Anti-Apoptotic Signaling in Multiple
Myeloma (MM) Cells: Therapeutic
Implications
Dharminder
Laurence
Chauhan
Catley
Jerome Lipper Multiple Myeloma Center
Dana Farber Cancer Institute,
Harvard Medical School, Boston

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Death Signal Transduction From Receptor to Nucleus
Apoptotic Signal
Mediators
Protein Tyrosine/Serine-threonine
Kinases and Phosphatases
Caspases
Proteases Family
Executioners
IAPs:Inhibitor of
Cyto-c
Caspase-8
Apoptosis Proteins
Caspase-3
Mito
Caspase-9
XIAP/Survivin
Smac
PARP
APOPTOSIS

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Two Major Caspase-activating Pathways
TRAIL
Stress Agent
Death Receptor
DR4/DR5
FADD
Caspase-8
Mitochondria
Caspase-9
Caspase-3
Caspase-3
DNA fragmentation >> Cell death

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Apoptotic Signaling Triggered by
Conventional and Novel Agents

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Bortezomib Induces Apoptosis in MM
via Both Intrinsic and Extrinsic Cell-
Death Pathways
Dex, Thalidomide
Bortezomib
DN-JNK
IL-6
SP600125
JNK
IL-6
Caspase-8, and -3
Mitochondria
Smac
Cyto-c
Caspase-9, and -3
Caspase -9, and -3
Apoptosis
Chauhan et al., J. Biol. Chem 2001; 2003

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Superoxide-Dependent and -Independent Mitochondrial
Signaling During Apoptosis
PS-341
2ME2
Dex
Thalidomide
IMiDs
NAC
NAC
Mitochondria
_
O2
MMP
Cyto-c
Smac
Chauhan et al., Oncogene (In press)
Caspases

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Apoptotic Signaling in MM cells:
Intrinsic and Extrinsic
Dex, Thalidomide
Bortezomib
Revlimid
DN-JNK
IL-6
SP600125
Thal
JNK
_
IL-6
m
O 2
Caspase-8, and -3
Mitochondria
IAP/XIAP
Smac
Cyto-c
Caspase-9, and -3
Caspase -9, and -3
Apoptosis

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Interaction of Smac and XIAP Prevents
Caspase-9 Repression by XIAP
Stress
Active
caspase-9
XIAP
Mitochondria
Smac
Caspase-9 activation
Liu Z., et al. Nature 2000, 408:1004; Wu G., et al. Nature 2000, 408:1008;
Du C, et al., Cell 2000, 102:33

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PS-341/Bortezomib: Mechanism of action
Bortezomib
Ubiquitin-Proteasome signaling pathway
Apoptosis
Growth & survival
Mitochondria - JNK/SAPK
NF-B
Mitochon
Cell Death
Chauhan et al., JBC 2003; Blood 2003; Oncogene 2003; Can Res 2003

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Bortezomib

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Structure and function of proteasome

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NPI inhibits all three proteasome
activities
Bortezomib/
NPI-0052
PS-341
NPI
NPI
Post-
1 Post-
2
glutamyl
Tryptic
glutamyl
Tryptic
7
3
7
3
PS
NPI
6
4
6
4
Chymo-
Chymo-
tryptic
tryptic
5
5

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NPI triggers apoptosis in Bortezomib-
refractory patient MM cells
n
tiota
Bortezomib
ne
Resistant cells
mg
raF
ANDe
tivlaeR
NPI (10 nM): -
+
-
+
- +
- +
-
+

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Targeting mechanisms mediating
Bortezomib-resistance
Oligonucleotide array analysis of Bortezomib-sensitive
DHL6 versus Bortezomib-resistant DHL4 cells
Genes elevated (238) in DHL4
versus. DHL6 cells
Hsp27:
1. Function similar to anti-apoptotic
protein Bcl2: Both block
mitochondria-mediated death
signals via cytochrome-c and
Smac release
2. Known in other cell systems
to confer drug-resistance
DHL6
DHL4

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Targeting mechanisms mediating
Bortezomib-resistance
Oligonucleotide array analysis of Bortezomib-sensitive
DHL6 versus Bortezomib-resistant DHL4 cells
Genes elevated (238) in DHL4
versus. DHL6 cells
Hsp27:
1. Function similar to anti-apoptotic
protein Bcl2: Both block
mitochondria-mediated death
signals via cytochrome-c and
Smac release
2. Known in other cell systems
to confer drug-resistance
DHL6
DHL4

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Anti-Sense-Hsp27 Restores Sensitivity in
PS-341/Bortezomib-Resistant DHL4 cells

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Overexpression of Hsp27 (WT) confers resistance
to PS-341 in PS-341-sensitive DHL6 cells

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p38 MAPK Inhibitor (SCIO-469) Enhances
Bortezomib/PS-341-Induced Cytotoxicity
MM.1S
120

SCIO-469 (nM)
100

0
l 80
100
tron
p38 MAPK
SCIO-469
o 60
200
c% 40
20
MAP kinase-activated
0
05
protein kinase-2
PS-341(nM)
(MAPKAPK-2)
Pat cells (PS-341 resistant)
120

l 100

tro 80

n

Hsp27
o 60
c% 40
20
0
020
40
PS-341 (nM)

