Wnt/
Wnt/ catenin
-
Signaling in
in
Multiple Myeloma
John D. Shaughnessy Jr. PhD
Myeloma Institute For Research and Therapy
University Of Arkansas For Medical Sciences
Little Rock, AR 72205
Osteolytic Lesions Adjacent to Focal Tumor Growth:
Dist
Dis inct Biology?
XRAY
Foc
Fo a
c l Lesion
MRI
High Level DKK1 Produced by MM Related to MM
Bone Disease
(Tian et
al., 2003). Fir
s
Fir t
s E
vidence
E
of
Potential Role of Wnt Signaling in MM
Log2 MAS5.0 DKK1
6
10
12
814
BM Bcells
16
BM PC
14
MGUS PC
WM PC
12
MMCL
KK1D 10
MM PC
MM PC FL
8
RL MM PC
MAS5.0
RL MM PC FL
6
g2
BM BX
Lo
4
MGUS BX
WM BX
2
MM BX
MM BX FL
RL MM BX
CD1
CD2
HY
LB
MF
MS
MY
PR
NPC
RL MM BX FL
MMCL
Focal
Random
Lesion
Iliac Crest
DKK1
H&E
400X
1000X
DKK1
400X
1000X
Bone Marrow Plasma Protein and PC Gene
Expression Are Correlated
n
DKK1 Affymetrix
~ 400 ng/ml
nLevels
Affymetrix
DKK1 ELISA
Protei
r = 0.64 (P < .0001)
d
an
eneG
alizedm
Nor
DKK1 normal = 10ng/ml
Tian et al., 2003
Wnt Signaling Antagonism Contributes to
Osteolytic Lesions in Myeloma
Wnt/DKK1 May be Primary Regulator of
Coupled Bone
Tu
rn
r ov
o e
v r
e
MyelomaDerived DKK1 Contributes to Bone Disease and
Cancer Progr
g ession
Osteolytic
Bone
Destruction
Suppression of Wnt Counterintuitive based on Paradigm that Wnt
is Oncogenic.
How co
u
co ld
l Cancer
be rel
re a
l te
a d
te to Suppr
ession
Suppr
of
Wn
t
Wn ?
Thought:
DKK1 may mask or reflect underlying Wnt activation?
Discovery that DKK1 Causes High Grade Pleiomorphic Sarcoma
Revelaed Wnt can b Tumor Suppressor in Mesenchymal Lineage
(Matushansky
(Ma
et al., JCI,
2007)
X
X
X
NPC
MGUSPC
SMMPC
MMPC
MB
BX
GUSB
MB
N
M
M
M
S
M
WNT2
WNT2B
WNT2B
WNT4
WNT4
WNT5A
WNT5A
WNT5B
WNT5B
WNT6
WNT6
WNT8A
WNT9A
WNT9A
WNT10A
WNT10A
WNT16
WNT10B
DKK1
WNT11
DKK2
WNT16
DKK3
DKK1
SFRP1
DKK2
SFRP2
DKK3
FRZB
SFRP1
SFRP4
FRZB
SFRP5
SFRP4
FZD1
FZD1
FZD2
FZD3
FZD3
FZD6
FZD4
FZD7
FZD6
FZD8
FZD7
LRP5
FZD8
LRP6
LRP6
WNT1, WNT3, WNT7A, WNT7B, WNT8B, WNT9B, FZD2, FZD4,
WNT1, WNT2, WNT3, WNT7A, WNT7B, WNT8A, WNT8B,
FZD5, FZD9, FZD10, SFRP2, SFRP5.
