Targeting The Stress-Induced Cytoprotective Chaperone, Clusterin, to

Overcome Treatment Resistance in Advanced Prostate Cancer

Martin Gleave MD, FRCSC, FACS

Professor, Department of Urologic SciencesUniversity of British ColumbiaB.C. Leadership Chair in Prostate ResearchDirector, The Prostate Centre at VGH

Disclosure of Conflicts of InterestPatent - OGX-011Founder - OncoGenex TechnologiesConsultant - CSO, OncoGenex Technologies

Castration Resistance and Prostate Cancer

HormoneTherapy

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? ? Molecular mechanisms

Seru

m P

SA

Tum

or Volum

e

1. Androgen receptor (AR) related• Overamplification (hypersensitive)• Mutations (promiscuous)• cross-talk - TK, PKA, AKT, STAT3 (phosphorylation, co-regulators)

2. Adaptation• Up-regulation of survival genes (Bcl-2, clusterin, Hsp27, YB-1)• Increased alternative GF pathways (her2/neu; IGF-1/IGFBP2&5; IL-6/STAT3)

High-Throughput Bioprofiling of Hormone-Treated Prostate Cancers to Identify Stress-Induced Targets

Gene expression profiling for Target I.D.

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0 5 10 15 20 25 30 35 40 45

Days

PSA

(ng/

mL)

Cx

AD/ +DHT

AD/ -DHT

AI/ -DHT

Tumour Models

Human CaP

Untreated 1 - 3 months NHT

6 months NHT 8 months NHT HRPC

Post-Hormone-Treated TMA

Tissue Bank:-Xenografts -Post-NHT-treated human CaP-pre-surgery NHT + Taxotere RP specimens-rapid autopsy HRPC specimens

TMA profiling for Target Validation

Hypothesis:Treatment-induced changes in gene

expression after hormone therapy render cells resistant to castration- and chemo-therapy.

++ PSA- Bcl-2

- EGFR- clusterin

- IGFBP 2&5- TGFβ

++IGFBP 3 & 4-YB-1

++survivin-Hsp27

+ PKC-α

- PSA++ Bcl-2++Bclx-L- EGFR

+++ clusterin++++IGFBP 5IGFBP 3 & 4

+c-myc+YB-1

-survivin+Hsp27

+ PKC-α

++ PSA++ Bcl-2++Bclx-L+ EGFR

+++ clusterin++ IGFBP 2++ IGFBP 5

++YB-1++ survivin+++Hsp27

Changes in Gene Expression After Castration Changes in Gene Expression After Castration and During AI Progression and During AI Progression

Androgen-dependent

Tumour regression

Androgen-independent

Clonal selectionAdaptive responses

Hormone withdrawal Progression

AI

ADAI AI

Untreated CaP 3 months NHT

6 month NHT+Tax HRPC

Untreated 1 - 3 months NHT

6 months NHT 8 months NHT HRPC

Post-Hormone-Treated TMA

TMA profiling for Target Validation

Clusterin

Vinculin

Docetaxel Rx (nM) 0 10 25 50 100 500

Docetaxel Rx in PC-3

Transcriptionally activated by- Hormone withdrawal (a.k.a. TRPM-2)- Chemotherapies- Radiotherapy - Targeted therapies (Herceptin, Velcade, OGX-225, etc)

Therapeutic Stress Increases Clusterin Levels in CaP Tumor Models and Human Tissues

Androgen Ablation inShionogi Tumors

GAPDH

Clusterin

Cancer Research 60; 170, 2000

0 3 7 21Days Post-Castration

SCLU-2: Stress-induced Cytoprotective Chaperone

1. Transcriptionally activated by HSF-1, repressed by p53

a. Increased by diverse array of therapeutic triggers (HT, CT, RT, velcade, herceptin)

b. Increased by cell survival factors like androgen, IGF-1

2. Intrinsically disordered and flexible protein

3. Potent inhibitors of aggregation of client proteins under stress conditions

4. Often associated with neurodegenerative diseases and cancer

5. Interact with and inhibit activated Bax; enhances NF-kB transcriptional activity

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Clusterin: Cytoprotective MechanismsClusterin: Cytoprotective Mechanisms

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.QuickTime™ and a

TIFF (LZW) decompressorare needed to see this picture.

