Supplemental Material Androgen Receptor Antagonism Drives Efficacy of CYP17 Inhibitors in CRPC John D. Norris1#, Stephanie J. Ellison1#, Jennifer G. Baker1, David B. Stagg1, Suzanne E. Wardell1, Sunghee Park1, Holly M. Alley1, Robert M. Baldi1, Alexander Yllanes1, Kaitlyn J. Andreano, James P. Stice1, Scott A. Lawrence1, Joel R. Eisner2, Douglas K. Price3, William R. Moore2, William D. Figg3, and Donald P. McDonnell1* 1 Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27710 2 Innocrin Pharmaceuticals, Durham, NC 27703 3 Genitourinary Malignancies Branch, National Cancer Institute, NIH, Bethesda, MD 20892 *To whom correspondence should be addressed: Donald P. McDonnell Email: [email protected] Phone: 919-684-6035 Fax: 919-691-7139 # Contributed equally to this work. 1 Supplemental Tables and Figures GR PR MR Enzalutamide > 20 μM 2.7 E-06 > 20 μM Seviteronel > 20 μM 9.0 E-06 > 20 μM Galeterone > 20 μM 1.9 E-06 > 20 μM Abiraterone > 20 μM 2.1 E-07 > 20 μM RU 486 7.9 E-09 1.0 E-11 ND Spironolactone ND ND 3.2 E-09 Supplemental Table 1. Effects of CYP17 inhibitors on transcriptional activation of GR, PR, and MR. CV1 cells were transfected with MMTV-luc, Renilla-luc, and either glucocorticoid (GR), progesterone (PR), or mineralocorticoid (MR) receptor constructs. Cells were then treated for 24 hours with increasing doses of the indicated antagonists in the presence of 0.1 nM of appropriate agonist: GR, dexamethasone; PR, R5020; MR, aldosterone. Luciferase values were measured and IC50 values are shown in the table. ND, not determined. 2 A B PSA-Luc CV1 VCAP LNCaP AR β-actin + Testosterone Supplemental Figure 1. Effects of CYP17 inhibitors on PSA-Luc (A) Protein extracts from CV1 cells transfected with wt-AR expression vector, MMTV-luc, and Renilla-luc were analyzed for AR expression using anti-AR antibody (N20). Protein extracts from VCAP and LNCaP cells are included as controls for endogenous AR expression. β-actin is included as a loading control. (B) CV1 cells were transfected with PSA-luc and treated for 24 hours with the indicated ligand in the presence of 1.0 nM testosterone. Cells were lysed and assayed for luciferase activity. Error is reported as SD from a representative experiment performed in triplicate. 3 A B 80000 150000 No Competitor No Competitor Enz, 0.2 μM Sevi, 3.0 μM 60000 Enz, 0.6 μM Sevi, 10 μM s 100000 s t i t i Enz, 2.0 μM n Sevi, 30 μM n U U t t 40000 gh i gh i L L 50000 20000 0 0 -14 -12 -10 -8 -6 -14 -12 -10 -8 -6 Log [Ligand] Log [Ligand] C D 80000 No Competitor 80000 No Competitor Gal, 1.0 μM Abi, 3.0 μM 60000 Gal, 3.0 μM 60000 Abi, 10 μM s s t t i Gal, 10 μM i Abi, 30 μM n n U U t 40000 t 40000 gh gh i i L L 20000 20000 0 0 -14 -12 -10 -8 -6 -14 -12 -10 -8 -6 Log [Ligand] Log [Ligand] Supplemental Figure 2. CYP17 inhibitors are competitive AR ligands. CV1 cells were transfected with wt-AR, MMTV-luc, and Renilla-luc, followed by treatment with increasing concentrations of R1881 and the indicated dose of competitor ligand (A) enzalutamide, (B) seviteronel, (C) galeterone, or (D) abiraterone. Luciferase activity was measured after 24 hour incubation. Error bars indicate SD of triplicate samples of a representative experiment performed in duplicate. Shifts in EC50 but not Vmax values indicate competitive antagonism. 4 KLK3 NKX3-1 25 10 s l 20 e 8 v Le 15 Veh A 6 Veh RN 10 T e 4 T v i t a l 5 e 2 R 0 0 i l i t i l i t h c z a r h c z a r Ve Bi En Sev G Ab O Ve Bi En Sev G Ab O Supplemental Figure 3. KLK3 and NKX3.1 expression in LNCaP cells. LNCaP cells were treated with vehicle, 10 nM testosterone (T), and 10 µM of the indicated ligand for 24 hours. Real-time PCR was performed to assess KLK3 and NKX3.1 mRNA expression. Error bars represent the SD from triplicate samples of a representative experiment performed in triplicate. 