þÿ0¹0é0¤0É

Overview of New Therapies
And Future Directions
Kenneth C. Anderson, M.D.
Jerome Lipper Multiple Myeloma Center
Dana-Farber Cancer Institute
Harvard Medical School

Novel Therapies Targeting Myeloma Cells
in the BM Microenvironment
Targeting MM Cell
IGF-1 inhibitors, CD40 Ab
17-AAG, PK11195, Smac mimetics
Telomestatin
BM
Targeting MM Cell
and
BM Milieu
Bortezomib/NPI0052
Thalidomide/Revlimid
stromal cells
SAHA/Tubacin
PTK787/GW
Targeting BM Milieu
IKK inhibitors
p38MAPK inhibitors

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

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

PTK787 Inhibits VEGF- Induced Effects in MM
Cells and the BM Microenvironment
Laminin,
Laminin,
BMSCs
fibronectin
VEGF
A. PTK
IL-6
IL-6
MM
cell
VEGF
VEGF, IL-6
VEGF
Increase of
angiogenesis
bone-resorbing
activity
Blood vessel
Proliferation,
Inhibition of
Osteoclast
migration
maturation
Dendritic Cell
B. PTK
Lin et al. Cancer Res 2002; 62: 5019-26

Mechanism of SAHA-Induced Cell
Death in MM Cells
SAHA
Bid
Bcl-2 Ca++
Calpain
Calpeptin
CELL DEATH
IL-6
Mitsiades et al: Blood May 2003

HDAC6 Inhibitor Tubacin Inhibits
Aggresome Formation and MM Cell Growth
misfolded protein
Ub
Ub
protein aggregates
(toxic)
Ub
Ub
proteasome
160
(48h MTT assay)
140
HDAC6
120
RPMI8226
100
l
LR5
tro 80
Tubacin
Ub
Ub
n
DOX40
oc 60
MM.1S
%
MM.1R
HDAC6
40
U266
20
0
0
1.25
2.5
5
10
20
Ub
Ub
Ub
Tubacin (µM)
aggresome

Targeting CD40-Mediated Signaling Cascades in MM
sCD40L
Anti-CD40
PI3K
AKT
n
src
nni
IKK
PS1145
294002
MEK K Raf
Wortma
LY
NF-B IB
PD 98059
MEK
SN50
ERK 1/2
NF-B target genes,
i.e., uPA
Cytokine secretion
mRNA transcription
protein synthesis Migration
Adhesion
Cytoskeletal change
Cell motility

IGF-1Mediated Signaling Cascades in MM
IGF-1

PI3-K
Shc
Ras
Sos
Grb2
IRS-1
Raf
SHP-2
Akt
SHP-2
MEK
NF-B
FKHR
GSK3
hTERT
ERK
?
?
NF-B
hTERT
cell adhesion
anti-apoptosis
cell cycle regulation
Mitsiades C et al. Oncogene 2002; 21: 5673-83

Mitsiades C et al, Cancer Cell 2004;221-230

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

7-mer Peptide from N-Terminus of Smac Binds XIAP
Triggers Caspase-9 Induced Apoptosis in MM
Mito
Smac mimetic
Smac
7
184
N-
Smac
-C
XIAP
BIR1
BIR2
BIR3
Ring
Caspase-9
Caspase-3
Apoptosis

Targeting Telomerase Activity in MM
Cells
IL6 IGF-1
Wortmanin
PS-1145
PI3-K
NKkB
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
Akiyama et al. Cancer Res 2003; 63: 18-21

IKK Inhibitor MLN120B Inhibits MM Cell Growth
in the BM Microenvironment
8 MLN120B
1200
7
0 µM
5 µM
MLN120B
6
10 µM
1000
)4
20 µM
0
0 µM
5
1
800
)
20 µM
(1x 4
600
CPM
(pg/ml
3
400
IL-6
2
200
1
0
0
BMSC
MM.1S
BM/MM.1S
BMSC
MM.1S
BM/MM.1S

p38 MAPK Inhibitor SCIO-469 Blocks IL-6
and VEGF Secretion From BMSCs
800
600
600
400
(pg/ml) 400
(pg/ml)
IL-6
VEGF 200
200
0
0
0
0.16
0.8
4
20
100
500
0
0.16
0.8
4
20
100
500
SCIO-469 (nM)
SCIO-469 (nM)
Hideshima et al, Oncogene 2004; in press