sCLU-2 is a COMMD1 and ubiquitin binding partner in cancer cells

Zoubeidi et al, 2007

Input IgG LNCLU2 LNmock

IP: CLU

COMMD1

Input LNmock LNCLU2

IP: COMMD1

CLU

Clu COMMD1 Merge

1. sClu-2 and COMMD1 co-localize in cytoplasm with a juxtanuclear aggregation

0 50 100 250 500 1000 (ng)

Clusterin

Clu transient transfection

Clusterin

COMMD1

Vinculin

COMMD1

sCLU-2

Vinculin

siRNA (25 nM) Scr Clu

Clu Knockdown increasesLevels of COMMD1 & total Iκ-Bα

I-κBα

2. sCLU-2 Levels Negatively Correlate with COMMD1 & Ik-B Levels

sCLU-2 Enhances TNF-a induced NF-kB Nuclear Translocation and Transcriptional Activity

Zoubeidi et al, 2007

10min (TNF-α) 0min (TNF-α)

LNCLU2

LNmock

p65-NF-κB

0100200300400500600700800900

1000

no treatment TNF-a

NF-

κB-lu

c ac

tivity

(fo

ld in

duct

ion/

cont

rol) LN-V

LN-CluLNCLU2

LNmock

1. sClu-2 Overexpression ↑↑ NF-kB Activity

0

0.2

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1

1.2

siRNA Scr siRNA Clu

NF-

kB-lu

c ac

tivity

(fo

ld in

duct

ion/

cont

rol)

PC3

siRNA Scr

siRNA Clu

PC3

p65-NF-κB

10min (TNF-α) 0min (TNF-α)

2. sClu-2 Knockdown ↓↓ NF-kB Activity

Clusterin Expression Levels Positively Correlate with NF-κB - regulated Genes

B Western Analysis

P T T-scr T-siRNA

66kDacIAP2

MCP-1 12kDa

7.5kDMCP-2

Perc

ent c

hang

e In

pro

tein

exp

ress

ion

20

100

80

60

40

cIAP2

MCP-1MCP-2

A

NGAL 845bp

sPLA2-IIa

2255bpSema3C

P T T-scr T-siRNA

Perc

ent c

hang

e In

mR

NA

exp

ress

ion

20

100

80

60

40

Northern Analysis

MIP3α 3000bp

Sema 3c

NGALsPLA2

MIP3α

854bp

* *

Ettinger et al, 2006

All Genes filtered1.5 Fold up in OE (red) 1.5 fold down in KD (blue)

OE KDAll Genes

NGAL

Clusterin

OE KD OE KD

Filtered Genes

sClu-2 Enhances COMMD1 and I-κBαDegradation by the Proteasome

Zoubeidi et al, 2007

I-κBα

p65

p50

I-κBα

p50

ubububP P

p65

nucleus

cytoplasm

20S

19S

19S

ububub

CLU

NF-ΚB

Stressp50

p65

NF-ΚB

COMMD1

P P

I-κBα

Functional Significance of sCLUFunctional Significance of sCLU--2 Over2 Over--expression expression in Prostate Cancerin Prostate Cancer

Cancer Research 60;2547, 2000; Clin Can Res 8:3276-84, 2002Cancer Research 60;170, 2000;

LNCaP over-expressing CLU

LNCaP-CluControls

0102030405060708090

100

0.0001 0.005 0.001 0.005 0.01 0.05 0.1 0.5 1

LNCaP/T1

LNCaP/C

% v

iabl

e ce

lls

Taxol concentration (nM)

IC50

LNCaPsCLU-2

• sCLU overexpression is antiapoptotic: confers broad spectrum treatment resistance including hormone, radiation, and chemo-therapy

CLU ASO (OGX-011) Suppress Clu Levels in PC-3 CellsMM Oligo OGX-011

Cel

l pro

lifer

atio

n co

mpa

red

to c

ontr

ol

Docetaxel concentration (nM)

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100

120

0 15 31 63 125 250 500 1000

PC3-R SiRNA Scr

PC3-R SiRNA Clu

OGX-011 Chemosensitizes PC-3 Cells to Docetaxel

Inhibition of Clusterin Expression Enhances Activity of Chemotherapy in Prostate Cancer Cells

