1. Intracrine Metabolism of Testicular Androgens by Castration-Recurrent Prostate Cancer – Are There Opportunities for Novel Treatments? James L. Mohler, MD Associate Director and Senior Vice President for Translational Research Chair, Department of Urology Professor of Oncology Roswell Park Cancer Institute Professor, Department of Urology University at Buffalo, State University of New York Buffalo, New York

2. Chemotherapy Simplified Docetaxel 1st line regimen No best second line regimen Sipuleucel-T (Provenge®) Asymptomatic or minimally symptomatic ECOG 0-1 2010 NCCN Guidelines Update Castration-Recurrent CaP

3. New Theories for Prostate Cancer (CaP) Recurrence Androgen Receptor (AR) responds to castration with molecular and biochemical alterations that cause hypersensitivity to low levels of ligand CaP responds to castration by synthesizing DHT from weaker androgens and/or cholesterol

4. AR Hypersensitized AR 10,000 times more sensitive in androgen-independent than androgen- sensitive CaP cell lines AR coactivators change from SRC-1 to TIF-2 in cell lines, xenografts, and clinical specimens AR phosphorylated by SRC or Ack1 tyrosine kinases Gregory CW, et al. Cancer Res. 2001;61(7):2892-2898. Agoulnik IU, et al. Cancer Res. 2006;66(21):10594-10602. Guo Z, et al. Cancer Cell. 2006;10(4):309-319. Mahajan NP, et al. Proc Natl Acad Sci U S A. 2007;104(20):8438-8443.

5. LNCaP cells (2 X 106 cells/injection) stably expressing caAck or vector control were injected subcutaneously into the flanks of castrated nude mice. Activated Ack1 Promotes Androgen-Independent Growth of LNCaP Xenografts Mahajan NP, et al. Proc Natl Acad Sci U S A. 2007;104(20):8438-8443.

6. New Theories for CaP Recurrence AR responds to castration with molecular and biochemical alterations that cause hypersensitivity to low levels of ligand CaP responds to castration by synthesizing DHT from weaker androgens and/or cholesterol

7. Tissue Androgen Levels using RIA in Benign Prostate (n = 32; gray) vs Castration-Recurrent CaP (n = 23; white) Mohler JL, et al. Clin Cancer Res. 2004;10(2):440-448.

8. LC-MS/MS of DHT and T in Benign Prostate Tissue DHT MW 291 T MW 289 Titus MA, et al. Clin Cancer Res. 2005 ;11(13):4653-4657.

9. Benign Prostate (n = 18) Castration-Recurrent CaP (n = 18) T (nM) DHT (nM) ADT T (nM) DHT (nM) 3.4 23.6 LHRH+flu 1.6 0.0 0 14.5 orch 3.7 0.0 1.2 16.8 orch+flu 13.6 4.9 1.8 11.3 LHRH 1.2 4.6 2.5 12 LHRH+flu 1.7 0.0 2.9 20.5 orch 3.8 7.8 13.0 17.1 LHRH 5.4 3.9 1.2 13.2 orch 8.6 6.7 2.9 9.8 1° hypogonad 9.8 2.8 1.4 14.3 flu 11.4 1.2 1.6 11.2 orch 1.1 0.0 2.0 6.5 orch 2.5 0.4 2.7 10.7 LHRH  DES 7.2 1.3 2.8 13.7 Lupron 0.0 0.0 2.8 13.7 orch 1.6 0.7 3.2 20.3 orch 6.7 5.2 3.3 38.3 DES  orch 9.1 1.5 3.9 12.4 flu  DES 1.1 0.0 Mass Spec 2.8 13.7 3.8 1.3 RIA 3.2 8.1 2.8 1.5 Titus MA, et al. Clin Cancer Res. 2005 ;11(13):4653-4657.

10. Testicular Androgen Levels in Castration-Recurrent CaP Titus MA, et al. Clin Cancer Res. 2005;11(13):4653-4657. Montgomery RB, et al. Cancer Res. 2008;68(11):4447-4454. Mohler JL, et al. Clin Cancer Res. 2004;10(2):440-448. Geller J, et al. Prog Clin Biol Res. 1979;33:103-111. Labrie F, et al. Br J Urol. 1989;63(6):634-638.

