CLU inhibition using OGX-011 synergistically enhances Zol activity in

osteosarcomaFrançois Lamoureux, Marc Baud’huin, Benjamin Ory, Martin E. Gleave,

Dominique Heymann and Françoise Rédini

INSERM UMR957Medicine Faculty, Nantes, FRANCE

Zoledronic Acid (ZOL)

• 3rd generation nitrogen containing bisphosphonate (high affinity for HAP).

• Inhibits bone resorption mediated by osteoclasts.

• Inhibits Farnesyl Diphosphate Synthase (mevalonate pathway) : inhibits prenylation of small GTPases.

• Direct antitumoral effect in vitro and in vivo (breast, prostate, pancreas, lung cancer, osteosarcoma).

• Other effects : inhibition of angiogenesis and cell migration.

• Clinical use : – Osteoporosis– Multiple myeloma– Prostate and breast bone metastases– Osteosarcoma : OS2006 protocol

Bone resorption

Tumor proliferation

Vicious cycle

Zoledronic acid

Clusterin (CLU)

Chi et al, Expert Opin. Investig. Drugs 2008

• CLU is a stress-induced cytoprotective chaperone activated by varied treatment stressors:- Chemo & Radio-therapy- Targeted therapies (Herceptin, Velcade, Hsp90 inhibitors, etc)

• CLU confers broad spectrum treatment resistance when highly expressed:1. Blocks apoptosis: Inhibits activated Bax2. Enhances survival signaling pathways: Enhances NF-kB activity and AKT signaling

• CLU expression is directly regulated by HSF-1

Rationale: Zoledronic acid induces Clusterin expressionin OS cell lines and in a OS xenograft model

HOS osteosarcoma xenograft model treated with 100μg/kg ZolZolCT

CLU

200µm 200µm

ZOL induces/increased CLU expression in human OS cells (HOS, SaOS2, MG-63, U2OS)

Clu

Vinculin

Zol (µM)0 3.1 6.2 12.5 25 50

CLU expression

60kDa

40kDa

03.

125

6.25

12.5 25 50

0

1

2

3MG63

CL

U m

RN

A e

xpre

ssio

n

Zol (μM)

CLU and resistance to Zoledronic acid

CLU overexpression protects OS cells from ZOL induced cell death

Zol-resistant MG63 osteosarcoma cells overexpress CLU

CLU

MG

63

MG

63

resist zol

Vinculin

60kDa

40kDa

***

0.01 0.1 1 10 1000

20

40

60

80

100

120

140

MG63MG63 resist

*******

Cel

l via

bilit

y (%

)

Zol (μM)

CLU

Actin

60kDa

40kDa

****

*

CLU

6

Antisense Clusterin: OGX-011 Product Description

MOEMOE P-S

2nd generation 21-mer MOE gapmer antisense oligonucleotide

• Advantages of 2'MOE analogues– Increased potency and resistance to degradation– Tissue half life ~ 7 days – Facilitates more convenient dosing regimen (once-weekly infusion)

O C

O

O

G

O

O

T

OO

O

O

A

O

OPO

O

OPO

A

OO

P

OO

P

OO

P

-S

-S

-S

-S

-S

OCH2CH2OCH3

OCH2CH2OCH3

H

H

OCH2CH2OCH3

CLU Knockdown by OGX-011 Clusterin

Vinculin

0 8 16 32 64 128 256 8 16 32 64 128 256 (nM)

Control SCR B OGX-011

CLU knockdown using OGX-011 synergistically enhances ZOL activity to inhibit OS cell proliferation

0 200 400 600 800 1000

0.2

0.4

0.6

0.8

1.0

Dose

Dose-effect curve

Effec

t

EC50 EC75 EC90

0

0.5

1

1.5

Com

bina

tion

Inde

x(C

I)

Ant

agon

ism

Syn

ergi

sm

OGX-011

OGX-011 + ZOL

ZOL

scrB OGX-011

scrB + Zol

OGX-011 +

Zol

0

50

100

150

200

250

300

350

400

450

Cas

pase

3/7

act

ivity

(%

)

***

*

OGX-011 ASO targets CLU

…and to induce apoptosis (HOS)

Chou-Talalay method: Constant ratio OGX-011ASO / ZOL for 48h

CLU knockdown re-sensitizes OS tumor cells to zoledronic acid

MG63 resistant and sensitive cells were treated twice with 300nM OGX-011 or control ScrB ASO + ZOL for 48h.