OGX-011 Enhances Taxol Activity in PC3 Tumours in vivo

PC3 Tumor Volume (cm3)

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1.6

1.8

2

0 1 2 3 4 5 6 7 8

Weeks

ASO Clusterin+ Taxol

MM Clusterin + Taxol

Clin Cancer Res 6:1655, 2000

OGX-011

OGX-225 100 nM+

Control ODN

OGX-225 100 nM+

Olig

ofec

tam

ine

OG

X-2

25 1

00 n

M

10 n

M

100

nM

500

nM

10 n

M

100

nM

500

nM

PC-3

Vinculin

CLU

60 kD

40 kD

Clusterin Clusterin is expressed in 65%is expressed in 65% of of Primary BreastPrimary Breast Cancers Cancers

So et al, Mol Cancer Ther, 2005

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0 0.7 1 1.4 2 2.5 2.7 3Log Paclitaxel concentration (nM)

Cel

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mpa

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to c

ontro

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s iRNA Clusterin

siRNA ScramOGX-011

ASO -Scram

Vinculin

+ O

GX

-011

+ Pa

clita

xel

+ Pa

clita

xel

+

OG

X-0

11

60 kDa

40 kDa

55 kDa

CLU ASO (OGXCLU ASO (OGX--011) Suppresses 011) Suppresses sCLU sCLU Levels andLevels andChemosensitizes Chemosensitizes MCFMCF--77 Xenografts Xenografts to to Paclitaxel Paclitaxel in vivoin vivo

In vitro In vitro

Mea

n T

umor

Siz

e (m

m3 )

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1 2 3 4 5S

()

OGX-011SCR

Weeks

* *

*

*

Paclitaxel

Paclitaxel

*

**

60 kDa

55 kDa

s-CLU

n-CLU

Vinculin

ASO SCR OGX-011In vivo In vivo

From Bench to Bedside: Translational Research in Action

sCLU sCLU as a Therapeutic Target:as a Therapeutic Target:Preclinical Studies For Proof of PrinciplePreclinical Studies For Proof of Principle

Of Mice and MenClusterin:•Stress-induced survival response•confers resistance•knockdown enhances chemo & HT in many tumor models

Antisense ClusterinAntisense Clusterin: OGX: OGX--011 Product Description011 Product Description

MOEMOE P-SLicensed from UBC for development by OncoGenex in collaboration with Isis

2nd generation antisense molecule4-13-4 21-mer MOE gapmer oligonucleotide

Advantages of 2'MOE analoguesIncreased potency and resistance to degradationFacilitates more convenient dosing regimen

once-weekly infusionJ Pharmacol Exp Ther. 298(3):934-40, 2001 O C

O

O

G

O

O

T

OO

O

O

A

OO

PO

OO

PO

A

OO

P

OO

P

OO

P

-S

-S

-S

-S

-S

OCH2CH2OCH3

OCH2CH2OCH3

H

H

OCH2CH2OCH3

CLU Isoform 1sCLU-1

CLU Isoform 2sCLU-2

Nuclear splice variant 1nCLU-v1ΔII

siRNA CLU ex3 siRNA CLU ex1

siRNA CLU ex3 OGX-011 (CLU ex2 ASO)

siRNA CLU ex3 OGX-011 (CLU ex2 ASO)

siRNA CLU ex1

I II III IV

IISpliced out

5’-UTR, v1ATG ATGATG

5’-UTR, v2

V VI VII VIII

II III IVATGATG

I III IV5’-UTR, v1

ATG ATG

siRNA CLU ex2

Nuclear localization signalER-targeting signal

Nuclear localization signalER-targeting signal

Nuclear localization signal

IX3’-UTR

V VI VII VIII IX3’-UTR

V VI VII VIII IX3’-UTR

siRNA CLU ex2

NCIC IND.153: Phase I PreNCIC IND.153: Phase I Pre--Surgery Surgery pkpk/pd /pd Trial of OGXTrial of OGX--011 011 -- Tissue Tissue PkPk datadata

