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
03.1
25 6.25
12.5 25 50
0
1
2
3MG63
CLU
mR
NA
expr
essi
on
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 EC900
0.5
1
1.5
Com
bina
tion
Inde
x(C
I)
Ant
agon
ism
Syn
ergi
sm
OGX-011
OGX-011 + ZOLZOL
scrB OGX-011
scrB + Zol
OGX-011 +
Zol
050
100150200250300350400450
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
05
10152025
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
2500ScrBScrB + 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