is shown alone (green), mEGFP merged with Hoechst stain (blue). Scale bar, 5 μm. e. Live imaging of mEGFP tagged AR in HEK293T cells. mEGFP signal is shown alone (green), mEGFP merged with Hoechst stain (blue). Scale bar, 5 μm. f. Live imaging of mEGFP tagged AR in H1299 cells. mEGFP signal is shown alone (green), mEGFP merged with Hoechst stain (right). Scale bar, 5 μm. g. Live imaging of mEGFP tagged AR in SF268 cells. mEGFP signal is shown alone (green), mEGFP merged with Hoechst stain (blue). Scale bar, 5 μm. h. Live imaging of mScarlet tagged AR in LNCaP cells. mScarlet signal is shown alone (magenta), mScarlet merged with Hoechst stain (blue). Scale bar, 5 μm. i. Schematic and sequencing validation of mEGFP knock-in strategy. j. Representative images of endogenous tagged AR–mEGFP treated with/without DHT stimulation. Scale bar, 5 μm (zoomed, 1 μm).

Source data

Extended Data Fig. 2 Active transcription markers can be detected in foci of AR splice variants.

a. Western blots showing the expression levels of cells expressing AR (full-length, FL) and AR (truncation)-EGFP-Flag. β-actin was used as control. Truncations containing LBD tend to have lower expression levels and we have adjusted the amounts before comparing the LLPS capability of various truncations. b. Time-lapsed images showing the FRAP experiments of AR(V7) puncta in LNCaP cells. Scale bar, 3 μm. (zoomed, 500 nm.) c. LNCaP cells transfected with AR(V7/V567es)–mEGFP were stained with anti-H3K27ac and anti-Pol II-S5P. Quantification of fluorescence intensity of AR(V7/V567es)–mEGFP and three markers along the line indicated in merged image were shown. Scale bar, 5 μm. Co-localization is assessed by Pearson’s correlation coefficient of the fluorescence intensity of each channel for all pixels and shown on the right of each group. NV7 = 82(H3K27ac), 95(PolII-S5P), NV567es = 104(H3K27ac), 120(PolII-S5P) cells. d. mNeonGreen MED1 knock-in HEK293T cells were transfected with AR(V7/V567es)-mTagBFP2 to examine the co-localization between MED1 and AR. Quantification of fluorescence intensity of AR(V7/V567es)-mTagBFP2 and mNeonGreen-MED1 along the line indicated in merged image were shown. Scale bar, 3 μm. Co-localization is assessed by Pearson’s correlation coefficient of the fluorescence intensity of each channel for all pixels and shown on the right of each group. NV567es = 91, NV7 = 89 cells.

Source data

Extended Data Fig. 3 Role of DBD in AR condensate formation and in vitro AR droplet formation.

a. Living-cell image of mEGFP labeled AR(WT) and AR(A574D) treated with DHT in LNCaP cells. Reconstructed images were shown and the puncta number and area of AR variants were analyzed. N = 100 cells. Scale bar, 2 μm. b. Time-lapsed images recording the fusion and fission events of AR(A574D) puncta in LNCaP cells. Scale bar, 5 μm. c. Images of AR(NTD), AR(DBD) and AR(LBD) in vitro phase separation. Each protein was mixed with a LLPS buffer containing 25 mM Tris pH8.0, 15%(w/v) PEG3350 and incubated for 10 min at 37 °C. Scale bar, 5 μm. d. Time-lapsed images of FRAP experiments in AR(NTD)-EGFP droplets. Scale bar, 2.5 μm. e. In vitro phase separation assays of AR(NTD)–mEGFP proteins under different concentrations of sodium chloride. Scale bar, 7.5 μm. f. Predictions of sequences mediating LLPS by catGRANULE score. G425-D496 showed the highest score. g. Western blots showing the expression levels of cells expressing AR (full-length, FL) and AR (deletions)-EGFP-Flag. GAPDH was used as control.