Chi et al, JNCI. 97:1287-96, 2005

OGX-011 Dose (mg)6404803201600

Con

cent

ratio

n of

OG

X-0

11 in

Pro

stat

e Ti

ssue

(µ

g of

OG

X-0

11 p

er g

ram

of p

rost

ate

tissu

e) 7

6

5

4

3

2

1

08040

Ptrend <.001

•25 men with localized CaP treated with 5 weeks of NHT + escalating doses of OGX-011

Chi et al, JNCI. 97:1287-96, 2005

IND.153: Target Regulation Data:Dose-dependent suppression of clusterin in Regional Lymph Nodes

Untreated

Controls

6 weeks NHT+ OGX-011(dose escalation) 160 mg

320 mg

480 mg

640 mg

80 mg

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450

No treatment >2 m NHT OGX011-40mg

OGX011-80mg

OGX011-160mg

OGX011-320mg

OGX011-480mg

OGX011-640mg

Treatment Grou

Clinical Proof-of-Concept:Dose-dependent Decreases in Clusterin Levels in RP Specimens using LCM and Real-Time PCR

Chi et al, J Nat.Canc.Inst. 97:1287-96, 2005

Clinical Trial Development with Clinical Trial Development with ClusterinClusterin ASO (OGXASO (OGX--011)011)

Established 640 mg once weeklyas phase II dose (OBD) based on

tissue pk and pd criteria

Completed:2 regimens found well tolerated -• OGX-011 + weekly Taxotere• OGX-011 + q 3 weekly Taxotere

• 30-60% decrease in serum CLU levels

• 10 pts Phase I• 70 pts Phase II• ASCO May 2007

NCIC IND.165:First Line mHRPC

Phase II RCT -Taxotere +/- OGX-011

NCIC IND.164: Phase II Metastatic Breast- Taxotere +

OGX-011

• 82/80 pts enrolled• ASCO May 2007

• 16 pts enrolled

Phase II stage III/IV NSCLC

Gem/cis + OGX-011

CUOG P-06a:2nd line mHRPC

Phase RCT Taxotere or mitoxantrone + OGX-

011

• 42/40 pts enrolled

NHTPre - surgery

Solid Tumors +docetaxel n = 35

Results as of May 24, 2007

Median Follow-up 12.7 months

Estimated Median Survival 14.1 months

Number of Patients Surviving ≥ 1 year 25/46 = 54% *

Number of Patients Surviving ≥ 18 months 8/22 = 36%

Number of Deaths 37/81 (46%)Median Progression-Free Survival (range) 4.6 months (0.06-15.6+)

Historical Controls* Phase 1 and 2 (n=81)

Median Survival 8.0 – 10.8 months 14.1 Months (estimated)

•Data from five randomized clinical trials using gemcitabine plus platinum-based chemoin 1st line NSCLC (1260 patients)

Phase 2 Study in 1st Line NSCLC:Treatment Schema

•81 pts with stage IIIB/IV NSCLC treated with gem/cis plus OGX-011

Laskins et al, ASCO, 2007

Randomized Open label, multicentre trial comparing docetaxel +/- OGX-011 in men with mHRPC (PI - K. Chi).

PSA Response Rates

BEST RESPONSE CRITERIA

Arm A(OGX-011 + Docetaxel)

N=40

Arm B(Docetaxel)

N=41

% Change in favor of OGX-

011

≥ 50% PSA Decline at 12 weeks 45% 34%

PSA Response (50% decline -confirmed)

50% 51% NA

22%

10%

32%

2%

32%

≥ 80% PSA Decline 38% 73%

PSA Progression (PSAWG Criteria)

0% 100%

PSA Non-Progression/Non-Response

45% 41%

Inevaluable 3% NA

NCIC IND.165: Taxotere +/- OGX-011 in First-Line mHRPC

Chi et al, ASCO, 2007

RECISTCRITERIA

Arm A(OGX-011 +

Docetaxel) n=26

Arm B(Docetaxel)

n=23

% Change in favor

of OGX-011Disease Control(CR+PR+SD)

92% 74% 24%

Inevaluable 4% 4% N/A

Complete Response 0% 0% N/APartial Response 19% 22% -14%Stable Disease 73%

9.7 months52%

7.6 months40% 28%

Progressive Disease 4% 22% 82%

Median PFS 7.3 months 5.9 months 24%

NCIC IND.165: Taxotere +/- OGX-011 in First-Line mHRPC

Chi et al, ASCO, 2007

NCIC IND.165: Taxotere +/- OGX-011 in First-Line mHRPC

Median for Arm A (OGX-011 + Docetaxel): 7.26 months (95%CI 5.22-9.33)Median for Arm B (Docetaxel): 5.85 months (95% CI 3.61-10.74)