11. Intracrine Metabolism of Testicular Androgens DHT from weak adrenal androgens DHT from cholesterol

12. Increased Levels of Enzymes that Make Testosterone Stanbrough M, et al. Cancer Res. 2006;66(5):2815-2825.

13. Intracrine Metabolism of Testicular Androgens DHT from weak adrenal androgens DHT from cholesterol

14. Testicular Androgen Production from Cholesterol LuCap xenografts and clinical specimens show up- regulation of key enzymes required for metabolism of progesterone to adrenal androgens and then testosterone (Montgomery, Cancer Res, 2008) LNCaP cells up-regulate enzymes required for cholesterol influx, synthesis, and metabolism to produce DHT by “back door” metabolism (Leon, Prostate, 2010) Montgomery RB, et al. Cancer Res. 2008;68(11):4447-4454. Leon CG, et al. Prostate. 2010;70(4):390-400.

15. Testicular Androgen Production from Cholesterol 14 C-cholesterol appears as 14 C-DHT in LNCaP cells thru up-regulation of StAR, the rate-limiting enzyme in steroid synthesis (Locke, Prostate, 2010) DHT synthesis persists in spite of CYP17A1 (ketoconazole) and 5α-reductase-2 (finasteride) inhibition in A-I LNCaP cells and C-R LNCaP xenografts (Locke, J Steroid Biochem Mol Biol, 2009) Locke JA, et al. Prostate. 2010;70(3):239-251. Locke JA, et al. J Steroid Biochem Mol Biol. 2009;115(3-5):126-136.

16. New Treatment Paradigm for Castration-Recurrent CaP Prevent synthesis of tissue androgens Enhance degradation of tissue androgens Inactivate or destroy AR Destroy prostate vasculature Prevent ligand-independent AR activation

17. Origin of Tissue DHT in Castration-Recurrent CaP

18. Barriers to Understanding Androgen Metabolism Mohler JL, Wilson EW, unpublished.

19. Adrenal androgen pathway Cholesterol pathway Backdoor pathway Modified from Locke JA, et al. Cancer Res. 2008; 68(15):6407-6415. Pathways to DHT Synthesis Intact pathway

20. A. Abiraterone B. TAK-700 C. VN124-1 D. MDV3100 E. All of the above What exciting new drugs are in clinical trials that: 1) block the metabolism of adrenal androgens into testicular androgens, or 2) block the affect of testicular androgens (better anti-androgen)?

21. A. Abiraterone B. TAK-700 C. VN124-1 D. MDV3100 E. All of the above What exciting new drugs are in clinical trials that: 1) block the metabolism of adrenal androgens into testicular androgens, or 2) block the affect of testicular androgens (better anti-androgen)?

22. CYP21 Inhibition Abiraterone – Attard, J Clin Oncol, 2008 – Cougar/Johnson & Johnson TAK-700 – Millenium/Takeda VN124-1 – Handratta, J Steroid Biochem Mol Biol, 2004 – Vasaitis, Mol Cancer Ther, 2008 – Tokai Pharmaceuticals Attard G, et al. J Clin Oncol. 2008;26(28):4563-4571. Handratta VD, et al. J Steroid Biochem Mol Biol. 2004;92(3):155-165. Vasaitis T, et al. Mol Cancer Ther. 2008;7(8):2348-2357.

23. Phase II Study of Dutasteride in Prostate Cancer Recurrent During Androgen Deprivation Therapy 25 evaluable men with asymptomatic castration-recurrent CaP (mean age 70, PSA 62, GS 8, and 15 M1b) Safety – Grade 3 or higher adverse events using NCI criteria in 8 men – All judged unrelated to treatment Responses – 14 progressed – 9 stable (3, 3, 3, 4, 4, 5, 5, 9, 9 mo) – 2 partial response [PSA decline > 50%] (5, 11 mo) Shah SK, et al. J Urol. 2009;181(2):621-626.