OGX-011 does not affect ZOL induced bone protection in the HOS xenograft model

200µm

ScrB

200µm

ScrB + Zol

200µm

OGX-011 + Zol

TRAP staining

0

5

10

15

20

25

35

ScrB ScrB + Zol

OGX-011 + Zol

BV

/TV

(%

)

n.s.

***30

ScrBScrB + Zol

OGX-011 + Zol Treatment

-100 μg/kg Zol (3 times/week)-20mg/kg ASO (ScrB or OGX-011): IP injection everyday the first week, then 3 times /week

OGX-011 potentiates ZOL activity in HOS xenograft model

0 10 20 30 400

500

1000

1500

2000

2500ScrB

ScrB + ZolOGX-011 + Zol

Tum

or v

olum

e (m

m3 )

Days

***

Ki67

ScrB ScrB + Zol OGX-011 + Zol

200µm 200µm 200µm

200µm 200µm 200µm

Clu

0 10 20 30 400

20

40

60

80

100

120

ScrBScrB + ZolOGX-011 + Zol

Sur

viva

l rat

e (%

)

Days

p<0.001

OGX-011 combined with ZOL delays tumor growth in OS xenograft model and enhances survival rate

% p

ositi

ve c

ells

ScrBSe-

ries1

0

20

40

60

80

100

***

% p

ositi

ve c

ells

Se-ries1

0

20

40

60

80

100

***

ScrB + Zol

OGX-011 + Zol

OGX-011 combined with ZOL:

1) Decreases Stress-induced CLU expression induced by ZOL

2) Decreases osteosarcoma cell growth in vitro and in vivo

3) Proof of principle to combine OGX-011 (phase II/III clinical trial in solid tumors) and zoledronic acid (phase III clinical trial in OS in France) in osteosarcoma treatment.

CONCLUSIONS

Bone resorptionTumor

proliferation

Vicious cycle

Zoledronic acid

Clu

Tumor Cells survival

OGX-011

Lamoureux F.

Ory B.

Baud’huin M.

Heymann D.

Gleave M.E.

Zoubeidi A.

Experimental therapeutic unit (Nantes)

Sources of funding

Acknowledgments

Rao et al. Human Pathology, 2012

extraosseous osteosarcoma

de-differentiated parosteal osteosarcoma

osteosarcoma samples displaying cytoplasmic and nuclear immunopositivity

Hsp90 Clu

Mass Spectrometry Analyses from tumor tissue

OS, ES and synovial sarcoma: 100% positive for Hsp908 of 14 OS were positive for CLU

Results were confirmed by immunohistochemistry (TMA included 75 specimens)

CLU in sarcoma

Clusterin plays a role in resistance to doxorubicin in OS cells(Lourda et al. Int. J. Cancer 2006)

Case age (y) sex Tumor location Primary/recurrence Histology Response to chimio

1 55 F sacrum Primary giant cell OS no chimio

2 56 F sacrum Recurrence giant cell OS no chimio

3 62 M pelvis Recurrence OS bad

4 38 M scaphoid Primary synovialsarcoma

5 18 M Primary OS good

6 22 M femur Primary OS bad

7 26 M femur Primary OS

8 27 M femur Recurrence OS bad

9 28 M femur Recurrence OS bad

10 80 F femur Primary OS on Paget bad

11 27 F femur Primary low grade OS no chimio

12 18 M Primary OS good

13 16 M femur Primary OS good

14 low grade OS no chimio

15 16 M humerus Primary OS good

10/15

CLU Expression in human osteosarcoma

Effect of Clu inhibition using OGX-011 in OS xenograft model

HOS model:

-2M HOS cells/ Nude mouse

-Treatment started when tumor is palpable

-20mg/kg ASO (ScrB or OGX-011): IP injection everyday the first week, and then 3 times /week

No effect of OGX-011 ASO on tumor progression