Targeting of VEGF- and PDGF signaling by
receptor tyrosine kinase inhibitors impairs
myeloma cell-induced neovascularisation in the
5TMM mouse models.
Ivan Van Riet
Department Hematology-Immunology
Vrije Universiteit Brussel, Belgium
Myeloma cell ­ endothelial cell communication:
a complex molecular interplay
Extravasation
Angiogenesis
VEGF-A
bFGF
HGF
Ang-1
TNF-
IL-1
TGF-
Receptor tyrosine kinases (RTKs) and their
inhibitors
·RTKs: group of transmembrane
signal transduction proteins involved
in a wide variety of cellular functions
including cell growth, differentiation
and angiogenesis
·Targets for novel anticancer agent
development
·Large number of small molecule
RTK inhibitors in phase I to III
clinical development
Myeloma cells produce endothelial cell-
activating factors
Myeloma cell
Endothelial cell
VEGF
bFGF
PDGF
PDGF-A (227 bp)
bFGF (237 bp)
PDGF-B (435 bp)
VEGF
(613 bp)
189
VEGF
(541 bp)
PDGF-C (177 bp)
165
VEGF
(480 bp)
145
mark
neg.
pos.co
EJM
Karpas
LP-1
MMs-1
MM5
U266
8226
VEGF
(408 bp)
PDGF-D (174 bp)
121
mark
neg.cont
pos.co
LP-1
MMs-1
MM5
8226
U266
Karpas
EJM
er
cont.
-1
nt.
er
-1
nt.
The 5TMM model
· 5TMM model includes several cell lines that originate from spontaneously
developed MM in eldery mice of the C57BL/KalwRij strain
· 5T2MM and 5T33MM lines best characterized and most commonly used
· Clinical characteristics of this model, including the presence of serum M-
component and the induction of osteolytic bone disease are similar to human
multiple myeloma
Asosingh K., Hematol. J., 1, 351-356, 2000
Vanderkerken K., Immunol. Rev., 194, 196-206, 2003
Angiogenesis in 5TMM model
· 5T2MM and 5T33MM cells induce angiogenesis
in vitro and in vivo
·Both 5TMM cell lines produce VEGF, bFGF and
PDGF
E. Van Valckenborgh et al., Brit. J.Cancer, 2002
· Tumor development in 5T2
MM model occurs in association
with an angiogenic switch
K. Asosingh et al., Blood, 2004
Angiogenesis in 5TMM model
·Part of the MM-associated micro-
vessels is functionally connected to
the systemic circulation
H. De Raeve
Raeve et al., Virchows Archiv., 2004
·Hypoxia in the bone marrow is
decreased during MM progression
K. Asosingh et al., Hematologica, in press
Involvement of RTKs in myeloma cell-induced
neovascularisation: a study in the 5TMM model
Myeloma cell
bFGF
VEGF
PDGF
SU6668
XX
X
Multi-targeted RTK inhibitors
SU11657
XX
SU10944
X
Specific RTK inhibitors
X
STI571
bFGF-R
VEGF-R1/2
PDGF-R
RTK inhibitors impair myeloma cell-mediated in vitro
bloodvessel formation in the rat aortic ring assay
CM 5T33MM + 10 M
CM 5T33MM
SU11657
In vivo inhibition of angiogenesis with SU-11657
and SU6668 in the 5T33MM model
5T33vvMM mice
60
P<0.0001
50
P<0.0001
40
D 30
MV
20
10
0
Naïve mice
5T33vv mice
5T33vv mice
5T33vv mice
Daily Injection with
(Untreated)
+
+
SU6668 (IP): 100 mg/kg
SU11657
SU6668
SU11657(orally): 40 mg/kg
(N=10)
(N=10)
(N=10)
(N=10)
MVD analysis by anti-CD31 staining on BM sections
At end stage disease in 5T33MM
control animals: evaluation
Of MVD in all groups
untreated 5T33MM mouse
SU 11657treated 5T33MM mouse
5T33MM model
In vivo inhibition of angiogenesis with SU10944
and STI571 in the 5T33MM model
5T33vvMM mice
30
P<0.0001
25
P<0.0001
D 20
MV 15
10
5
0
Naïve mice
5T33vv mice
5T33vv mice
5T33vv mice
(Untreated)
+
+
SU10944
STI571
Daily Injection with
(N=10)
(N=10)
(N=10)
(N=10)
SU10944 (orally): 150 mg/kg
STI571 (orally): 50 mg/kg
STI571 (Glivec) inhibits MM cell induced
neovascularisation
At end stage disease in 5T33MM
control animals: evaluation
Of MVD in all groups
In vivo inhibition of angiogenesis with SU11657 in
the 5T2MM model
5T2MM mice
Detectable M
Daily Injection with
SU11657(orally): 40 mg/kg
component (w7-8)
After 4 weeks: evaluation
Overall survival
of MVD in all groups
100
p< 0,0001
30
)% 80
25
P<0.001
(
ility
20
ab
60
group
D
b
15
1
MV
rop
10
40
2
aliv
5
rvu
20
SU11657-treated
0
S
Naïve mice
5T2MM mice
5T2MM mice
untreated
0
(Untreated)
+
SU11657
0
10 20 30 40 50 60 70 80
(N=10)
(N=10)
Time (days)
(N=10)
Median survival 27 vs 55 days
In vivo inhibition of angiogenesis with SU11657 in
the 5T2MM model
Bone marrow
100
p< 0,0001
5T2MM
)
fibroblasts
%
80
(
PDGFR- (399 bp
b )
ility
ab
60
group
b
PDGFR- (367 bp)
1
rop 40
2
al
VEGF
VEG R-1
R
(317
(3
bp)
iv
rvu
20
SU11657-treated
S
VEGFR-2 (538
(53 bp
b )
untreated
0
0
10 20 30 40 50 60 70 80
Time (days)
Lin et al., Canc Res, 2002
Podar et al., Blood, 2004
RTK inhibitors in 5TMM model: Conclusions
MM cell - induced neovascularisation involves VEGF-A and
PDGF signaling as important triggering mechanisms
Targeting VEGF and PDGF receptors might be an efficient tool
for anti-angiogenesis therapy in MM
ClassIII/V tyrosine kinase receptor inhibitors, like SU11657,
affect endothelial cell activity but might target also other cells of
the microenvironment (stromal fibroblasts, osteoclasts ?) and MM
cells directly.
Targeting MM with RTK inhibitors can contribute to disease
control
Karin Vanderkerken, PhD
Thiery Facon, MD,PhD
Kewal Asosingh, PhD
Xavier Leleu, MD
Isabelle Vande Broek, MD
(Lille, France)
Els Van Valckenborgh
Angelo Willems
Rik De Raeve, MD
Nicole Arras
(Antwerp, Belgium)
Barbara Stein
Ben Van Camp, MD, PhD