At Risk0

20

40

60

80

100

Arm A: OGX-011 + DocetaxelArm B: Docetaxel

0.04041

5.02118

10.024

15.000Arm B

Arm A

Time (months)

Mature Data

Progression Free Survival

Chi et al, ASCO, 2007

Indicators of Anti-cancer Activity

Consistent trend in favor of OGX-011/docetaxel arm:More patients with a 50% decline in PSA within the first 12 weeks More pts (38% vs 22%) with >80% decline in PSA; less pts (0 vs 10%) with primary PSA progression as best response Longer time on treatment and a greater median # of treatment cycles.Higher frequency and longer duration of stable measurable disease.Lower frequency of progressive disease as “best response”. Longer time to progression

NCIC IND.165: Taxotere +/- OGX-011 in First-Line mHRPC

Cel

l pro

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PC3-R MM

PC3-R ASO

PC3 MM

PC3 ASO

OGX-011 in docetaxel-refractory HRPC:CLU knockdown chemosensitizes taxane-resistant PC3-dR cells to docetaxel

docetaxel +/- OGX-011

0

20

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120

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PC3-R MM

PC3-R ASO

PC3 MM

PC3 ASO

CLUVinculin

PC-3

PC-3R

MM OGX01150 200 500 50 200 500 O

onl

y

CLUVinculin

In vitro Docetaxel concentration (nM)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4 5 6 7 8 9

weeks

PC-3R, MM

PC-3R, OGX-011

PC-3, MM

PC-3, OGX-011

* *

In vivo

* - chemotherapy treatment

Tum

or V

olum

e

Phase II Feasibility Trial of OGX-011 in 2nd Line Therapy in HRPC:

RANDOMIZE

Arm ADocetaxel (75 mg/M2 IV) q 21 days and OGX-011 (640 mg IV) weekly plus Prednisone (5 mg po bid) daily

Arm BMitoxantrone (12 mg/M2 IV) day 1 and OGX-011 (640 mg IV) weekly plus Prednisone (5 mg po bid) daily.

Continue treatment until disease progression or unacceptable toxicity. If removed from treatment for any reason, follow until death

PR

D OI G

S RE EA SS SE I

ON

S FU OR LV LI OV WA L U

P

n = 41

HRPC Patients Who Had Disease Progression

During or Within 6

Months of 1st

Line Docetaxel Treatment

n = 19

n = 22

Study treatment ongoing in 11 (26%) of patients

OGX-011 in 2nd Line Therapy in HRPC:Chemosensitizes taxane-resistant patients to docetaxel

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4500

PSA

Valu

e

Start 1st line therapy

End 1st line therapy6 cycles

Start 2nd line therapy

Off Study Treatment for disease progression

5 Cycles

Time (2 Weeks per mark)

Patient: 009-002

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Time (2 Weeks per mark)

PSA

Valu

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Off Study Treatment for

disease progression

6 cycles

Start 1st line therapy

End 1st line therapy5 cycles

Bone Scan, PSA Progression

Start 2nd line therapy

Patient: 025-002

Summary: Clusterin as a Therapeutic Target in HRPC

sCLU is a stress-activated cytoprotective chaperone that is highly expressed in HRPC

Over-expression of sCLU-2 confers broad spectrum treatment resistanceInhibits protein aggregation, facilitates proteasome degration of ubiquitinated proteinsInteracts and and inhibits activated Bax, preventing cytochrome C releaseIncreases NF-kB transcriptional activity

CLU knockdown using OGX-011Enhances treatment-induced apoptosis in vitro and in vivoPre-clinical proof-of-principle in prostate, breast, lung, urothelial, melanoma, renal cell

OGX-011, a 2nd generation ASO potently suppresses target CLU levels >90% in human CaP tissues

Anti-cancer activity observed in multi-centre Phase II trials in breast, HRPC, lungPhase III registration trial in second-line HRPC set to begin in 2008