WNT9B, WNT10B, WNT11, FZD5, FZD9, FZD10, LRP5
Wnt3a Can
Can Induces Stabilization of
of -catenin in
MM Cell Lines
3h
6h
3500
s
Wnt3a
+
+
3000
Unit
Con
2500
Wnt3a
Non-p-
2000
catenin
ciferaseu 1500
L 1000
Tubulin
500
Relative
0
pTOPFLASH
pFOPFLASH
H929
Qiang et al. Oncogene 2003
Wnt3a has
has no effect on proliferation in
in
multiple myeloma cells
1.2
rWnt3a(-)
0.7
Pt#1
rWnt3a(+)
0.6
1
Pt#2
0.5
0.8
0.4
0.3
570 0.6
OD570
D 0.6
DO
0.2
0.4
0.1
0
0.2
cm
cm
(-)
(+)
29
3a
t3a
3a
0
L9
nt3a
nt3a
H929
INA6
OPM-2
W
rW
rWnt
Qiang et al, Blood, 2005 and 2008b
Wnt3a induces stabilization of -catenin in MM
cell lines
d
an
i
pr mary Disease
Time
3hr
6hr
3hr
6hr
Wnt3a
+
+
++
MM144
OPM-2
EJM
H929
-Tubulin
B Pt# D62
D70
D73
D81
D82
D95
Wnt3a
++
+
+
+
+
-catenin
-Tubulin
Qiang et al. Blood 2008
Dkk-1 Inhibits Wnt-3a induced activation
of -catenin/TCF in MM
Dkk1
++
+ +
3000
Wnt3a
+ +
+
++
2500
seUnits 2000
catenin
1500
Lucifera 1000
500
nonp
p
ative
0
catenin
Rel
PEF/ PEF/ Dkk- Dkk- Dkk- Dkk-
pFOP pTOP
1/
1/
2/
2/
pFOP pTOP pFOP pTOP
Tb
Tub l
u ilin
Qiang et al. Oncogene 2003
Overexpression of Wnt3a in myeloma cells actives
canonical Wnt signaling
signaling, but Not Induces
Induces
Proliferation in vitro
EV
Wnt-3a
1
Anti-HA
0.8
0.6
570 0.4
Free--catenin
OD 02
0.2
0
Tubulin
48h
72h
Qiang et al, Blood, 2005 and 2008b
TT2
1.0
3.0
0.8
25
2.5
e
e
2.0
lu
0.6
Aliv
va P 1.5
0.4
portion
P=
P = 0.05
g10
05
g
o
Lo
Pr
HR = 1.5
1.0
0.2
signature > 1.1 (42/131)
0.5
signature < 1.1 (87/217)
0.0
0.0
0
20
40
60
80
100
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Months from Start of Therapy
1.0
3.0
0.8
2.5
e
0.6
Aliv
2.0
alue
ion
va
0.4
P = 0.18
P
1.5
oport
HR = 2.5
Pr
Log10
1.0
0.2
signature > 0.77 (2/23)
signature < 0.77 (50/250)
05
0.5
0.0
0.0
010
20
30
40
50
Months from Start of Therapy
1.0
1.5
2.0
While Wnt can Stimulate catenin Stabilization and Low
Level TOPFLASH Activity
in MMCL
...
Whole Transcriptome Analyses in Primary Disease has
Revealed No Evidence of a Significant Clinically Relevant
Wnt/catenin Signature
Possible Reasons:
High DKK1
High NF
kB
High Level RUNX3
High Level Notch
High Proteasome Activity
Over Expression of Cadherins
Loss of RB1
Loss of CDK8
Wnt3a Induces Increase in Active Form of -
catenin and Activation of LEF-1/TCF
Transcription in Osteoblast cells
8 hr
24 hr
Wnt-3a
++
P<0.001
500000
C2C12
450000
FOPflash
TOPFlash
400000
hFOB1.19
Unit 350000
300000
ferase 250000
MG63
Luci 200000
150000
Saos-2
100000
Saos
Relative
50000
0
L929
Wnt-3a
Non-phosphorylated -catenin
Qiang et al, Bone. 2008;42(4):669-80
Wnt3a increases OPG mRNA and Protein in
Osteoblast Progenitors
g
60
2500
48hrs
72hrs
GAPDH 50
/ml)
48hrs
72hrs
o
g 2000
to 40
(n
1500
30
relativeA
Protein 1000
A 20
G
mRN 10
OP
500
0
OPG
0
Wnt-3a
0
12.5
25
50
100
Wnt-3a
0
65
6.5 12.5 25
50
100
ng/ml
ng/ml
Qiang et al, Blood 2008a
blockage of Wnt signaling by Dkk1 or sFRP1
in myeloma cells has no
no effect
effect on
proliferation in myeloma cells
Treatment of MM cells with sFRP1 or Dkk1 had not effect on proliferation
Overexpression of Dkk1 in myeloma cell lines did not have any effect on
Proliferation or survival
These results suggest th
that endogenous canonical Wnt signaling pathway
is not required for MM growth
Qiang et al Oncogene 2003,
Colla et al. Blood 2007
Conflicting data on effect of Canonical Wnt
Signaling on
on MM
MM Growth
Growth
Conflicting data on effect of Canonical Wnt
Signaling on
on MM
MM Growth
Growth
·
No APC or -catenin (Qiang et al., 2003; our unpublished data)
·
High
g levels of DKK1 and FRZB, potent
,p
inhibitors of Canonical Wnt
Signaling, synthesized and secreted by MM tumor cells
·
Wnt3a fails to induce MM proliferation
·
LiCl, anti-DKK1, and Wnt3a suppresses MM growth in SCID-Hu
·
No evidence of canonical Wnt/-catenin/TCF transcriptional
signature in primary MM
Conflicting data on effect of activation of
Canonical Wnt Signaling
Signaling on
on MM
MM Growth
Growth
PNAS 2007
PNAS 2007
Why only two lines?