Source data

Extended Data Fig. 4 AR phase separation underlies mutation mediated-antiandrogen resistance.

a. Living-cell imaging of LNCaP cells expressing AR(WT) and AR(T878A)-EGFP treated with hydroxyflutamide (Hof) in the absence/presence of DHT. Scale bar, 3 μm. b. Box plot showing puncta number in LNCaP cells expressing AR(WT) and AR(T878A)-EGFP treated with hydroxyflutamide in the absence/presence of DHT (N = 71-200 cells). c. Transactivation reporter (ARE) assays of HEK293T cells expressing AR(WT) and AR(T878A) treated with hydroxyflutamide in the absence/presence of DHT. Data are presented as the mean ± SD, n = 3 biologically independent samples per group. d. Transactivation reporter (ARE) and puncta number statistical analyses of HEK293T cells expressing AR(WT) and AR(T878A) treated with varying concentrations of 17β-estradiol (E2) and progesterone. Data are presented as the mean ± SD, n = 3 biologically independent samples for E2 treatment and 2 for progesterone treatment. N = 200 cells for puncta analysis. e. Representative images of LNCaP cells expressing mEGFP tagged AR variants treated with E2 and progesterone were shown. Scale bar, 3 μm. f. Living-cell imaging of LNCaP cells expressing AR(WT), AR(T878A) and AR(W742C)-EGFP treated with enzalutamide (Enza) in the absence/presence of DHT. Scale bar, 3 μm. g. Box plot showing puncta number in LNCaP cells expressing AR(WT), AR(T878A) and AR(W742C)-EGFP treated with enzalutamide in the absence/presence of DHT (N = 73-200 cells). h. Transactivation reporter (ARE) assays of HEK293T cells expressing AR(WT), AR(T878A) and AR(W742C) treated with enzalutamide in the absence/presence of DHT. Data are presented as the mean ± SD, n = 3 biologically independent samples per group.

Source data

Extended Data Fig. 5 NTD inhibitor EPI001 exhibits weak inhibition on condensates of AR resistant mutations.

a. Living-cell imaging of LNCaP cells expressing AR(WT), AR(T878A), AR(W742C) and AR(F877L/T878A)-EGFP treated with EPI001 upon DHT stimulation. Scale bar, 2.5 μm. b. Box plot showing puncta number in LNCaP cells expressing AR(WT), AR(T878A), AR(W742C) and AR(F877L/T878A)-EGFP treated with EPI001 in the absence/presence of DHT (N = 50–100 cells). c. Transactivation reporter (ARE) assays of HEK293T cells expressing AR(WT), AR(T878A), AR(W742C) and AR(F877L/T878A) treated with EPI001 in the absence/presence of DHT. Data are presented as the mean ± SD, n = 3 biologically independent samples per group.

Source data

Extended Data Fig. 6 Identification of ET516 as a potential AR inhibitor.

a. Schematic representation of image-based LLPS screen combined with two parallel reporter assays. b. Three-dimensional scatter plot depicting the overall effects on both puncta, transactivation reporter and proliferation of compound library. The high content imaging features were first reduced by principal component analysis (PCA). The dissimilarity was calculated as spatial Euclidean distance to the center of vehicle group using first three components of principal component analysis from high content imaging features. The ARE/PSA reporters were averaged to one dimension. c. Principal component analysis of compound library screening using high content imaging data. All imaging features were extracted with Operetta CLS built-in methods. d. Pairwise scatter plot of ARE/PSA reporter and image-based puncta number (filtered by normalized CTG > 0.7). The linear regression line was added with 95% confidence interval (line range). Pearson correlation coefficient (R) and two-tailed p-value were also calculated and labeled. e. Compounds ranked by the antagonizing effects of AR activity. The inhibitory activity was calculated as normalized spatial distance to vehicle group by merging image level dissimilarity, transactivation reporter and proliferation data in (c). f. Immunoblot analysis of LNCaP cells expressing AR–mEGFP treated with 20 μM ET516 for indicated time points.

