Intracrine Metabolism of Testicular Androgens by Castration-Recurrent

Prostate Cancer – Are There Opportunities for Novel Treatments?

James L. Mohler, MDAssociate Director and Senior Vice President for Translational Research

Chair, Department of UrologyProfessor of Oncology

Roswell Park Cancer InstituteProfessor, Department of Urology

University at Buffalo, State University of New YorkBuffalo, New York

• 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

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

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.

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.

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

Tissue Androgen Levels using RIA in Benign Prostate (n = 32; gray)

vsCastration-Recurrent CaP (n = 23;

white)

Mohler JL, et al. Clin Cancer Res. 2004;10(2):440-448.

LC-MS/MS of DHT and T in Benign Prostate Tissue

DHTMW 291

TMW 289

Titus MA, et al. Clin Cancer Res. 2005 ;11(13):4653-4657.

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 LHRHDES 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 DESorch 9.1 1.5 3.9 12.4 fluDES 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.

Testicular Androgen Levels in Castration-Recurrent CaP

Mass Spec RIA Titus 2005 Mohler 2004

T DHT T DHT AS-BP (n=18) 2.75 13.7 AS-BP (n=30) 3.26 8.13 CR-CaP (n=18) 3.75 1.25 CR-CaP (n=15) 2.78 1.45

Montgomery 2008 Geller 1979 T DHT T DHT AS-BP (n=6) 0.04 1.92 AS-BP (n=17) - 17.6 AS-CaP (n=4) 0.23 2.75 CaP orch ± DES (n=9) - 4.47 CR-Met CaP (n=8) 0.74 0.25 CaP DES 1 mg (n=6) - 12.4

Labrie 1989 T DHT human CaP (n=?) - 18.6 orch (n=5, 2-12m) - 9.29

orch+fl (n=4, 2m) - ND 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.

Intracrine Metabolism of Testicular Androgens

• DHT from weak adrenal androgens

• DHT from cholesterol

Increased Levels of Enzymes that Make Testosterone

Stanbrough M, et al. Cancer Res. 2006;66(5):2815-2825.

Intracrine Metabolism of Testicular Androgens

• DHT from weak adrenal androgens

• DHT from cholesterol

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.

Testicular Androgen Production from Cholesterol

• 14C-cholesterol appears as 14C-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.

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

Origin of Tissue DHT in Castration-Recurrent CaP

Barriers to UnderstandingAndrogen Metabolism

Mohler JL, Wilson EW, unpublished.

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

A. AbirateroneB. TAK-700C. VN124-1D. 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)?

A. AbirateroneB. TAK-700C. VN124-1D. 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)?

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.

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.

Gene NCBI Blast search revealed a gene located

at 4q12 spanning3.1 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

5α-Reductase Type 3

AS-CaPAS-BP CR-CaP

qRT-PCR

IHC

Type 1 Type 2 Type 3

AS-CaP 0.71 ± 0.55 0.047 ± 0.032

0.69 ± 0.36

CR-CaP 0.26 ± 0.11 0.003 ± 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.

5α-Reductase Type 3NOT inhibited by finasteride or dutasteride

Metabolism of T to DHT (pmol/mg/min)

0

250

500

750

1000

Control 5 nM Dutasteride

CWR22 CWR22R

Titus MA, Mohler JL, unpublished.

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.

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

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.

∆TR LTR∆U3LTR∆U3

pTK511

LacZ LTR∆U3LTR∆U3

pTK478

∆TR LTR∆U3LTR∆U3

pTK511

∆TR LTR∆U3LTR∆U3 ∆TR LTR∆U3 LTR∆U3LTR∆U3 LTR∆U3

pTK511

LacZ LTR∆U3LTR∆U3

pTK478

LacZ LTR∆U3LTR∆U3 LacZ LTR∆U3 LTR∆U3LTR∆U3 LTR∆U3

pTK478

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

Median Tumor Volume(to 1st Death)

Kafri, Wilson, UNC, Titus, Mohler, RPCI

Survival

Delta TR + T LacZ + T0

20

40

60

80

100

Day

s

(P = 0.033)

Kafri, Wilson, UNC, Titus, Mohler, RPCI

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

Prostate Endothelial Cells Express Androgen Receptor

Organ-Specific in Clinical Specimens

AS-Benign AS-CaP Kidney

DHT Translocates AR to Nucleus in Primary Cultures of Human ProstateEndothelial Cells

- DHT

+ DHT

AR / vWF

Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.

Human Prostate Endothelial Cellsare Androgen Responsive

Endothelial Apoptosis Peaks on Day 2 after Castration in Human Prostate Xenografts

Control Castration (d2)

CD

34

MVD

Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.

Endothelial Cell AR is Functional andTargetable in vivo

Fluorescent PlateletsAdhere to DamagedEndothelium

Ad-ARE-MMTV

Adenoviral Vectors Administered i.v. Confirm Endothelial Cell Uptake and AR Function

Castration (d2)

Control

CD

34

Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.

ImmunoAnalysisO. Harris Ford, III, MSSwaroop Singh, PhDDiana Mehedint, MDAntony Jeyaraj, PhDDesok Kim, PhD Andrew B. Smitherman, MD

MutationsSheila GreeneOlga Kozyreva, PhDYousef Sharief, PhD

Androgen ReceptorFrank S. French, PhDElizabeth Wilson, PhDPeter Petrusz, PhDSusan J. Maygarden, MDMichael J. Schell, PhD Christopher W. Gregory, PhD

NIEHSKenneth Tomer, PhDFred LihPat StocktonJulie F. FoleyGordon Flake, MD

SupportNCINIADODGSK

Roswell ParkGary J. Smith, PhDAlejandro Godoy, PhDViviana Montecinos, PhD Michael Moser, PhDGregory Wilding, PhDShaozeng Zhang, MD, PhD

Androgen Metabolism Mark Titus, PhDElzbieta Kawinski, PhDCarol WrzosekYun Li

Gene TherapyTal Kafri, MD, PhDBrian Ziethamel