Is this specifc?
High Concentrations of PFK115584
Induces MM
Cell Kill
Enzastaurin Triggers catenin Accumulation
Raab et al, Blood 2009
Ectopic Expression of catenin Induces Cell Death Via
Induction of
c
Jun and
Raab et al, Blood 2009
Catenin contributes to enzastaurininduced
cell dt
dea h
th via id
ind t
uc i
tion f
o cJun and p73
Enzastaurin (M)
Raab et al, Blood 2009
Enzastaurin Inhibits Phosphorylation of catenin,
thereby
y inducing
g accumulation of bcatenin, leading
g to
UPR and Apoptosis via cJunstabilization of p73
DKK1??
Raab et al, Blood 2009
In Vivo Studies Show that Increasing Wnt Signaling
Results in
Suppression
of Tumor Growth
1. Directly Increase Wnt signaling by overexpression of Wnt3a
in myeloma cells or administration of Wnt3a in bone
marrow microenvironment using SCIDhu mice as a model
(Qiang et al Blood, 2003).
2. Indirectly increasing Wnt signaling by administration of
antiDkk1 in bone marrow microenvironment using SCID
rab mice as a model (Yaccoby et al 2007)
3. Indirectly Increasing Wnt signaling via Lithium Chloride
a GSK3beta inhibitor (reducing phosphorylation of
catenin) using 5TGM model (Edwards et al 2008)
Wnt-3a Increases Free -catenin in MMCL and
Prevents Tumor-Induced Bone Destruction
Vector
Wnt-3a
Pre-
Pre MM
MM
Pre-
Pre MM
MM
ild
ector
W
V
Wnt-3a
Mouse 1
Mouse 1
Anti-HA
Free--catenin
Mouse 2
Mouse 2
Tubulin
T
Mouse 3
Mouse 3
Qiang et al., Blood , 2008b
MMCL Expressing Wnt-3a Exhibits Reduced
Tumor Growth and Bone Loss In-
In vivo
EV
W3
/ml) 60
50
(mg
60
40
l)
30
nIg
50
m
a 20
m
10
40
Hum
(mg/
0
0
30
-20
change)
nIg
20
-40
(%
W3
Huma 10
-60
EV
BMD
-80
0
50
EV
W3A
01 23 456 7 89 10 11
40
Weeks
bone
30
mm 20
2
10
Cells/
0
Qiang et al., Blood , 2008b
Osteoclasts
Osteoblasts
Although Reducing Tumor Growth in
Bone Wnt3a Has no Effect on
Subcutaneous Growth in SCID Mice
EV
W3A
EV
W3A
Qiang et al., Blood , 2008b
Wnt-3a Has Bone Anabolic and Anti-Myeloma
Effects on Primary My
yyeloma
Cont CM
500
l)
Wnt3a CM
40
4 0
m
0
m 300
Pre-RX
Final
(ug/ 200
Ig 100
0
Cont CM
-2
02
468 10
weeks
6
4
Wnt CM
hange)c 2
0
(%
-2
BMD -4
Con CM
Wnt3a CM
Qiang et al., Blood , 2008b
Lithium Chloride increase b-catenin activity
and reduced tumor growth
growth in
in 5TGM
5TGM myeloma
myeloma
bearing mice
In vitro assay
Serum IgG2bk concentration
Edwards et al, Blood, 2008
Autocrine Suppression of Wnt by DKK1 Promotes
Proliferation and Blocks Differentiation of MSC
stationary
ecells
-1]
e
log
[Dkk
viabl%
lag
time (d)
U
P
Gregory et al JBC, 2003
DKK1 Induces MSC to Produce High Levels of
Myeloma Growth Factor IL-
IL 6
-
MSC1
MSC2
Supernatant Supernatant
Log
Stat
Log
Stat
52
dimer
52
31
26 kDa
17
11
IL-6 Western on medium
(normalized by cell number)
Gunn et al., Stem Cell, 2006
MSC Conditioned Media Activates DKK1
Expression by MM Cell Lines
DKK-1
100
100
n
FRZB DKK-1
90
90
FRZB
80
80
no )
APDH)
70
70
essi DH
xpressio
G
r
G
p P
x
E
60
60
fE yGA
by
b
of
50
50
itso ed