24. Gene NCBI Blast search revealed a gene located at 4q12 spanning 3.1 kb with possible AP-1 sites Protein Widely distributed protein (318 aa) with homology to Type 1 (20%) and Type 2 (25%) isozymes 5α-Reductase Type 3

25. AS-CaP AS-BP CR-CaP qRT-PCR IHC Type 1Type 2Type 3 AS-CaP0.71 ± 0.550.047 ± 0.032 0.69 ± 0.36 CR-CaP0.26 ± 0.110.003 ± 0.006 0.56 ± 0.15 5α-reductase activity shifts from Type 2 in AS-BP → Type 1 in AS-CaP → Type 3 in CR-CaP Godoy A, Mohler JL, unpublished.

26. 5α-Reductase Type 3 NOT inhibited by finasteride or dutasteride Metabolism of T to DHT (pmol/mg/min) Titus MA, Mohler JL, unpublished.

27. 5  -reductase-3 immunostaining in androgen-stimulated benign prostate (AS-BP), androgen-stimulated high grade intraepithelial neoplasia (AS-HGPIN), androgen-stimulated CaP (AS-CaP), and castration-recurrent CaP (CR-CaP) tissue sections Godoy A, Mohler JL, unpublished.

28. New Treatment Paradigm for Castration-Recurrent CaP Prevent synthesis of tissue androgens Enhance degradation of tissue androgens Inactivate or destroy AR Destroy prostate vasculature Prevent ligand-independent AR activation

29. Inactivate AR Using Antiandrogens Old and relatively ineffective – Flutamide – Bicalutamide – Nilutamide New and perhaps more effective – Small molecule AR antagonist (MDV3100) Tran, Science, 2009 Medivation, Inc. – AR-specific histone deacetylase inhibitors Vorinostat, panobinostat, romidepsin ie, Welsbie, Cancer Res, 2009 Tran C, et al. Science. 2009;324(5928):787-790. Welsbie DS, et al. Cancer Res. 2009;69(3):958-966.

30. Lentiviral vectors containing Delta TR (AR dominant negative) or LacZ (control) “Destroy” AR using AR Dominant Negatives or si/shRNA Kafri, Wilson, UNC, Titus, Mohler, RPCI

31. Median Tumor Volume (to 1st Death) Kafri, Wilson, UNC, Titus, Mohler, RPCI

32. Survival Delta TR + T LacZ + T 0 20 40 60 80 100 Days (P = 0.033) Kafri, Wilson, UNC, Titus, Mohler, RPCI

33. New Treatment Paradigm for Castration-Recurrent CaP Prevent synthesis of tissue androgens Enhance degradation of tissue androgens Inactivate or destroy AR Destroy prostate vasculature Prevent ligand-independent AR activation

34. Prostate Endothelial Cells Express Androgen Receptor Organ-Specific in Clinical Specimens AS-BenignAS-CaPKidney DHT Translocates AR to Nucleus in Primary Cultures of Human Prostate Endothelial Cells - DHT + DHT AR / vWF Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.

35. Human Prostate Endothelial Cells are Androgen Responsive Endothelial Apoptosis Peaks on Day 2 after Castration in Human Prostate Xenografts ControlCastration (d2) CD34 MVD Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.

36. Endothelial Cell AR is Functional and Targetable in vivo Fluorescent Platelets Adhere to Damaged Endothelium Ad-ARE-MMTV Adenoviral Vectors Administered i.v. Confirm Endothelial Cell Uptake and AR Function Castration (d2)Control CD34 Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.

37. ImmunoAnalysis O. Harris Ford, III, MS Swaroop Singh, PhD Diana Mehedint, MD Antony Jeyaraj, PhD Desok Kim, PhD Andrew B. Smitherman, MD Mutations Sheila Greene Olga Kozyreva, PhD Yousef Sharief, PhD Androgen Receptor Frank S. French, PhD Elizabeth Wilson, PhD Peter Petrusz, PhD Susan J. Maygarden, MD Michael J. Schell, PhD Christopher W. Gregory, PhD NIEHS Kenneth Tomer, PhD Fred Lih Pat Stockton Julie F. Foley Gordon Flake, MD Support NCI NIA DOD GSK Roswell Park Gary J. Smith, PhD Alejandro Godoy, PhD Viviana Montecinos, PhD Michael Moser, PhD Gregory Wilding, PhD Shaozeng Zhang, MD, PhD Androgen Metabolism Mark Titus, PhD Elzbieta Kawinski, PhD Carol Wrzosek Yun Li Gene Therapy Tal Kafri, MD, PhD Brian Ziethamel