Influence of Tumor Microenvironment on Drug
Response and Resistance: a Potential New
Target for Drug Development in Myeloma
Melissa Alsina, M.D.
William S. Dalton, PhD, MD
H. Lee Moffitt Cancer Center and
Research Institute
Unicellular drug resistance
The Tumor Microenvironment:
A Heterologous Network of Survival Signals
Myeloma Cell
VLA-4,
VLA-6
LFA-1
VLA-5VLA-1
v 3
NCAM VLA-4
IL-6
NCAM VCAMICAM
FN
CO LM VN
Bone Marrow Stroma
CAM-DR in 8226 Myeloma Cells
50%
30%
40%
cells
cells
ic
ic
20%
*
30%
*
20%
Apoptot
Apoptot
10%
%
%
10%
0%
0%
Suspension FN adhered
Suspension
FN adhered
1 µM Doxorubicin
50 µM Melphalan
* P<.05 by Student's T-test; effect reversed by addition of 4
and 5 blocking Ab
Damiano et al, 1999. Blood. 93:1658
Co-Culture
Co-Culture of myeloma cells and BMS cells
inhibits drug induced apoptosis
100
Suspension
90
80
51.8%
Co-cultured
70
with stroma
(TUNEL)
M1
60
50
40
22.8
%
30
apoptosis
M1
% 20
10
0
1 µM2 µM4 µM
Mitoxantrone
Cell Adhesion Mediated Drug Resistance (CAM-DR)
(CAM-DR)
in Hematopoietic Cell Lines
Myeloma
Cell line
Cytotoxic agent
8226
VP-16
·Damiano et al Blood 93:1658-67, 1999
Doxorubicin
·Hazlehurst et al Oncogene 38:19-27, 2000
Melphalan
Radiation
U266
Mitoxantrone
·Damiano et al Leuk. Lymphoma 38:71-81, 2000
Melphalan
Leukemia
Cell line
Cytotoxic agent
K562
Mitoxantrone
·Damiano et al Leukemia 15: 1232-9 2001
Melphalan
STI571
AG957
Ara-C
Vincristine
Radiation
U937
Mitoxantrone
·Hazlehurst et al Blood 98:1897-903. 2001
VP-16
Patient Specimen
0.1% PolyHEMA
Fibronectin
+ 200 µM Melphalan
FN Adhesion Protects Myeloma Patient
Specimens From Melphalan-Induced Apoptosis
100
90
Sus
80
FN
70
60
apoptosis
50
40
Specific 30
% 20
10
0
12
345
678
9
10
Melphalan [200 µM]
Mechanisms of CAM-DR
Question: Does cell adhesion to FN alter
gene expression of genes that may
influence cell survival or drug response
(de novo drug resistance)?
Genotypic and Phenotypic
Comparisons Between de novo and
Acquired Drug Resistance
Question: Are the mechanisms determining
initial drug response (or de novo drug
resistance) similar to mechanisms that
determine acquired drug resistance?
Hypothesis: CAM-DR is a form of de novo drug
resistance that eventually allows cells to
acquire a complex drug resistant phenotype.