Rationally Based Ongoing
Clinical Protocols Combining
New and Conventional Agents
Based on Cell Signaling:
Bortezomib and dexamethasone
Thalidomide/Revlimid and dexamethasone
Based on Proteomics:
Bortezomib and DNA damaging agents Doxil
and melphalan

Dex Augments Thal/Revlimid-Induced
Growth Inhibition in MM
30000
Dex
25000
0 nM
1 nM
10 nM
20000
100 nM
)401 15000
(x
CPM
10000
5000
0
Control
Thal
Revlimid
Hideshima T et al, Blood 2000; 2943-50

Proteomics: Preclinical Rationale for
Combination Therapy in Clinical Trials (2002)
1. Low dose Bortezomib enhances sensitivity to DNA
damaging chemotherapy
2. Low dose Bortezomib can restore sensitivity to DNA
damaging agents
120
(-)
100
Dox
80
(+)
60
survival% 40
20
0
0
2
5
10
Bortezomib
(nM)
Mitsiades N et al. Blood 101:2377

Rationally Based Ongoing Clinical
Protocols Combining New Agents
Based upon gene profiling:
Bortezomib and Hsp90 inhibitor 17 AAG
Based upon cell signaling
Bortezomib and Revlimid
Bortezomib and HDAC inhibitor
Based upon correlative science:
Bortezomib and p38 MAPK inhibitor
SCIO 469

Clinical Application: Coupling PS-341 with
Hsp90 Inhibitors (NCI) Augments
MM Cell Death (2002): Clinical Trial (2003)
50
PS341
0 2 4 8 16 24 h
45
40
Hsp90
35
30
death 25
Hsp70
20
Cell 15
% 10
Hsp27
5
0
l
1
4
34
366
83664
Contro
PS-
C6
S
NSC68
N
1 +
PS-34
Mitsiades N et al. Proc Natl Acad Sci USA 2002 99: 14374

Combination of Bortezomib +Revlimid
50
Patient Cells
40
Revlimid
100
0 uM
5 uM
80
30
lo
death
60
contr
20
%
cell
40
%
20
10
0
010
20
Bortezomib (nM)
0
h)
nM
(72
3
3
ID
h)+PS341
IM
PS341
(72
3
ID
IM

HDAC6 Inhibitor Tubacin Inhibits
Aggresome Formation and Augments Btezomib-
Induced Cytotoxicity in MM Cell Growth
misfolded protein
Ub
Ub
protein aggregates
(toxic)
(MM.1S, 24h MTT assay)
Ub
120
Ub
proteasome
100
HDAC6
Bortezomib
80
Velcade
ontrol
0 nM
c 60
5 nM
Tubacin
Ub
%
Ub
10 nM
40
HDAC6
20
0
05
10
Ub
Tubacin (µM)
Ub
Ub
Aggresome

Gene Microarray Predicts Clinical Response to
Proteasome Inhibitor
response
non-response
Fc alpha receptor
Hrk activator of
apoptosis
N-ras
Hsp 27
Programmed
cell death 10
Cancer/testis antigen 2
Ubiquitin carrier protein

p38 MAPK Inhibitor (SCIO-469) Enhances
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
0
MAP kinase-activated
05
protein kinase-2
PS-341(nM)
(MAPKAPK-2)
Pat cells (PS-341 resistant)
120

100
l

tro 80

n

Hsp27
o 60
c% 40
20
0
020
40
Hideshima et al, Oncogene 2004; in press
PS-341 (nM)

Targeting Host-Tumor Interactions
in Myeloma Therapies
1. A new treatment paradigm targeting both the
tumor cell and its bone marrow micro-
environment can overcome drug resistance
2. Ongoing signaling, gene microarray and
proteomics studies will define novel targets
in the tumor cell and its bone marrow
microenvironment to provide for new, more
specific and less toxic, myeloma therapies

United Nations Against Myeloma
Jerome Lipper Bench to Bedside Research Team
Kenneth Anderson
Masaharu Akiyama
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