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The Influence of the Tumor Microenvironment on
Drug Resp
gponse and Drug Resistance: A Potential
New Therapeutic Target for Myeloma
William Dalton PhD, MD
H. Lee Moffitt Cancer Center and
Research Institute
Tampa, Florida
Florida

Proposed model of
of the
the influence of
of BM
BM Microenvironment
Microenvironment on
on MM
MM
cell drug response and acquired drug resistance.
Drug Exposure
A.
Apoptosis
B.
Mutations
MM cells
Bone Marrow Stroma:
De novo drug resistance
Acquired Drug Resistance and
1) Soluble Effectors and 2) Direct Contact
BM independent Growth

The Tu
T mor
u
Microenvironment:
A Heterologous Network of Survival Signals
Ml
Myeloma C l
e l
ll
VLA-4,
LFA-1
VLA-5
VLA-6
VLA-1

v 3
NCAM VLA-4
IL-6
NCAM VCAMICAM
FN
CO LM VN
Bone Marrow
Marr
Stro
Str ma

CAM-DR in 8226 Myeloma Cells
50%
30%
40%
cells
cells
tic
20%
*
tic
30%
o
*
o
*
20%
Apopt
Apopt
10%
%
%
10%
0%
0%
Suspension FN adhered
Suspension
FN adhered
1 M Doxorubicin
50 M Melphalan
* P<
P 0
. 5
05 by
by Student's
Student' T-test; effect
effect reversed by addition of 4
and 5 blocking Ab
Damiano et al, 1999. Blood. 93:1658

Patient Specimen
Specimen
01
0. %P
1%
olyHEMA
PolyHEMA
Fibronectin
+ 200 M Melphalan

FN Adhesion Protects Myeloma Patient
Specimens From
From Melphalan
Melphalan Induced
-
Apoptosis
100
90
Sus
80
FN
ptosis 70
60
apo
50
ificc 40
Spe 30
% 20
10
0
12
345
678
9
10
Melphalan [200 M]

Bim, Bcl-2 Interacting Mediator of Cell
Death
Classification of Bcl-2 Proteins
Bcl-2 Protein Family
Anti-apoptotic members
Pro-apoptotic members
Eg: Bcl-2, Bcl-xL
Multi-domain or Bax-like proteins
BH3-only proteins
Eg: Bax, Bak
Eg: Bim, Bid, Bad, Blk

Bim levels
levels are
are reduced in 8226 cells on adhesion to
to
Fibronectin compared to Suspension or LR5 cells
Hazlehurst et al. Cancer Research, 2003

Hematopoietic tumor cell lines with divergent
genetic background show reduced Bim levels
when adhered to FN (BJH,2007).
H929
U937
K562
us
N
us
N
us
N
S
F
S
F
S
F
BimEL
BimL
BimS
-actin

Bim half-life
Bead only 0 hr
FN 1hr Sus 1hr FN 2hr Sus 2hr
FN 4hr
Sus 4hr
FN 6hr
Sus 6hr
100
90
FN
SUS
80
70
ion
dat
60
rag
50
e
td
40
nerc
30
Pe
20
10
0
01
23
4
5
6
Time / hours
·Bim is degraded faster on adhesion to FN

Bim degradation associated with FN adhesion
is bl
blocked by
t
pro eosomal inhibit
hibitors
A.
1hr
2hr
4hr
6hr
Bead only
FN
SSS
FN
FN
FN
S
Bim
B.
G-132M
G-132
M
M
+
+
C-Lac
MG-132
C-Lac
C-Lac
MG-132
VC
+
+
+
C-Lac
VC
+
+
us
us
us
us
N
N
N
N+
S
S
S
S
F
F
F
F
BimEL
-actin

Conclusions
· Reduction of Bim levels is sufficient to
cause di
drug resistance
· Bim levels in FN adhered cells are
regulated in part by increased rate of
protein degradation
· Proteosome inhibitors are able to block
Bim degradation
· Clinical trials combining proteosome
inhibitors with other drugs are warranted

