Potential Mechanisms of Drug
Resistance towards Thalidomide (Thal)
and its Immunomodulatory Derivatives
(IMiDs) in Multiple Myeloma Cells: Role
of C/EBP
Introduction
·BM from MM patients show an increase in MVD which
correlates with disease activity (Vacca et al.,1999)
·Thalidomide demonstrated to have anti-angiogenic effects
(D`Amato et al., 1994)
·Thalidomide used as single agent achieved overall response
rate in refractory myeloma of 32 % (Shingal et al., 1999)
·Thalidomide has severe side effects and patients experience
a development of drug resistance after certain time
Development of Immunomudolatory
Derivatives of Thalidomide
Immunomudolatory Derivatives
Thalidomide
(IMiDs, CC-4047)
Presumed mechanisms of IMiDs (CC-4047, CC-5013):
· anti-angiogenic properties
· direct anti-tumor effects
· modulation of immune effector functions (T-/NK-cells)
·less side effects than thalidomide
·no cross resistance with thalidomide
Treatment with CC-4047 induces remissions
in SCID-mice with MM.1R tumors
CC-4047
Thal
Control
3500
1
0,9
3000
0,8
2500
0,7
2000
0,6
Rate
0,5
1500
0,4
1000
0,3
Survival 0,2
500
0,1
0
0
5
152535
455565
0
25
50
75
100
125
Days of treatment
Days of treatment
Lentzsch et al, Cancer Research, 2002
CC-4047 is a stronger angiogenesis
inhibitor than Thalidomide
Mouse corneal micropocket assay
2
mm
3,00
in
2,50
Area
2,00
*
Vessel
1,50
*
cedu
1,00
d
In
0,50
bFGF
0,00
Control
Thal
CC-4047
Lentzsch et al, Cancer Research, 2002
Evidence for clinical activity of CC-4047
in Multiple Myeloma
·Open, single-center, phase 1 dose escalation study of CC-4047 by
Schey et al. in relapsed and refractory MM
·13 of 24 patients (54%) showed decrease of paraprotein >50%
17 of 24 patients (71%) showed decrease of paraprotein >25%
·Responses were observed regardless the number of prior therapies
and therapies with thalidomide
·Evidence for direct anti-myeloma effect and for in vivo T-cell
costimulation
Scheyat al.,JCO, 2004
Heterogeneous response of Multiple Myeloma
cell lines to CC-4047
Resistant
Sensitive
120
Control
to
100
80
Comparede
60
Uptak
40
20
3H-Thymidine
0
-9
.1R
AS
SV
.1S
I-8226
INA-6
U266
M
M
HSS
ARH-77
IM
M
M
ANBL-6
%
RPM
Lentzsch et al, Cancer Research, 2002
Aim of the study
· Test effects of CC-4047 on a wide variety of
hematopoietic cell lines
· Understand mechanisms underlying
resistance/sensitivity to CC-4047
· Identify genes regulating resistance/sensitivity to
CC-4047
Effect of CC-4047 on hematopoietic cell lines
Proliferation measured by 3H-Thymidin incorporation under treatment with 100 µM
CC-4047 for 48 hours
Resistant cell lines
Sensitive cell lines
H9
T-cell leukemia
L591
Hodgkin
L363
Hodgkin
MM1R
Myeloma
L1236
Hodgkin
DHL1
ALC
L540CJ
Hodgkin
Daudi
Burkitt
DEL
ALC
ANBL6
Myeloma
JB-6
ALC
MM1S
Myeloma
Namalva
Burkitt
HSS
Burkitt
BJAB
Burkitt
AS
Myeloma
RPMI
Myeloma
INA6
Myeloma
ARH77
Myeloma
XG1
Myeloma
U266
Myeloma
H929
Myeloma
IM9
Myeloma
SV
Myeloma
Reh
Pre-B-All
Gene array studies to identify genes
regulating sensitivity/ resistance to CC-4047
Method
·MM.1S cells were cultured in RPMI 1640 media containing 10%
FCS in the presence of either CC-4047 (100 µM) or DMSO
(0.1%) for 48 h
· mRNA expression was measured by oligonucleotide microarray
analysis using the Affymetrix GeneChip system
·Results were compared to control (DMSO 0.1% treated cells) and
expressed as fold change
Oligonucleotide Array Expression
Analysis
Clone identification
Gene/Clone match
Fold change Up
regulated
U 66198
Human fibroblast growth factor homologous factor 2 (FHF-2)
3.1
U 49392
Human allograft inflammatory factor-1 (AIF-1)
4.6
U 11690
Human faciogenital dysplasia (FGD1)
4.8
AF 044197
Homo sapiens B lymphocyte chemoattractant (BLC)
7.0
L 07594
Human transforming growth factor-beta type III receptor (TGFß-rec.)
