The Role of Wnt Signaling in
Myeloma and Its Interaction with
Marrow Stromal Cells
Erming Tian
Donna D. and Donald M.
Lambert Laboratory of
Myeloma Genetics
Multiple Myeloma
Diff-Quik® staining of myeloma BM aspirate
Lytic Bone Lesions in Myeloma
Normal
FDG PET
Myeloma
FDG PET
Normal vs. Myeloma Bone Turnover
Osteoclast
Normal
Coupled
Bone
Osteoblast
Turnover
Bis-Phosphonates
stop lesions.... but
RANKL
no repair!
MIP1a
?
Myeloma
Bone
DKK1 Gene Contributes to
Myeloma Bone Disease
Donna D. and Donald M.
Lambert Laboratory of
Myeloma Genetics
DKK1 Gene Contributes to
Myeloma Bone Disease
H : µ = µ
Myeloma
O
1
2
With Focal Lesions
H : µ µ
A
1
2
x = 35.99 ng/ml
s = 68.83
p-value = 0.005
n = 75
p = 0.0015
p < 0.0005
Myeloma
Normal
W/O Focal Lesion
Donors
x = 13.70 ng/ml
x = 8.91 ng/ml
s = 11.09
s = 4.21
n = 47
n = 14
p = 0.0038
Normal vs. Myeloma Bone Turnover
Osteoclast
Normal
Coupled
Bone
Osteoblast
Turnover
RANKL
MIP1
DKK1
Myeloma
Bone
Soluble Inhibitors sFRP and DKK Block
Canonical Wnt Signaling
No Signaling
Active Signaling
WNT
sFRP
DKK1
WNT
FZD
FZD
LRP
LRP
APC
AXIN
GSK
-catenin
LEF-1
-catenin
TCF
U
TCF/LEF
P
Mutations in LRP5 Cause Hereditary Bone
Metabolism Defects
LRP Loss-of-Function = Osteoporosis
LRP Gain-of-Function = High Bone Mass
(Gong et al., Cell 2001)
(Boyden et al., NEJM 2002)
WNT
T
T
normal
FZD
LRP5
APC
AXIN
GSK
-catenin
TCF
U
P
Osteoblast Inactivation
Osteoblast Hyperactivation
Transgenic Models Support Role for DKK1
in Bone Turnover
Col1A1 driven Dkk1 mice develop osteoporosis and
exhibit dwarfism, short limbs, and loss of digits.
Li et al., Abstract 1017, ASBMR, 2004.
Guo et al., Abstract 1018, ASBMR, 2004.
Model of DKK1 in Myeloma Progression
MM Stem
Cell?
IL-6
Osteoclast
RANKL
DKK1
Marrow
Stem Cell
Reya et al., Nature, 2003
Hem. Stem Cell
U
P
OB
Niche?
N-cadherin
Calvi et al., 2003,
Zhang et al.,2003
Osteoblast
Myeloma Therapy Via Osteoblast
Differentiation?
Monoclonal
Antibody
Soluble
MSC
LRP
Transplant
PTH
Osteoclast
RANKL
U
P
Li
Proteasome
Inhibition
Osteoblast
DKK1 Antibody Therapy
In Collaboration with Josh Epstein and Shmuel Yaccoby
Donna D. and Donald M.
Lambert Laboratory of
Myeloma Genetics
Mouse Model for Human Myeloma
Pre-tumor
Tumorgenic
Testing the Role of DKK1 in Myeloma
Experimental Design
SCID-rabbit mice were engrafted with CD138+ selected
primary myeloma cells expressing DKK1 (assessed by
microarray).
