New Therapeutic Approaches in
Myeloma Bone Disease
Claire M. Shipman
University of Texas Health Science Center at San Antonio
Bone and Bone Marrow Interactions in
Multiple Myeloma
Myeloma Cells
Myeloma cells
Stimulate
Promote
Bone
Growth +
Resorption &
Osteoclasts
Survival
Inhibit Bone
Formation
Bone
Osteoblasts
Osteoclasts
Trabecular Bone
The RANKL/OPG System in Multiple Myeloma
Myeloma Cells
·RANKL expression, by
stromal cells/osteoblasts
and/or myeloma cells
RANKL
OPG
·OPG
expression
by
stromal cells/osteoblasts
and in serum
RANK
BMSCs/
Osteoblasts
Osteoclasts
Trabecular Bone
Targeting the RANKL/OPG system can prevent
myeloma bone disease
Control
5T2MM
5T2MM + Fc.OPG
Croucher et al. 2001
Additional Effects of OPG
· Survival factor for endothelial cells in vitro (Malyankar
et al 2000)
· Increased tumour growth in breast cancer (Fisher et al
2002)
· Soluble decoy receptor for TRAIL (Emery et al 1998)
TNF-related
TNF-related Apoptosis Inducing Ligand (TRAIL)
·Type 2 transmembrane protein
·Expressed by activated T cells, monocytes
·Induce myeloma cell apoptosis in vitro
·Anti-myeloma activity in vivo
·Inhibit the anti-osteoclastogenic effect of
Ashkenazi, 2002
OPG in vitro
TRAIL & OPG in the Bone Marrow
Microenvironment
DcR1 DR4/5
DcR2
OPG
TRAIL
APOPTOSIS
Can OPG function as a survival factor for myeloma cells
by preventing TRAIL-induced apoptosis?
Human myeloma cells do not express
or release OPG
OPG (ng/ml)
40
MG63 osteoblast-
600
Control
like cells
30
OPG
500
20
10
400
0
300
100
101
102
103
Log FITC fluorescence
200
NCI H929
100
myeloma cells
40
0
30
Control
MG63
NCI
RPMI
JJN-3
OPG
20
H929
8226
10
Myeloma cell lines
0
100
101
102
103
Log FITC fluorescence
OPG may be associated with patients with MGUS
OPG expression
Multiple Myeloma
Intracellular detection of OPG
1
2
OPG
20
3
Control
OPG
15
4
10
MGUS
Counts
1
5
2
0
100
101
102
103
3
log FITC fluorescence
4
5
MGUS,
<10%
plasma
cells by haematological
6
examination
detected,
not detected
Human Myeloma Cells Express TRAIL Death
Receptors
Sensitive to TRAIL
Resistant to TRAIL
NCI H929
RPMI-8226
JJN-3
DR4
DR4
DR4
Counts
Counts
Counts
Log FITC fluorescence
Log FITC fluorescence
Log FITC fluorescence
DR5
DR5
DR5
Counts
Counts
Counts
Log FITC fluorescence
Log FITC fluorescence
Log FITC fluorescence
TRAIL-induced
TRAIL-induced apoptosis of myeloma cells is
prevented by OPG
Control
%
apoptosis
80
60
*
40
50ng/ml TRAIL
*
*
20
*
0
*
control
vehicle 10ng/ml 100ng/ml 500ng/ml 1000ng/ml
*
*
OPG
OPG
OPG
OPG
*
*
+ 50ng/ml TRAIL
NCI H929 myeloma cells
OPG inhibits TRAIL-induced
TRAIL-induced apoptosis of primary
myeloma cells
Bone marrow mononuclear cells
Control
50ng/ml TRAIL ± 500ng/ml OPG, 24h
TRAIL
%
Labelled with;
apoptosis
p<0.05
p<0.05
40
anti-immunoglobulin light chain - PE
In situ nick translation assay - FITC
35
30
10%
+ve
25
scatter
Side
20
15
Ig light chain
10
35%
5
apoptosis
untsCo
0
Control
OPG
Apoptosis
TRAIL-induced
TRAIL-induced apoptosis of myeloma cells is
prevented by OPG released from osteoblasts
% apoptosis
80
p<0.05
70
p<0.05
60
50
40
30
20
10
0
Control
Vehicle
MG63 MG63 c.m.
con.
+ anti-OPG
media
+ TRAIL
NCI H929 myeloma cells
TRAIL cannot induce apoptosis of myeloma cells
in myeloma/osteoblast cocultures
% NCI H929
apoptosis
p<0.01
60
50
40
30
ns
20
10
0
Control
TRAIL
Control
TRAIL
alone
coculture
NCI H929 myeloma cells cultured alone or in the presence of a normal human
osteoblast cell line hPOB-tert
A DR5 agonist can induce apoptosis of myeloma
cells in myeloma/osteoblast cocultures
% NCI H929
apoptosis
p<0.01
35
p<0.01
30
25
20
15
10
5
0
Control
anti-DR5
Control
anti-DR5
alone
coculture
NCI H929 myeloma cells cultured alone or in the presence of a normal human
osteoblast cell line hPOB-tert
Endog
Endo enous
g
OPG may function as a survival factor
for myeloma cells in the BM microenvironment
Myeloma
Cells
Monocytes
/ T cells
MYELOMA
APOPTOSIS
BMSCs/
Osteoblasts
TRAIL
OPG
DR4
DR5
Acknowledgements
University of Oxford
Rachel Locklin
Philippa Hulley
Ermanno Federici
Graham Russell
University of Sheffield
Peter Croucher
University of Texas HSC at San Antonio
Greg Mundy