Isogenic Myeloma Model for Studies of de novo
and Acquired Melphalan Resistance
8226/LR5 cell line:Acquired
Melphalan Resistance
(Bellamy et al Cancer Research, 1991)
· Chronically exposed RPMI 8226 myeloma cells to
melphalan
· 6 fold resistant to melphalan
·Reduced DNA crosslinks
·Increased Non Protein Sulfhydryl (NPSH) levels
CAM-DR in 8226 Myeloma Cells
70
60
8226/Sus
8226/FN
50
8226/LR5
40
Apoptosis
30
20
Specific
10
%
0
20
40
60
80
100
120
Melphalan [uM]
(Damiano et al, BLOOD, 93:1658, 1999)
Comparison of de-novo and Acquired Melphalan
Resistance: Crosslinks
Acquired
de novo
60
80
8226/Sus
8226/Sus
50
8226/LR5
60
8226/FN
40
30
40
20
Cross-links
Cross-links 20
10
DNA
DNA 0
0
-10
-20
20
40
60
80
100 120
20
40
60
80
100 120
Melphalan [uM]
Melphalan [uM]
8226/Sus vs LR5
8226/Sus vs FN
Comparison of de-novo and Acquired Melphalan
Resistance: Mitochondrial Perturbations
HCl-ETOH
50 uM Melphalan
40
Sus
FN
LR5
Sus
FN
LR5
35
8226/Sus
8226/FN
Procaspase-3
Staining 30
8226/LR5
6
25
DiOC 20
of
15
Procaspase-7
Loss 10
5
Percent
0
25
50
100
Melphalan (µM)
Caspase-9
-actin
Summary Functional Comparisons of
Acquired Vs De-novo Melphalan
Resistance
· Acquired resistance: correlates with
decreased melphalan induced
crosslinks, increased gluthathione
levels, mitochondria protection
· De-novo resistance: Correlates with
mitochondria protection despite equal
melphalan induced crosslinks
Genomic Comparison
Acquired Vs de-novo Melphalan Resistance
· Genomic profiling
Affymetrix HG-133A chip
· Preformed 4 independent experiments
8226/S cells cultured in suspension-reference
population
8226/LR5 cultured in suspension
8226/S adhered to FN 24 hrs
Microarray analysis
· Affymetrix suitworks 5.0
Change between comparison probe set p<0.006
(Wilcoxon signed rank test).
· Probe set- significantly changed relative to
suspension 3 out of 4 experiments performed.
· FN
72 probe sets changed
· LR5
1764 probe sets changed
· Common
25 probe sets in common
Summary Comparison Genotypic
Data
· Acquired melphalan resistance-complex
changes in the transcriptome.
Changes in the FA/BRCA1 pathway
Several changes in the G1/S checkpoint
Gluthathione metabolism
Amino acid transport
· Cells adhered to FN less genotypically complex
Reduced Bim expression.
· Despite divergent phenotypes
Common genes include cholesterol metabolism, G-
protein signaling, adhesion molecules.
Validation of Microarray Analysis: Adhesion
to FN Downregulates BIM Protein Levels
8226/Sus
8226/FN
8226/LR5
BimEL
BimL
Bims
-Actin
The Role of Bim in CAM-DR
· Preclinical studies with 8226 myeloma
cells demonstrates decrease in Bim with
FN adhesion.
Determine universality of this observation
Determine how FN adhesion regulates Bim levels
Prevent downregulation of Bim
Evaluate as new drug target
Conclusions
· Acquired melphalan resistance is phenotypically
and genotypically more complex than CAM-DR
multiple mechanisms primarily related to decreased DNA
damage
· CAM-DR less complex and occurs despite similar
amounts of DNA damage
protection related to reduced mitochondrial damage and
caspase activation, possibly by reducing BIM
Reduced DNA damage is a late event
· CAM-DR protects cells from initial melphalan
cytotoxicity allowing cells to acquire a more
complex drug resistance phenotype related to
decreased DNA damage
Potential Clinical Relevance of CAM-DR
in Myeloma
· The tumor microenvironment prevents tumor cell
apoptosis.
· The bone marrow microenvironment may serve as a
sanctuary for hematological malignancies.
· Tumor cell adhesion is a form of de novo drug
resistance.
· Inhibiting cell adhesion or signaling associated with
cell adhesion may enhance therapeutic effect.
· New drug development in myeloma should include
CAM-DR
Cytotoxicity of Bortezomib in 8226 Cells
Adhered to FN vs Maintained in Suspension
80
70
Suspension
60
Fibronectin
50
Positive
40
30
20
Annexin-V
10
%
0
5 nM
10 nM
20 nM
50 nM
100 nM
Bortezomib
The Dalton Lab and Friends
Acknowledgments
Lori Hazlehurst, PhD
Jeff Painter
Ken Shain, PhD
Terry Landowski, PhD
Lee Wisner
Jonathon Storey
Sarah Saporta
Rich Jove, PhD
Dan Sullivan, MD
Melissa Alsina MD