its
Un aliz
n
zed
n
40
40
e
tiv rm
U
30
(no
30
elaR
20
20
lative
(normali 10
10
Re
0
0
CM
MSC
CM
MSC
XG1
XG1 ANBL6 ANBL6
low
high
( - )
(+)
( + )
( - )
(+)
( + )
Gunn et al., Stem Cell, 2006
Conditioned Media from MSC Supports
Growth of IL-6-Dependent
p
MM Cell Line
4000
Negative IL6
de 3500
IL6 +
0.2 CM
3000
0.4 CM
ecoverR
06C
0.6 M
CM
2500
0.8 CM
Cells 2000
1.0 CM
1500
1000
500
0 0
20
40
60
80
100
120
Time in Culture (hours)
Gunn et al., Stem Cell, 2006
Anti-IL-6 Blocks Viability of XG-1
Cells Grown in MSC CM
6000.0
Lm 5000.0
per 4000.0
red 3000.0
recove 2000.0
lls
Ce 1000.0
0.0
MSC CM
CM
-
++
+
+
0
0
.10
5
10
Conc. Ab.
(mg/mL)
Gunn et al., Stem Cell, 2006
Anti-DKK1 mAb (BHQ880) as a Potential
Therapeutic for Multiple Myeloma.
·
anti-DKK1 neutralilizing antibody (AB) enhances OB differentiation and reduces IL-6
production in MSC
·
OB differentiation of MSC reduced in the presence of IL-6-dependent, DKK1 +ve INA-6
·
AB able to restore OB activity in a dose-dependent manner
·
No direct effect of AB on growth or survival of human MM cell lines
·
AB-induced growth inhibition of MM cells cultured with BMSC associated with
downregulation of IL-6
·
INA-6 MM cells in SCID-hu murine model revealed direct correlation between the level of
soluble human DKK1 in murine blood and tumor growth.
·
AB increased trabecular bone, number of OB and osteocalcin within 1 month
·
AB suppressed MM growth after 4 weeks
Fulciniti et al. Blood, Volume 110, issue 11, 2007
Summary
1. Canonical and Noncanonical Wnt pathway components expressed by Myeloma
cells and Bone Microenvironment.
2. DKK1 production
pr
by
MM cells may
ma pla
y
pla ro
l
ro e in
bone disease
.
3. Antagonizing DKK1 or augmenting Wnt has Anabolic and AntiTumor Activity
4. Wnt3a induces stabilization of catenin and TCF transcriptional activity
indicating that canonical Wnt signaling is activated in myeloma cells
5. Activat
a i
t on
o of
o can
ca oni
o ca
c l
a Wnt
t sign
sg alin
a
g
g did
dd not
ot show
so subs
sub equen
eque t
t prolif
po er
e at
a i
t on
o or
o
GEP effects indicating that canonical Wnt signaling not likely in primary disease
6. Anti MM effects of Wnt inhibiting small molecule have been reported but in
vivo activity remains t
o be tb
esta li
bli h
s
d
e
7. Inhibiting PKC kills MM, in part, through stabilization of catenin.
8. Although not formally proven, numerous mechanisms could be actively
suppressing catenin nuclear activity in MM
Model of Focal Disease Growth in Multiple Myeloma
Marrow Stem Cell
Osteoclast
OB
Osteoblast
Niche
OPG
RANKL
Hematopoietic
Stem Cell
Marrow
Marr
St
em
St
Cell
Marrow Stem Cell
DKK1
IL6
BCAT
NCAD
MM
Focal PCT
A389
A602
B361
B366
B957
C143
C144
C648
C649
C707
C711
C725
C764
C787
C842
catenin
Tubulin
C272
C277
C297
C394
C451
C596
C647
C859
C877
C884
C890
C890
C891
C922
C950
catenin
Tubulin
12000
CDH2 (203440_at)
10000
CDH1 (201131_s_at)
Signal
ed
8000
6000
Normaliz0
4000
MAS5.
