Molecular Mechanisms
Involved in Homing and
Migration of Plasma Cells in
Response to CXCR4
April 12, 2005
Homing of plasma cells (PC)
· BM microenvironment supports PC survival and
proliferation
· MM is usually wide spread in all BM
· 80% of patients have circulating PC
· Indicating (re)circulation of PC and (re) entrance
in the BM
· The mechanism by which PC migrate to specific
microenvironment niches is not well identified
CXCR4/SDF-1
· Adhesion, migration, proliferation and
survival
· G-protein coupled receptors
· Trafficking of B cells
· Unresponsiveness to SDF-1 leads to
the accumulation of plasma cells in the
spleen of NOD/SCID mice
CXCR4/SDF-1
· Dissemination of malignant cells in both
hematological malignancies and solid
tumors
· CXCR4 neutralization in vitro and in vivo:
­ Inhibition of migration
­ Apoptosis
­ Decreased proliferation
CXCR4/SDF-1 in MM
· Expression and Migration
­ High SDF-1 expression in BM
­ Low level of CXCR4 expression on MM cells in the BM
­ MM cells migrate in response to SDF-1
· Confining cells in BM
­ SDF-1 upregulates VLA-4
· Proliferation and survival
­ Promotes proliferation
­ Protects against Dex-induced apoptosis
­ Upregulates IL-6 and VEGF in BM
PNAS Oct 2003, Blood Jan 2001, JCO March 2004, Mol Cancer Ther. May 2002
Objectives
· Role of SDF-1/CXCR4 in MM migration and
homing
· Factors regulating PC entry and mobilization
from the BM
· Downstream effectors of SDF-1 dependent
migration in MM
Importance
· Therapeutic implication
­ Delay/prevent disease progression in
early MM
­ Mobilize MM cells from the BM into PB
­ Disrupt the BM microenvironment of MM
· AMD3100
· Regulators of downstream pathways
Proposed model of SDF-1 dependent homing of
PC into and out of the BM
Vascular niche
SDF-1 gradient
PC
CXCR4
internalization
Osteoblastic niche
Migration
Adhesion
Invasion
IL-6
Re-circulation
+VLA4, MMP9
VEGF
Proliferation
Survival
?anti-CXCR4
CXCR4 surface expression in MM
n 80
o
-
59.5
60
ressi
exp 40
27.1
% 20
iande 0
M
MM
PB
pg/mL
SDF-1 levels in MM
7000
6000
5000
4000
3000
2000
1000
0
12
MMPB
NPB
34
MMBM
NBM
ELISA performed on samples obtained from patients
Internalization of CXCR4 in response to SDF-1
A
B
Post
Pos
cont
c
r
ont o
r l
o
SDF
SD -
F 1
-
Pre-
Pr SD
e-
F
SD -
F 1
-
CXCR4
CX
Surf
Sur ac
f e
ac CXCR4
Sf
CX
C C
X R
C 4
R
No SDF-1
50nM SDF-1 30 min
Pre-SDF-1
Pre-SDF-1
Post-SDF-1
Post-SDF-1
Migration of MM to SDF-1
300
250
PB levels
200
150
control
BM level
of 100
%
50
0
0
1020
30
5075
100
SDF-1 (nM)
MM.1S
U266
No migration of MM.1S cells without SDF-1
Migration of MM.1S cells with 20nM continuous SDF-1
SDF-1
CXCR4
PLC
DAG
Ras
Ca++
Gi
PKC
Rac
Raf
PKC
Ca++
Rho
PI3K
Ca++ Ca++
MEK
Ca ++ Ca++
mTOR
FAK
NFkB
ERK1
PYK2 Paxilin
ERK 2
CRK
p130Cas
Anti-CXCR4 inhibit migration
MM.1S stimulated with patients bone marrow plasma
120
100
100
80
BM plasma only
60
Control
46
39.4
With anti-CXCR4
36
of
40
200ug/ml
13.2
%
20
0
0
CTRL
NBM
MM BM
123
SDF-1 20nM
Molecular mechanisms regulating CXCR4 dependent migration
7
% cells
migrating
6
5
4
3
2
1
0
1234
CTRL
Anti-CXCR4
LY294002
Rapamycin
20ug/ml
50uM
25nM
+SDF-1 30nM
Molecular mechanisms of SDF-1 dependent migration
120
100
80
60
control% 40
20
0
C T R L
A nti-C XC R 4
A nti-C XC R 4
L Y
LY
PD
PD
0
12
10ug/m l
20
12
3456
ug/m l
25uM
50uM
25uM
3456
7
50uM
7
LY294002
PD 098059
MEK downstream of PI3K in SDF-1 dependent migration
120
100
80
60
Control% 40
20
0
CTRL
Anti-CXCR4
LY
PD
LY+PD
12345
20ug/ml
25uM
25uM
Anti-CXCR4 abrogates cytoskeletal changes with SDF-1
PI3K and MEK inhibitors abrogate cytoskeletal changes
Before SDF-1
After SDF-1 50nM (30min)
SDF-1 induces invasion in MM
250
200
l
ro 150
ont
C 100
%
50
0
SDF-1 (nM)
123456
CTRL
1
10
20
30
100
Molecular mechanisms of invasion
120
100
l
80
ro
ont
60
C% 40
20
0
SDF-1 (20nM)
12345
CTRL
Anti-CXCR4
LY
PD
LY+ PD
20ug/ml
25uM
25uM
Conclusion
· Circulating PC express higher CXCR4 as
compared to BM
· Internalization of CXCR4 in response to high
SDF-1
· SDF-1 gradient induces/inhibit migration
· CXCR4 is important for migration and invasion
· PI3K and MEK are important for SDF-1
dependent migration
· MEK is downstream of PI3K in MM
Future direction
· CXCR4 siRNA in vitro and in vivo
· Anti-CXCR4 in vivo
· Other molecular pathways regulating SDF-1
dependent migration
· Effect of CXCR4 inhibition on proliferation, adhesion,
apoptosis, angiogenesis, OCL
· Preclinical development of anti-CXCR4 in MM
· Mechanisms of migration of other chemokine
receptors-CXCR3
Effect of CXCR4 inhibition on angiogenesis
VEGF
Control
Suramin
CXCR4 inhibitor 10 ug/ml
LY
PD 25uM
50uM
Acknowledgement
University of Pittsburgh
Mayo Clinic
·
Michael Timm, BS
·Ujjal Singha, PhD
·
Terrie Kimlinger
·Ganwei Lui, PhD
·Yazan Alsayed, MD
Investigators:
·Lanie Kasdan, MD
·
Thomas Witzig, MD
·
Karen Hedin, PhD
·
Philip Greipp, MD
Investigators:
·David Roodman, MD, PhD
Grant Support
·Suzanne Lentzsch, MD, PhD
·MMRF
·IMF
·ASH Scholar