9.7
Down regulated
X 52560
Human gene for nuclear factor NF-IL6 (C/EBPß)
3.9
M 15024
Human c-myb
3.5
U 67369
Human growth factor independence-1 (Gfi-1)
2.5
J 03040
Human SPARC/osteonectin (HUMSPARC)
2.4
CAAT/enhancer-binding protein (C/EBP)
·
C/EBP plays an important role in regulation of cell proliferation and
differentiation
·
Mice deficient in C/EBP show impaired generation of B lymphocytes
suggesting that C/EBP plays an important role in B lymphopoiesis
·
C/EBP is regulated by IL-6, IL-1, TNF-a, LPS therefore was called NF-
IL6
·
C/EPB increased the IL-6 promoter activity in MM cells
·
C/EBP might play an important role in the pathogenesis of MM
Protein expression of C/EBP is down
regulated in MM cell lines sensitive to CC-4047
H929
HSS
AS
DMSO
CC-4047
Thal
DMSO
CC-4047
Thal
DMSO
CC-4047
Thal
-C/EBP
Induction of C/EBP by IL-1 can not prevent
down regulation of C/EBP protein expression in
MM cell lines sensitive to CC-4047
MM.1S
MM.1S + IL-1
DMSO
CC-4047
Thal
DMSO
CC-4047
Thal
-C/EBP
Protein expression of C/EBP is not affected in
MM cell lines resistant to CC-4047
L363
U266
L1236
DMSO
CC-4047
Thal
DMSO
CC-4047
Thal
DMSO
CC-4047
Thal
-C/EBP
Conclusion I
·CC-4047 inhibits the growth of hematopoietic cells and
angiogenesis to a greater extent than thalidomide
·Phase1 study showed response rate of 70% in relapsed and
refractory myeloma
·Resistance of cells to CC-4047 cannot be overcome by higher
dosages of CC-4047
·Therefore specific mechanism and/or characteristics of resistant
cell lines confer resistance to CC-4047 and probably to CC-
5013
Conclusion II
·Protein expression of C/EBP is down regulated with
treatment with CC-4047 in MM cell lines sensitive to CC-
4047
·C/EBP expression is unaffected by CC-4047 in cell lines
resistant to CC-4047
·Induction of C/EBP by IL-1 cannot prevent down
regulation of C/EBP by CC-4047 in MM cell lines sensitive
to CC-4047
·C/EBP might confer resistance toward CC-4047
Future questions
·
Does C/EBP overexpression confer resistance to CC-4047
in MM cell lines?
·
Does specific blocking of C/EBP in MM cells increase
sensitivity to CC-4047?
·
Correlation between C/EBP expression and drug resistance
to other frequently used drugs in the treatment of MM?
·
Does C/EBP provide a marker to predict responsiveness to
IMiDs?
Acknowledgment
·University of Pittsburgh Cancer
·Dana-Farber Cancer Institute,
Institiute, Pittsburgh
Harvard Medical School, Boston
Gulsum Anderson
Richard LeBlanc
Irene Ghobrial
Kenneth Anderson
David Roodman
·University Medical Center
·Children`s Hospital,
Charitè,
Harvard Medical School, Boston
Campus Buch, Berlin, Germany
Mike Rogers
Martin Janz
Robert D`Amato
Ralf Bargou