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Mitochondrial peripheral Benzodiazepine
receptor (mt PBR)
Mitochondrial specific
PS-341
agent: PK-11195
PBRs: Protective
function as Bcl2
Mitochondria
Enhance anti-MM activity
Overcome PS-341-resistance
Reduce PS-341 toxicity

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Mitochondrial peripheral Benzodiazepine
receptor (mt PBR) as therapeutic target
PBRs: mitochondrial pores; overexpressed in cancer cells,
including MM; function similar to Bcl2 i.e, block apoptosis
PK-11195: PBR antagonist-sensitizes AML (MDR+) cells to
chemotherapy; non-toxic to normal myeloid cells
(Cotter et al., 2002)
p-glycoprotein-mediated
Facilitate apoptosis >
drug-effux >> Ara-C toxicity
opening mitochondrial
-pore/ (PTPC)
PK-11195: Small molecule- Orally available; stable in vivo and
well-tolerated (Cotter et al., 2002)

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Apoptotic signaling mechanism of PS + PK
PS-341 + PK-11195
DN-JNK
JNK
Bax
Bax-/-

-
m
O2
Mitochondria
Smac
Cyto-c
Caspase-9
Caspase-8
Caspase-3
Apoptosis

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SN38-induced Apoptotic Signaling
Hours
0
2
4
6
8
16
24
p-JNK
JNK
Hours Fas +ve
p21
24
81%
caspase 8 FL
8
80%
CF
4
70%
caspase 3 FL
2
68%
CF
0
63%
isotype
PARP FL
CF
tubulin

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Augmentation of SN38-induced cytotoxicity
SN38 0 nM
110%
SN38 2nM
SN38 0 nM
SN38 4 nM
100%
l)
SN38 2 nM
l)
y
ro
SN38 4 nM
90%
it
y
ro
80%
it
s
s
nt
n
n
cont
e
co
60%
lD
of
70%
lDe
of
a
n
a
n
tic
tio
io
p
c
tic
40%
p
ct
O
ra
50%
O
ra
(F
(F
20%
0%
30%
050
100
00.25
0.5
NU1025 (mM)
CH11 (mcg/mL)

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CD95L
FADD
MORT1
CD95
DISC
c-FLIP
JNK
Caspase-8
CPT
Topoisomerase
Caspase-3
PARP

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CD95L
Fas activators
FADD
MORT1
CD95
DISC
c-FLIP
JNK
Caspase-8
CPT
Topoisomerase
Caspase-3
PARP
PARP inhibitors

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Conclusion
Understanding cellular molecular events during apoptosis
gives important information to guide future therapy directed
at overcoming drug resistance
Novel therapeutic targets may initiate apoptosis as well as
releasing the block to apoptosis further downstream
Apoptotic signaling events may guide future combinations
of novel therapies with conventional therapies

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Collaborations
Kenneth C. Anderson
Stanley Korsmeyer
Laurie Catley
Anthony Letai
Teru Hideshima
Cancer Immunology &
Klaus Podar
AIDS, Dana Farber Cancer
Renate Burger
Institute, Harvard Medical
Robert Schlossman
School, Boston
Nikhil Munshi
Roger Davis
Paul Richardson
UMass Medical School
Worcester, MA
Steven Rosen & Nancy Krett
Robert H. Lurie Comprehen-
Sive Cancer Center,
Chicago, IL
Medical Oncology, Dana Farber
William S. Dalton
Cancer Institute, Harvard Medical
H. Lee Moffitt Cancer Center,
School, Boston
Tampa, FL

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Bone marrow microenvironment and
drug-cytotoxicity
Growth/survival
IL-6
MM cells
IGF
Chemotherapy-induced
Cytotoxicity: e.g Dex
BMSCs

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Interaction of MM Cells and Their
BM Microenvironment

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Gene Expression Analysis in MM Patient versus
Syngeneic Twin
F
Mcl-1 antisense
GFR3 I
Anti-apoptosis
T
n
ra
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n
i
n
s
b
fo
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o
A
T
a
r
t
n
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i
tiatio
on
l
Mcl-1, Dad-1
t
FG
o
i
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s
m
FLIP
FR3
en e
ifferen

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scr ivi
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An
ct
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Tran A
ge
Ant
nesi
Ubiquitin/
i
s
me
-angi
Genes modulated in MM
proteaso
Ag
og
e
e
nt
n
Function of gene products
i
s
c
Proteasome
Targeted therapies
Inhibitor ­ PS-341

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IGF-1Mediated Signaling Cascades in MM
IGF-1

PI3-K
Shc
Ras
Sos
Grb2
IRS-1
Raf
SHP-2
Akt
SHP-2
NF-B
MEK
FKHR
GSK3
hTERT
ERK
?
?
NF-B
hTERT
cell adhesion
anti-apoptosis
cell cycle regulation

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Targeting Telomerase Activity in
MM Cells
IL6 IGF-1
Wortmanin
PS-1145
PI3-K
NF-B
LY 294002
Arsenic Trioxide
hTERT Transcription
Phosphorylated
Akt
hTERT Protein
hTERT Protein
Phosphorylation
(unphosphorylated)
p21
Hsp90
Geldanamycin
p53
Protein Folding
Telomerase Activity
Telomestatin, Porphyrins
Anti-sense Telomerase
Anti-telomerase RNAi

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