DKK1 neutralizing antibodies (R&D Systems) were injected
S.C. into area surrounding implanted bone
Polyclonal Ab: 50 µg/injection/3 times a week
Monoclonal Ab: 100 µg/injection/day
Testing the Role of DKK1 in Myeloma
Experimental Data Analysis
Human Ig and BMD were measured pre Rx and end of each
experiment (varying from 5-7 weeks) (histological sections)
Mature osteoblasts were identified by osteocalcin IHC
Osteoclasts were identified by TRAP+ and multinucleation on
sequential sections in myelomatous rabbit bone from control
and -DKK1 treated mice
Inhibition of DKK1 Associated with Increased Numbers
of Osteoblasts and Reduced Numbers of Osteoclasts
Con trol
Con trol
60
p < 0.0009
60
A n ti-D K K 1
p < 0.0007
A n ti-D K K 1
bone
40
40
myeloma
20
p < 0.003
20
/
Cells
0
0
Osteoclasts
Osteoblasts
Osteoclasts
Osteoblasts
Pt H
Pt D
Control
Anti-DKK1
Blocking DKK1 Associated with Reduced Bone
Loss and Tumor Burden
Pt D
Pt H
Rx)
Rx)
)
50
300
Untreated
pre 250
Anti-DKK1
pre
of p 200
of 30
10
150
(%
Rx)
( 100
(%
6
g
Ig
50
10
2
pre
0
0
Ig
Rx)
of
-50
-2
-5
-10
-10
(%
Human
-6
pre
-15
Human
-10
of
BMD
-20
(%
-25
BMD
Control
Anti-DKK1
Blocking DKK1 is Associated with
Reduced Bone Loss and Tumor Burden
Summary of 7 Experiments
C ontro l
A n ti-D K K 1
500
400
Rx
300
pre
20
200
of%
10
100
Rx
0
0
pre
of
-10
%
-20
hIg
BM D
Effect of Anti-DKK1 on Osteoblast and
Osteoclast Activities
Pt. G
TRAP
Osteocalcin
OC
Control
Anti-DKK1
OC
OB
Effect of Anti-DKK1 on Osteoblast and
Osteoclast Activities Con't
Pt. G
TRAP
Osteocalcin
Control
OC
OB
OB
Anti-DKK1
OC
Increases in Mature Osteoblasts and Reduced
Osteoclasts in DKK1 Treated Animals
Anti-DKK1
Control
Osteocalcin IHC
Osteocalcin IHC
OB
OB
MM
MM
Pt D
TRAP
TRAP
MM
OC
OC
MM
Osteoblasts Induce MM Cell Apoptosis In
Anti-DKK1-Treated Animals
Osteocalcin IHC
Osteocalcin IHC
OB Zone
Pt D
Apoptotic MM?
Apoptotic MM?
Viable MM
Conclusions
· DKK1 production by myeloma (through unknown
mechanisms) contributes to bone defects
· MSC CM contains soluble factor that activates DKK1 in MM
Cells
· DKK1 induces IL-6 production by MSC which in turn promotes
myeloma cell proliferation
· Blocking IL6 function decreases MM Cell viability
· Inhibition of DKK1 in myelomatous bone is associated with:
1) Increased osteoblast numbers and bone formation
2) Reduced osteoclast numbers activity
3) Inhibition of tumor growth
Conclusions
· DKK1 can be activated by genotoxic and non-genotoxic
agents in other tumor cells
· DKK1 can be activated in tumor and non tumor cells by
thalidomide and other drugs but not the proteasome inhibitors
· DKK1 is thought to be mediator of Thalidomide embryopathy
Donna D. and Donald M.
Lambert Laboratory of
Myeloma Genetics
John Shaughnessy
Bart Barlogie
Christopher S Adams
Elias Anaissie
Adam Hicks
Athanasios Fassas
Yongsheng Huang
Frits van Rhee
Bob Kordsmeier
Raymond Thertulien
Chris Randolph
Guido Tricot
Owen Stevens
Jeffrey Sawyer
David R Williams
Maurizio Zangari
Yan Xiao
Hong Wei Xu
Joshua Epstein
NCI
Shmuel Yaccoby
YaWei Qiang
Ichiro Hanamura
Stuart Rudikoff
James P Stewart
Fenghuang Zhan
Tulane
Carl Gregory
Darwin Prockop