++ PSA- Bcl-2

- EGFR- clusterin

- IGFBP 2&5- TGFβ

++IGFBP 3 & 4-YB-1

++survivin-Hsp27

+ PKC-α

- PSA++ Bcl-2++Bclx-L- EGFR

+++ clusterin++++IGFBP 5IGFBP 3 & 4

+c-myc+YB-1

-survivin+Hsp27

+ PKC-α

++ PSA++ Bcl-2++Bclx-L+ EGFR

+++ clusterin++ IGFBP 2++ IGFBP 5

++YB-1++ survivin+++Hsp27

Changes in Gene Expression After Castration Changes in Gene Expression After Castration and During AI Progression and During AI Progression

Androgen-dependent

Tumour regression

Androgen-independent

Clonal selectionAdaptive responses

Hormone withdrawal Progression

AI

ADAI AI

Post-docs

Hide MiyakeMoto Muramaki

Isis PharmaceuticalsBrett Monia

PI’sKim ChiLarry GoldenbergColleen Nelson Paul RennieAlan So

Sue EttingerAmina Zoubeidi Eliana BeraldiRichard SoweryPathology

Antonio HurtadoLadan Fazli David HuntsmanTorsten NeilsenTed Jones

Grant FundingNCICNIH SPOREDoDPCF

CollaborationsUW - B. VessellaP. Lange, P Nelson, Tia HiganoU Montreal - F. Saad

OncoGenex Tech. Inc.Scott Cormack

NCIC IND GroupElizabeth EisenhauerLesley Seymour

OGX-011: Safety Profile in >270 Patients

Well tolerated in all Phase 1 and Phase 2 studies to date Safety profile of OGX-011 in combination with docetaxel vsdocetaxel alone

Increase in Grade 1 or 2 AE’s events (fever, rigors/chills and sweating during the loading-dose week and sensory neuropathy during therapy)lymphopenia was more prevalent in the OGX-011 + docetaxel arm (no clinical sequelae)No increase in SAEs in the OGX-011 + docetaxel arm

OGX-011 in combination with gemcitabine/platinum-based or mitoxantrone regimens

Safety profile similar to that expected for regimen (no increase in expected rate of Grade 3 or higher AEs)

OGX-011 Mechanism of Action

OGX-011

↑ nCLU

⇑ Cytochrome C ↓ NF-κB ⇑ ER Stress ↓ ProteasomeActivity

⇑ Bax ⇑ COMMD1⇑ Protein Aggregation ↓ UBP

↓ sCLU ↑ Apoptosis

↑ Apoptosis

Alternative splicing of Clusterin mRNA

Clusterin: Isoforms and Splice Variants

CLU Isoform 1sCLU-1

CLU Isoform 2sCLU-2

Nuclear splice variant 2nCLU-v2ΔII

Nuclear splice variant 1nCLU-v1ΔII

siRNA CLU ex3

siRNA CLU ex3 siRNA CLU ex1

siRNA CLU ex3 OGX-011 (CLU ex2 ASO)

siRNA CLU ex3 OGX-011 (CLU ex2 ASO)

siRNA CLU ex1

I II III IV

IISpliced out

5’-UTR, v1ATG ATGATG

5’-UTR, v2

V VI VII VIII

II III IVATGATG

I III IV5’-UTR, v1

ATG ATG

5’-UTR, v2III IVATG

siRNA CLU ex2

Nuclear localization signalER-targeting signal

Nuclear localization signalER-targeting signal

Nuclear localization signal

Nuclear localization signal

IISpliced out

IX3’-UTR

V VI VII VIII IX3’-UTR

V VI VII VIII IX3’-UTR

V VI VII VIII IX 3’-UTR

siRNA CLU ex2

Fold

Cha

nge

CLU Isoform 1

EtOH R1881

Fold

Cha

nge

0

0.5

1

1.5

2

2.5

CLU Isoform 2

EtOH R1881

clusterin

tubulin

Vehi

cle0.

01 n

M

0.1

nM

1 nM

10 n

M

60 kDa

40 kDa

1 nM R1881

1 μM Casodex

48 ho

urs

60 ho

urs

72 ho

urs

ClusterinmRNA

- + + - + + - + +

- - + - - + - - +

TotalRNA

CLU is an androgen-regulated gene

Cochrane et al, JBC, 2006