Pathophysiology of Myeloma Bone Disease
Peter Croucher
Academic Unit of Bone Biology,
Section of Musculoskeletal Science,
Uni
it
vers y f
o Sh
Sh f
e fi
ffield M d
e i
di
l
ca S h
c
l
oo , Sh
Sh f
e fi
ffi ld
e , UK
Myeloma Bone Disease
Bone Lesions
Characteristics
· Osteolytic Bone Lesions
· Osteoporosis
· Bone Pain
· Fracture
· 16x greater than expected
(Melton et al 2005)
· 69% pathological - 2 fold
increase in OP fracture (Melton
et al 2005)
Fractures
Cellular Mechanisms:
Lessons from Bone Histomorphometry
Osteoclast:
· Il
Increase osteoclast
CFU-GM
Myeloma Cells
formation.
+
Mesenchymal
· Increased
+
Stem Cells
ostl
teoclast acti
tivit
ity
Pre-osteoclast
-
+
Osteoclast
-
-
Osteoprogenitor
Osteoblast:
· Early increase in
bone formation.
Osteoblasts
· Later suppression
of bone formation
Bone
Cellular Mechanisms:
Lessons from Bone Histomorphometry
Osteoclast:
· Il
Increase osteoclast
CFU-GM
Myeloma Cells
formation.
+
Mesenchymal
· Increased
+
Stem Cells
ostl
teoclast acti
tivit
ity
Pre-osteoclast
-
+
Osteoclast
-
Osteoprogenitor
Osteoblast:
· Early increase in
bone formation.
· Later suppression
of bone formation
Bone
Cellular Mechanisms:
Lessons from Bone Histomorphometry
Molecular Mediators
· Lymphotoxin
CFU-GM
Myeloma Cells
+
· IL-1
Mesenchymal
· TNF
+
Stem Cells
Pre-osteoclast
· HGF
-
+
· MIP1 /
Osteoclast
-
· RANKL / OPG
Osteoprogenitor
· SDF1
· Wnt antagonists
· IL-3
· IL-7
Bone
Myeloma Cells Promote RANKL and Block OPG
Expression in the Bone
Bone Microenvironment
C
· Murine and human myeloma
IH
cells upregulate expression of
RANKL in strom
o al
a cell
ce s (Oyajobi et
et al
RANKL
1998 Pearse et al 2001; Guiliani et al 2001)
· Human myeloma cells promote
RANKL expression T cells
ISH
RANKL expression T-cells -
L
mediated by IL-7 (Guiliani et al 2002)
RANK
· Myeloma cells downregulate
yg
OPG expression in stromal cell,
endothelial cells and osteoblasts
(Pearce et al2001; Guiliani et al 2001; Shipman
IHCG
et al 2003)
PO
Myeloma Cells May Express RANKL Directly
RANKL Staining of
-ve control
Myeloma Cells
· CD38+++/CD45+ cells express
RANKL - FAC
FA S
C /
S RT
R PCR (Farrugia et
al 2003)
+ve control
· CD138+ / cell lines express
p
RANKL - RTPCR/In situ/IHC (Lai et
al 2004)
Sezer et al (2002) Blood 99:4646-4647
· CD138+ express RANKL
RANKL - ICC
(Yaccoby et al 2004)
Isotype
cells
ofs
RANKL
· Induce osteoclast formation in
Number
vitro, directly
Log Fluoresence
Croucher et al (2001) Blood 98:3534-3540
Myeloma Bone Disease is Associated with
Increase sRANKL in Mice
Serum sRANKL (ng/ml)
0.5
p<0.005
0.4
0.3
0.2
0.1
0
Naive 5T2MM
Naive 5T33MM
Naive
5T2MM
Naive
5T33MM
Serum OPG (ng/ml)
20
Naive
15
10
5T2MM
5
0
Naive 5T2MM
Naive 5T33MM
5T33MM
Fc.OPG Prevents the Development of
Myeloma Bone Disease
Control
Fc.OPG
0 weeks
8 weeks
12 weeks
5T2MM model
Inject 5T2MM cells
Paraprotein
Sacrifice
Treatment
Group 1 = naïve
Group 2 = 5T2MM + vehicle
Group 3 = 5T2MM + Fc.OPG (30mg/kg, 3/week)
Naive
20
p<0.005
ns 15
5T2MM + vehicle
lesio 10
of
No
5
5T2MM + Fc.OPG
0
Naive
Veh
Fc.OPG
+5T2MM
Croucher et al Blood 2001
OPG Peptidomimetics Prevent the Development of
Myeloma Bone Disease
Naive
5T2MM+Veh.