High Dose Melphalan + Bortezomib
· PI: Melissa
Melissa Alsina MD
· Correlative Science:
Bim levels in myeloma cells b f
e ore
d
an
f
a tfter
bortezomib (Hazlehurst, Cancer Res
2003;BJH 2007)
Fanconi Anemia gene and protein expression
levels before and after bortezomib (Chen
(Chen,
BLOOD, 2005)

1 integrin-mediated adhesion to fibronectin enhances IL-6
mediated Stat3 signaling in MM cells
Kenneth H. Shain, Danielle Yarde, Mei Huang, Richard Jove, and William S. Dalton
Manuscript submitted
submitted

IL-6 Signal Transduction
IL6R
GP130
JAK2
STA
ST T
SAP
A
3
K
Ras
3
3
T
T
A
A
ST
ST
MAPK
Apoptosis
Mitogenesis

Activation of STA
ST T
A 's
' in Patients
with M
Myeloma
l
(I
((Immun
(I
it
iity,
ity 1999)

Collaboration between Soluble Factors and
It
Integrins to Promote Cl
Ce lll Survival and G
t
row h
th
FN
IL-6

gp130
Intergins
IL-6R/gp80
P
Vin
P
P
P
Paxillin Talin P
p80
-Y-
P
Src-Family TKs
p110
Pyk2
-Y-
p80
P p80
(c-Src, Lyn, Fyn,
yy
Hck)
p110
P
p130
--Y SHCP
P
Grb2
(?)
Sos
P
RelB
PPP
PPP
P
P
ERK
ER 1/2
ERK
P -S-
ER 1/2
P
P
-S-
P
Stat3
Cell Growth and Survival

Adhesion of the RMPI 8226 MM cell line to FN
enhances IL
IL--66 signaling and Stat3 activation
Sus
FN
A. +IL-
+IL 6:
0
01
0.1
1
10
0
01
0.1
1
10
(ng/ml)
p-STAT3
STAT3
n
4
e
latio
3
2
Increas
1
ldo Phosphory 0
F
00.1
1
10
0
0.11
10
IL-6 (ng/ml)
Stat3
B.
3.5
Sus
FN
3
crease)
++
--
nI 2.5
Stat3
(Fold
2
1.5
1
DNA-Binding3 05
0.5
Stat
0
-
+
-
+
+IL-6
-
++
-
IL-6 (ng/ml)
Sus
FN

The collaboration between 1
integrin adhesion and
IL
IL--66 is observed in all MM cell lines examined
MM1.S
U266
H929
S
S
FN FN
S
S
FN FN
SS
S
S
FN
FN FN
FN
-
+
-
+
-
+
-
+
-
+
-
+
1.0 ng/ml IL-6
p-STAT3
STAT3

t
t
ta
n
ta
S
2
5
S
6
n
tio
n
tion
i
a
1.5
4
5
i
la
Stat3
on
se
y 4
3
ti
r
1
ease
a
o 3
in
2
h
cr
phoryl
e
ryl
crea
p 2
0.5
o 1
h
In
os
In
os
1
h
p
h
0
ld
P
0
creas
ld
0
P
n
os
Susp-
Susp+IL-6
FN -
FN+IL-6
o
Sus p -
Sus p +IL-6
FN -
FN+IL-6
o
S usp-
S usp+IL-6
FN -
FN+IL-6
I
F
F
Ph
Fold

FN adhesion facilitates 1) the pre-loading of Stat3 on gp130
and 2) increased receptor phosphorylation following IL-
IL 6
stimulation
IL-6R
rhIL-
rhIL 6
gp130
IL-6R/gp80
rhIL-6
Sus:
P
P
P
-Y-
p80
P p110
--Y SHC
P
P
Grb2
P
Sos
PPP
P -S- P
P -S-P
P
ERK1/2
ERK
Stat3
gp130
rhIL-6
IL-6R/gp80
rhIL-6
FN:
P
P
P
P
P
P
X
X
P - P
P
-Y-Y P
-
P
P
P
P
Stat3
Stat3
-S-
P
P
-S-
P
P
Stat3