5 100 80 G1 S 60 G2/M 40 Percentage of Cells 20 0 Veh T Enz Sevi Gal Abi Ort + T Supplemental Figure 4. CYP17 inhibitors prevent entry of prostate cancer cells into S phase. LNCaP cells were treated with vehicle, 10 nM testosterone (T), and 10 µM of the indicated ligand for 24 hours. DNA was stained with propidium iodide and cell cycle analysis was performed using flow cytometry. Data are presented as percentage of cells in G1, S, or G2/M phase. Representative data are shown from an experiment performed in triplicate. 6 DU145 20000 Veh Enz 15000 Sevi U Gal F 10000 R Abi 5000 0 0 2 4 6 Days of Treatment Supplemental Figure 5. CYP17 inhibitors have minimal effects on AR-negative DU145 cell growth. DU145 cells were treated with 10 µM of the indicated compound and analyzed for cell growth by Hoechst dye (DNA content) on 1, 3, and 5 days post-treatment. Error bars represent SD of triplicate samples of a representative experiment performed in duplicate. 7 KLK3 NKX3-1 15 8 s s l l e e v v 6 Le Le 10 A A Veh Veh 4 RN RN e e T T v v i i t t 5 a a l l 2 e e R R 0 0 i l i t i l i t h c z a r h c z a r Ve Bi En Sev G Ab O Ve Bi En Sev G Ab O Supplemental Figure 6. KLK3 and NKX3.1 expression in LNCaP-AR cells. LNCaP-AR cells were treated with vehicle, 1.0 nM testosterone (T), and 10 µM of the indicated ligand for 24 hours. Real-time PCR was performed to assess KLK3 and NKX3.1 mRNA expression. Error bars represent the SD from triplicate samples of a representative experiment performed in triplicate. 8 A B WT T877A 40000 Enz 25000 Enz Bic Bic 20000 30000 Sevi Sevi s s t t i Gal i n n 15000 Gal U U t 20000 Abi t Abi gh gh i i 10000 L L 10000 5000 0 0 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 Log [Ligand] Log [Ligand] C D W741C F876L 40000 20000 Enz Enz Bic Bic 30000 15000 Sevi Sevi s s t t i i n Gal n Gal U U t 20000 Abi t 10000 Abi gh gh i i L L 10000 5000 0 0 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 Log [Ligand] Log [Ligand] Supplemental Figure 7. CYP17 inhibitors do not display agonist activity on wild-type or mutant AR. CV1 cells were transfected with MMTV-luc, Renilla-luc, and either (A) wt-AR, (B) AR-T877A, (C) AR-W741C, or (D) AR-F876L expression plasmids. Cells were then treated for 24 hours with increasing doses of indicated ligands in the absence of R1881 and luciferase activity was measured. Error bars represent SD of triplicate samples for a representative experiment performed in duplicate. 9 KLK3 NKX3-1 80 8 s s l l e e v v 60 6 Le Le A A Veh Veh 40 4 RN RN e e T T v v i i t t a a l l 20 2 e e R R 0 0 i l i t i l i t h c z a r h c z a r Ve Bi En Sev G Ab O Ve Bi En Sev G Ab O Supplemental Figure 8. KLK3 and NKX3.1 expression in LNCaP AR-F876L cells. LNCaP AR F876L cells were treated with vehicle, 3.0 nM testosterone (T), and 10 µM of the indicated ligand for 24 hours. Real-time PCR was performed to assess KLK3 and NKX3.1 mRNA expression. Error bars represent the SD from triplicate samples of a representative experiment performed in triplicate. 10 A LNCaP-AR B LNCaP-F876L 16000 18000 16000 14000 14000 U U F 12000 F R R 12000 10000 10000 8000 8000 -16 -14 -12 -10 -8 -16 -14 -12 -10 -8 Log [Ligand] Log [Ligand] Supplemental Figure 9. R1881 promotes growth of AR overexpression and enzalutamide- resistant CRPC models. (A) LNCaP-AR or (B) LNCaP-F876L cells were treated with increasing doses of R1881. Following seven-day incubation, cell growth was measured by assessing DNA content using Hoechst dye. Error bars represent SD of triplicate samples from a representative experiment performed in duplicate. 11 A LNCaP 1000 Veh Sevi, 15 mg/kg r ) 800 o 3 Sevi, 50 mg/kg m m u Sevi, 100 mg/kg T (m 600 e m age r 400 e olu v a V A 200 0 0 7 14 21 28 Days of Treatment B KLK2 SGK1 1.5 1.5 n n o o i i ss ss e e r r 1.0 1.0 p p x x E E ed ed z z li li a a 0.5 0.5 m m r r o o N N P < 0.01 P < 0.01 0.0 0.0 e l e l Vehicl Vehicl Seviterone Seviterone TMPRSS2 DBI 1.1 1.5 n n o o i i 1.0 ss ss e e r r 1.0 p p x x 0.9 E E ed ed z z 0.8 li li a a 0.5 m m r r o o 0.7 N N P < 0.05 P < 0.05 0.6 0.0 e l e l Vehicl Vehicl Seviterone Seviterone Supplemental Figure 10.