5T2MM+OP3-4
RANKL
OPG
OP3-4
P<0.001
0 weeks
8 weeks
12 weeks
P<0.001
p<0.05
12
5T2MM model
sions 10
eL
Inject 5T2MM cells
Paraprotein
Sacrifice
8
of
6
Treatment
4
mber
Group 1 = naïve
u
Group 2 = 5T2MM + vehicle
N
2
Group 3 = 5T2MM + OP3-4 (120µg/kg - 2/weekly)
0
Naïve
5T2+con.
5T2+OP3-4
OP3-4 = Tyr-Cys-Glu-Ile-Glu-Phe-Cys-Tyr-Leu-Ile-Arg
Targeting RANKL and the Development of Myeloma
Bone Disease
Strategy
Model
Bone
Tu
T mour
u
Reference
Disease
Burden
Fc.OPG
5T2MM
Croucher et al 2001
RANK.Fc
5TGM1
Oyajobi et al 2000
SCID/Hu
Pearse et al 2001
ARH-77
Pearse et al 2001
OPG mimetics
5T2MM
Heath et al 2007
Lentiviral OPG
ARH-77
Doran et al 2004
KS-12-BM
Rabin et al 2007
Macrophage Inflammatory Protein-1
(MIP-1
-
1 )
and Bone Destruction
· MIP-1 identified as a potential osteoclastogenic factor
(Choi et al 2000)
· Anti-sense inhibition of, and antibodies to, MIP1-
prevents bone destruction in vivo (Choi et al 2001, Oyajobi et al
2003)
· MIP-1 effect may be RANKL dependent (Abe et al 2002;
Oyajobi et al 2003) independent (Han et al 2001).
· Targeting CCR1 reduced myeloma bone disease in vivo
(Menu et al 2006)
· MIP-1 identified as a downstream target of FGFR3 (Masih-
Khan et al 2006)
Increased Osteoclast Activity in Myeloma:
A Role
Role for Stromal-
Stromal Derived
-
Factor
Factor-1
-
1
5T2MM cells promote increased
resorptive activity
Control
· SDF-1 promotes
Rt
Resorp i
tion (%)
(%)
osteoclast chemotaxis and
activity but not number (Yu et
20
al 2003, Grassi et al 2004)
· SDF-1 expressed by
myeloma cells stimulates
+5T2MM
10
osteoclastic activity (Zannettino
et al 2005)
0
Con
5T2MM
Summary
PTHrP
IL7
IL3
HGF
· RANKL signaling
signaling plays
plays a critical
critical
role.
·
RANKL
Other molecules promote
resorption via RANKL
RANKL, lth
a
h
oug
MIP1
SDF-1
RANKL independent pathways
may also contribute (MIP1).
· Regulate osteoclast formation
and others activity.
· Do all patients use
use identical
pathways/molecules?
· Molecular subtypes determine
kt
key pa h
thways
Wnt Signalling and Bone Formation
in Myeloma
RANKL- `key'
· Dkk-1 over-expressed in
A. Wnt Signalling
B. Inhibition of Wnt Signalling RANK -ll
mye `loma ce l
receptor
ll 's
d
an is
Wnt
increased in the bone marrow
sFRP
OPG -
(T `Inhibitor
ian et al
'
2003).
Wnt
L
Frizzled
L
Frizzled
kk1
R
D
R
· Dkk-1 produced by myeloma
P
P
cells inhibits bone formation
(Tian et al 2003).
catenin
catenin
· Serum Dkk-1 concentrations
Proteosome
in patients with myeloma
TCF catenin
TCF
(Politou et al 2006, Terpos et al 2006).
Target
Target
N
N
Cell
Cell
· Anti-Dkk-1 prevents myeloma
bone disease in the SCID-rab
mice (Yaccoby
(Y
et al 2006).