The progression of the proposed models of
Environment Mediated Drug Resistance or
EMDR.
Model
A.
"Suspension"
RM
B. IL-6
C.
"CAM-DR"
FN
D.
MM Cell
FN+IL-6
Growth
E.
&
"EMDR"
Survival
SF
F.
MM cell
SF+A
HS-5 cell
G.
"in vivo"

EMDR in different BMS co-
co culture systems
A. 10 uM Melphalan
B. 10 nM Mitoxantrone
40
s
50
35
45
40
30
apoptosi
apoptosis
35
25
30
20
25
specific
gspecific
15
20
Dru
Drug
15
10
%
%
10
5
5
0
0
RM
TW
SF+A
RM
TW
SF+
SF A
Dexamthesone
C.
Dexamethasone
D.
E. 45
60
RM
RM
RM
40
tosis
TW
60
tosis
SF + A
CM
p 50
p
35
o
o 50
40
30
ap
ap 40
25
30
30
20
ecific 20
ecific 20
15
10
sp 10
sp 10
%
%
5
0
0
0
5nM
20nM
100nM
5nM
20nM
100nM
Dex
Melphalan

S l
o bl
u e factors produced by BMS
BMS cells induces Stat3
phosphorylation
8226
RM
TW
Time (hours)
2
4
8
2
4
8
p-Stat-3
Stat 3
-

MIP-1
MM Cells
TNF-
ECM
IL-6
ECM
IL-
BMSCs
Physical Determinants
Soluble Determinants
MIP-1, IL-6, VEGF
IL-6
IL-6
FGF
TNF-
HGF
VEGF
IGF-1
TGF-
VEGF
IL-1

Bone Marrow Microenvironment Influence
on Ml
Myeloma Growth
th
d
an Survi l
va
·Physical Factors: ECM with Integrins
yg
G0/S1 Myeloma Growth arrest due to inc. p27
(Oncogene, 2003)
Resistance to apoptosis:
pp
dec Bim (Can. Res 2003;
(; BJH 2007)
inc non-can NFB (Oncogene 2003)
· Physical Factors (ECM & BMS) + Soluble Factors (IL-6,
others)
IL-6 overcomes growth arrest mediated by integrins (FN)
by inc STAT3 and ERK signaling
Resistance to apoptosis: inc Notch1 (BLOOD 2004)
inc Stat3 (submitted)

RZ3 and HYD1 but not the scrambled peptide
(HYDS) block adhesion of
of 8226 cells to HS-5
stroma cells.
80000
70000
60000
50000
FU
40000
30000
20000
10000
0
3
rol
Z
R
ontC
/ml
lHYD1
HYD1
YDS1
YDS1
l
l
H
m
H
ug
m
l
l
5
m
m
ug/5
ug/
20
ug/5
ug/
20

HYD1 reverses resistance associated with
co-culturing 8226 cells with BMS cell line.
45
*
Si
Suspension
40
Co-culture
35
*
capoptosis 30
specifi 25
lan
20
Melpha 15
10
5
0
no peptide
HYD1 [50ug/ml]
HYD1S [50ug/ml]

OVERALL CONCLUSIONS
CONCLUSIONS
· Bone Marrow Microenvironment confers a
temporary survival for Myeloma cells against all
forms of cell death (extrinsic-Fas and intrinsic-
drugs, radiation)
· Protection is mediated by combination of
physical and soluble factors
factors (CAM-DR; EMDR)
· Working Hypothesis: Targeting bone marrow
microenvironment, integrin receptors (HYD1)
and soluble factors will enhance treatment
effectiveness and reduce minimal residual
disease and onset of acquired drug
drug resistance
resistance.

Acknowledgments
Lori Hazlehurst, Ph.D.
Mark Meads, Ph.D.
Vasco Oliveira, Ph.D.
Ken Shain, Ph.D., M.D.
Linda Mathews
Tint Lwin, M.D., Ph.D.
Rich Jove Ph.D.
Lia Perez, M.D.
Zhi-Wei Li, Ph.D.
Melissa Alsina, M.D.
UNC Qing Chen, Ph.D.
Mohamad Hussein, MD
H. Lee Moffitt Cancer Center and Research Institute

Full Disclosure: The Dalton Backyard
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Gators: Fl
Florid
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