2000
0
catenin
/+
+
++
+++
++++
(n=17)
(n=11)
(n=17)
(n=16)
(n=11)
3000
ed
2500
_at) 2000
rmalizo 440
o
3
N
1500
(20 1000
500
MAS5.0
CDH2
0
1
9
1
7
4
8
5
1
0
5
7
ARP
H92
P05
P97
P73
P09
P97
P40
P83
P64
P92
N dh
ca
erin
130Kd
catenin
92Kd
tubulin
A)
C)
22%
%
8
0.3%
8
3
3
%
3
CD
CD
PE
PE
ECD
ECD CD45
FITC
FITC Mo IgG
ECD
ECD CD45
Mo
hCDH2 +
FITCMo IgG
B)
D)
15.4%
77.6%
8.5%
D38
38D
79.4%
C
D
PE
C
PE
ECDCD45
Mo hCDH2 +
ECDCD45
Mo hCDH2 +
FITCMo IgG
FITCMo IgG
v2
PBBC
BPC
MMCL
U95An
TBC
MGUS
o
WM
TPC
MM
Signal
CLL
H2DC
16
)
14
_at
12
10
MAS5.02 01131_s
8
2 (
Log
6
CDH1
4
2
C
1
2
F
S
Y
L
Y
CD
CD
HY
LB
M
M
M
PR
NP
MMC
16
14
12
_at)
10
MAS5.0
03440
2
2
8
2
(2
6
Log
CDH2
4
2
C
1
2
Y
B
F
S
Y
R
L
R
C
LB
NP
CD
CD
H
M
M
M
P
MM
16
16
t)
14
14
t)
12
12
10
10
MAS5.0
8
MAS5.0
8
(201015_s_a
(203038_a
Log2
P
6
g2
6
o
K
6
JU
Lo
4
PTPR
4
2
2
CD1
CD2
HY
LB
MF MS
MY
PR
NPC
MCL
CD1
CD2
HY
LB
MF MS
MY
PR
NPC
MCL
M
M
16
IQGAP2
5.0
t) 14
4.0
AP2
0
4_a
G
0
.0
G
7 12
IQ
3.0
/10
10
MAS5
(2034
2.0
8
Signal
Log2
1.0
6
AS5.0
203474_at
R2= 0.81
IQGAP2 4
M
0.0
2
0
20
40
60
80
100 120 140
Protein expression
CD1
CD2
HY
LB
MF MS
MY
PR
NPC
(summed peptide intensity/1000)
MMCL
2500
_at)
2000
1500
Normalized
(203440
1000
2
S5.0
H
500
MA
CD
0
ARP1
JJN3
ARP1
JJN3
t
Ex
w
Control
CDH2
Control
CDH2
CDH2wt
(130 kDa)
CDH2Ex
(130 kDa)
(31 kDa)
-catenin
(92 kD
kDa)
-actin
(42 kDa)
9000
8000
CDH2 (203440
t)
_a
7000
CDH1 (201131_s_at)
Signal
6000
5000
Normalized
4000
5.0
3000
MAS
2000
1000
0
MS
MF
LB
CD
MS HMCL
CDH2
WHSC1
3.0
+ 3.0
Is the Suppression of Wnt/-
cati
tenin Nl
Nuclear Functi
tion a
Pathogenetic
g
Mechanism in
Multiple Myeloma and the Bone
Microenvironment?
A)
RB1
C)
4000
3500
FISH13
RB1
FISH13
RB1
FISH13
RB1
3000
2500
Signal 2000
1500
1000
500
0
B)
FISH13
No FISH13
Whole
0.40
MM 4
chromosome
shut off?
MM 3
0.30
MM 1/2
0.20
0.10
D)
0.00
1234567
89
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Signal
RB1
-0.10
2500
Mean
2000
-0.20
1500
-0.30
Signal
No FISH13 / high RB1
1000
an
-0.40
RB1
FISH13 / low RB1
500
No FISH13 / low RB1
Me
-0.50
0
Cen
Tel
TBC
TPC
BPC
32 Ch13 copy # sensitive genes