Source data

Extended Data Fig. 7 ET516 binds to a redistributed conformation of AF1.

a. Representative western blot showing thermostable AR in HEK293T cells expressing AR-EGFP following heat shock in the presence of 10 μM ET516 with 100 nM DHT. GAPDH was used as loading control. b. Melt and shift curve of AR in HEK293T cells expressing AR–mEGFP treated with ET516 (blue) and DMSO control (black) in the presence of 100 nM DHT. Data are represented as mean ± SEM, n = 3. c. Microscale thermophoresis experiments performed with AR (NTD, 1-555aa)–mEGFP proteins incubated with varying concentrations of ET516/Enzalutamide. mEGFP proteins were used as negative control. MO.Affinity Analysis software was used to fit the data. d. Steady-state fluorescence spectra of AR-AF1(144-450aa) protein(0.3 mg/mL) in 25 mM Tris-HCl pH8.0, 150 mM NaCl buffer treated with ET516. e. Thermal shift assay in AR AF-1(144-450aa, 0.3 mg/ml) incubated with ET516. The heating process increased from 25 to 81.5°C at a ramp rate of 1°C/min. Ratio of emission intensities (Em350nm/Em330nm) was recorded and plotted as a function of temperature. The fluorescence intensity ratio and first derivative were analyzed from PR.ThermControl. f. Principal component analysis (PCA) of Tau5_R2R3 structures extracted from GaMD trajectories. Tau5_R2R3 structures extracted from MD simulations at 2 ps intervals are merged and projected onto PC axes for the first two PCs. Color indicates the reweighted free energy using cumulant expansion to the second order.

Source data

Extended Data Fig. 8 ET516 specifically repress AR-dependent signaling and inhibits cell proliferation of prostate cancer expressing mutants resistant to antiandrogens.

a. GSEA enrichment plots comparing DHT(1 nM)-stimulated LNCaP cells to vehicle cells showing positive enrichment for gene set corresponding to AR target genes. b. Differential regulated signal pathway in ET516 treated LNCaP cells compared to DHT-stimulated control cells. LNCaP cells were treated with ET516 two hour prior to the DHT stimulation for 10 hours. c. GSEA enrichment plots comparing ET516 treated LNCaP cells to control cells showing no obvious enrichment for gene set corresponding to ER and p53 target genes compared to AR target genes. d. ET516 was subjected to a panel of transcription factor reporter assays. Data are presented as the mean ± SD, n = 3 replicates. e. A panel of condensates assays to test the specificity of ET516. Scale bar, 5 μm. ET516 was added to a series of phase-separated condensates for 2 hours and the puncta number of ET516/vehicle was analyzed. Data are presented as the mean ± SD, n = 4–7 biologically independent samples per group. In d-e, hydrocortisone(10 nM), E2(10 nM) and IL-6(4 ng/ml) were used to activate glucocorticoid receptor, estrogen receptor and Stat3, respectively. YAP condensates were induced with TEAD co-transfection. f. Images of LNCaP cells expressing AR(F877L/T878A) treated with DHT combined with DMSO (vehicle), ET516 and enzalutamide (Enza) for 8 days (Top). Brightfield images of LNCaP cells expressing AR(V7) treated with DMSO (vehicle), ET516 and enzalutamide (Enza) for 8 days (Bottom). Scale bar, 100 μm. g. Representation of hollow fiber assay for evaluating the efficacy of ET516, EPI001 and enzalutamide. h. Efficacy results of hollow fiber assay that treated with (ET516, 30 mg/kg, p.o., BID; EPI001, 30 mg/kg, p.o., BID; Enzalutamide, 30 mg/kg, p.o., QD) for 10 days, Data are presented as the mean ± SD, N = 6 mice each group.

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Extended Data Fig. 9 AR V7 exhibits weaker LLPS capability and less sensitive to ET516 compared with full-length.

a. Fluorescence images showing the distribution of AR(FL)–mEGFP and two splice variants AR(V7/V567es)–mEGFP. Scale bar, 3 μm. Statistical analysis of the puncta number was shown on the right. N = 100 cells. b. Representative scheme showing the statistics of puncta fluorescence contrast and the analysis results of AR full length and splice variants. N = 150 cells. c. Box plot showing puncta number in LNCaP cells expressing AR(V7)–mEGFP treated with indicated concentrations of ET516 (N = 100 cells). d. Transactivation reporter (ARE) assays of HEK293T cells expressing AR(V7)–mEGFP treated with indicated concentrations of ET516. Data are presented as the mean ± SD, n = 3 biologically independent samples per group.

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Xie, J., He, H., Kong, W. et al. Targeting androgen receptor phase separation to overcome antiandrogen resistance. Nat Chem Biol 18, 1341–1350 (2022). https://doi.org/10.1038/s41589-022-01151-y

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