Transcript Diapositive 1

CLU inhibition using OGX-011
synergistically enhances Zol activity in
osteosarcoma
François Lamoureux, Marc Baud’huin, Benjamin Ory, Martin E. Gleave,
Dominique Heymann and Françoise Rédini
INSERM UMR957
Medicine 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.
Zoledronic acid
•
Clinical use :
– Osteoporosis
– Multiple myeloma
– Prostate and breast bone metastases
– Osteosarcoma : OS2006 protocol
Vicious
cycle
Tumor
proliferation
Bone
resorption
Clusterin (CLU)
•
CLU is a stress-induced cytoprotective chaperone activated by varied treatment stressors:
-
Chemo & Radio-therapy
Targeted therapies (Herceptin, Velcade, Hsp90 inhibitors, etc)
•
CLU expression is directly regulated by HSF-1
•
CLU confers broad spectrum treatment resistance when highly expressed:
1.
2.
Blocks apoptosis: Inhibits activated Bax
Enhances survival signaling pathways: Enhances NF-kB activity and AKT signaling
Chi et al, Expert Opin. Investig. Drugs 2008
Rationale: Zoledronic acid induces Clusterin expression
in OS cell lines and in a OS xenograft model
ZOL induces/increased CLU expression in human OS cells (HOS, SaOS2, MG-63, U2OS)
CLU expression
0 3.1 6.2 12.5 25 50
2
Zol (µM)
60kDa
Clu
1
40kDa
Vinculin
50
25
12
.5
3.
12
5
6.
25
0
0
CLU mRNA expression
MG63
3
Zol (μM)
HOS osteosarcoma xenograft model treated with 100μg/kg Zol
CT
Zol
CLU
200µm
200µm
CLU and resistance to Zoledronic acid
Zol-resistant MG63 osteosarcoma cells
overexpress CLU
140
***
* ******
100
60kDa
CLU
40kDa
Vinculin
80
60
40
MG63
MG63 resist
20
0
0.01
0.1
1
10
MG63 resist zol
MG63
Cell viability (%)
120
100
Zol (μM)
CLU overexpression protects OS cells from ZOL induced cell death
CLU
**
60kDa
40kDa
CLU
Actin
**
*
Antisense Clusterin:
OGX-011 Product Description
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
O O
P
-S
Control
0
SCR B
8
16 32 64 128 256
OGX-011
8
16 32 64 128 256 (nM)
Clusterin
CLU Knockdown by OGX-011
P-S
MOE
MOE
A
O
OCH2CH2OCH3
O
O O
P
-S
O C
H
O
Vinculin
OO
P
-S
O G
H
O
OO
P
-S
T
O
OCH2CH2OCH3
O
O
OO
P
-S
A
OCH2CH2OCH3
O
6
CLU knockdown using OGX-011 synergistically enhances ZOL activity to
inhibit OS cell proliferation
Chou-Talalay method: Constant ratio OGX-011ASO / ZOL for 48h
OGX-011 ASO
targets CLU
Antagonism
Dose-effect curve
0.6
ZOL
OGX-011 + ZOL
0.4
0.2
0
200
400
600
800
1000
1.5
1
Synergism
OGX-011
Combination Index(CI)
0.8
0.5
0
EC50
EC75
EC90
Dose
…and to induce apoptosis (HOS)
Caspase 3/7 activity (%)
Effect
1.0
600
***
500
400
300
200
100
0
*
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
ScrB
+ 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
n.s.
35
***
30
BV/TV (%)
ScrB
25
20
15
10
5
0
ScrB ScrB OGX-011
+ Zol
+ Zol
TRAP staining
ScrB
200µm
OGX-011 + Zol
ScrB + Zol
200µm
200µm
OGX-011 potentiates ZOL activity in HOS xenograft model
2500
ScrB
ScrB + Zol
OGX-011 + Zol
2000
120
Survival rate (%)
Tumor volume (mm3)
OGX-011 combined with ZOL delays tumor growth in OS xenograft model and enhances survival rate
1500
***
1000
500
0
0
10
20
30
200µm
ScrB
ScrB + Zol
OGX-011 + Zol
40
20
OGX-011 + Zol
200µm
100
80
***
60
40
20
Clu
200µm
200µm
% positive cells
100
0
200µm
10
20
Days
Ki67
200µm
60
0
% positive cells
ScrB + Zol
p<0.001
80
0
40
Days
ScrB
100
80
***
60
40
20
0 ScrB
ScrB OGX-011
+ Zol
+ Zol
30
40
CONCLUSIONS
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.
Zoledronic acid
Vicious
cycle
Tumor
proliferation
OGX-011
Clu
Tumor
Cells survival
Bone
resorption
Acknowledgments
Lamoureux F.
Ory B.
Baud’huin M.
Heymann D.
Gleave M.E.
Zoubeidi A.
Sources of funding
Experimental therapeutic unit (Nantes)
CLU in sarcoma
Mass Spectrometry Analyses from tumor tissue
OS, ES and synovial sarcoma:
100% positive for Hsp90
8 of 14 OS were positive for CLU
Results were confirmed by immunohistochemistry (TMA included 75 specimens)
extraosseous
osteosarcoma
de-differentiated
parosteal osteosarcoma
Rao et al. Human
Pathology, 2012
Hsp90
Clu
osteosarcoma samples displaying cytoplasmic and
nuclear immunopositivity
Clusterin plays a role in resistance to doxorubicin in OS cells
(Lourda et al. Int. J. Cancer 2006)
CLU Expression in human osteosarcoma
10/15
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
Primary
OS
good
low grade OS
no chimio
OS
good
femur
14
15
16
M
humerus
Primary
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