+ve Formation
-ve Formation
9
6
2
Heterogenous Expression of Dkk1
BMSC MG63 SaOS
OPM-2
XG1
JJN-3
NCIH92
RMI822
DKK1
DKK2
y
DKK2
in M eloma Cells
DKK3
DKK4
sFRP1
DKK-1 (ng/ml)
sFRP2
Anti-Dkk1
Isotype
sFRP3
6
sFRP4
5
FZD1
FZD2
4
FZD3
FZD4
3
FZD5
FZD6
2
FZD7
FZD8
1
FZD9
FZD10
0
Kremen1
XG1
OPM2
JJN3 NCIH929 RPMI
Kremen2
8226
LRP5
Norrin
Sclerostin
Wnt1
Wnt2
Differentiation of SaOS2 cells
Wnt2B
Wnt3
4
Wnt3A
Wnt4
l/mg)
3
Wt
Wn 5A
t5A
m
Wnt5B
2
0BM P -2
Wnt6
(U/
Wnt7A
1
50 ng B M P -2
Wnt7B
Wnt8A
0
Wnt8B
lp:protein
Wnt9A
a
Wnt9B
Wnt10A
Wnt10B
Wnt11
Wnt16
Dkk1 ng/m l
Additional Regulators of Osteoblast Differentiation
· sFRP2 rather than Dkk1 regulates
Control
JJN-3
RPMI-
NCI-
8226
H929
osteoblast differentiation in vitro
(Oshima et al 2005).
Alk.
Phos.
· IL-7, rather than soluble wnt
antagonists, inhibit osteoblast
Calcium
diff
dif erentiation in myeloma (Giuliliani et
al 2005).
Collagen
· Cell contact through VLA4
decreases Runx2 activity (Giuliani et al
2005).
Total
· IL-3 increased in bm of patients
p
with myeloma and blocks
osteoblast differentiation in vitro
Soluble IL-6R released from
(Ehrlich et al 2005).
myeloma cells
cells can stimulate
stimulate
osteoblast differentiation (Karadag et al
· HGF regulates osteoblast
2000).
differentiation (Standal et al 2007).
Summary
Mesenchymal
Stem Celllls
IL-6/sIL6R
+
· IL-6 signalling may promote
early recruitment to the lineage
Dkk1
sFRP2
· Wnt signalling
signalling antagonists
IL7
Osteoprogenitor
-
(Dkk1, sFRP2), IL-7, IL-3 &
IL3
HGF are all implicated in the
inhibition of differentiation.
HGF
VLA-4
Pre-osteoblast
· Direct induction of osteoblast
apoptosis may also contribute.
Apoptosis
· Limited functional data to
support a causal role in vivo
5T2MM cells inhibit bone formation
Control
80
Naïve Mice
70
5T2MM Bearing Mice
/mm)
60
(o 50
tNs 40
a
30
5T2MM
20
Osteobl
10
0
0
2
4
5
67
89
11
12
Weeks
Control
5T2MM
60
Naïve Mice
5T2MM Bearing Mice
**
50
Surface
40
de
30
20
Mineralis%
10
0
0
2
4
5
6
7
8
9
11
12
Weeks
Stimulating Osteoblastic Bone Formation with
Soluble Activin
Activin Receptor Prevents Bone
Bone Disease
Osteoblast No
Osteoclast No
0 weeks
8 weeks
12 weeks
p<.001
ns
30
p<.001
p<.001
3.0
25
2.5
5T2MM model
20
2.0
5T2MM cells
Paraprotein
Sacrifice
(/mm)
(/mm)
15
1.5
b.N
c.N
O 10
O 10
1.0
Treatment
5
0.5
Group 1 = naïve
Group 2 = 5T2MM + vehicle
0
0
Naïve +Veh +RAP
Naïve +Veh +RAP
Group 3 = 5T2MM + RAP-011 (120µg/kg - 2/weekly)
+5T2MM
+5T2MM
Naïve
5T2MM+Veh
5T2MM+RAP011
Poster 319
Conclusions
RANKL
MIP1
· Direct and indirect effects
SDF-1
Myeloma Cells
IL-7
contribute
IL3
Dkk1
HGF
sFRP2
· Many molecules - yet
PTHrP
IL-7
functional data in vivo is
IL3
HGF
limited.
· Spacial and temporal
Stromal Cells,
regulation
g
is unclear
T-cells,
Osteoclasts
Endothelial Cells
· Relationship with
molecular subtypes is
unclear
· Identification of pathways
Osteoblasts
has provided new targets
pg
Bone
Acknowledgements
University of Sheffield
University Pennsylvania
Les Coulton
Rachmandran Murali
Debby Heath
Shelly Lawson
Amgen
Clive Buckle
Colin Dunstan
Andy Chantry
Bill Dougall
Evy De Leenheer
Orla Gallagher
Acceleron
Holly Evans
Jas Seehra
Free University Brussels
Karin Vanderkerken
Angelo Willems
Willems