MODULATION OF SIGNALING CASCADES
BY INHIBITORS
INHIBIT
OF HISTONE
HIST
DEACETYLASE
AND AKT
T. Hideshima, H. Ikeda, C. Mitsiades, D. Chauhan,
J.E. Bradner, N.C. Munshi, P.G. Richardson,
KC
K.C. A d
n erson
Jerome Lipper Myeloma
y
Center
Dana-Farber Cancer Institute

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Signaling Cascades in MM Cell
migration
in the BM Microenvironment
g
in the BM Microenvironment
GSK-3
SC
FKHR
PKC
MM
Survival
Caspase-9
Anti-apoptosis
Akt
NF-B
Cell cycle
mTOR
PI3-
PI3 K
Bd
Bad
Bcl-xL
Survival
JAK/STAT3
Mcl-1
Anti-apoptosis
Cytokines
Raf
MEK/ERK
proliferation
Bcl-xL
Survival
IL-6, VEGF
NF-B
IAP
Anti-apoptosis
IGF-1, SDF-1
Cyclin-D
Cell cycle
BAFF, APRIL
BSF-3
TNF
Proliferation
TGF
MEK/ERK
TGF
p27Kip1
At
An i
ti
ti
-apoptosis
VEGF
Smad, ERK
Adhesion
cytokines
NF-B
adhesion
LFA-1
molecules
ICAM-1
NF-B
MUC-1
BMSC
NF-B
VCAM-1
Fibronectin
VLA-4

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HDACs
HDAC inhibitors
Nuclear membrane
HDACs (Class II)
Ac
Ac
HDAC
Histone
Histone
Ac
HAT
Tr
T anscription
ON
OFF

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Classification of histone deacetylase
Class I Class II Class III* Class IV
HDAC1 HDAC4
SIRT
SIR 1
T
HDAC11
HDAC1
HDAC2 HDAC5
SIRT2
HDAC3 HDAC6
SIRT
SIR 3
T
HDAC8 HDAC7
SIRT4
HDAC9
SIRT5
HDAC10 SIRT6
SIRT7
*Class III HDACs are homologs of the yest protein Sir2.

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Synergistic anti-tumor activity by HDAC6 inhibitors plus proteasome inhibitors
Protein
Ub
Ub
protein aggregates
(toxic)
Ub
Ub
Ub
Ub
Proteasome
HDAC6
Bortezomib
Ub
Ub
NPI-0052
Tubacin
LBH589
HDAC6
dynein
Ub
Ub
Aggresome
HDAC6
Ub Ub
dynein
Ub Ub
Ub
Ub
Microtubule
Autophagy
Hideshima et al, PNAS 2005;102: 8567.
Hideshima et al, Clin Cancer Res;2005;11: 8530
Catley et al, Blood 2006;108: 3441.

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An acetylation site in the middle domain of Hsp90 regulates chaperone function.
Scroggins BT, Robzyk K, Wang D, Marcu MG, Tsutsumi S, Beebe K, Cotter RJ, Felts S,
Tof
To t D, Karnitz L,
L Rosen N, Neckers L.
L
Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Mol Cell. 2007, 25(1):151-9.
HDAC6 inhibition enhances 17-AAG--mediated abrogation of hsp90 chaperone function
in human leukemia cells.
Rao R, Fiskus W, Yang Y, Lee P, Joshi R, Fernandez P, Mandawat A, Atadja P,
Bradner JE, Bhalla K.
Medical College of Georgia (MCG) Cancer Center, Augusta, GA 30912, USA.
Blood. 2008, 112(5):1886
1
-93.

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Tubacin Increases Hsp90 Acetylation and
Phosphorylates Akt
IP:Hsp90
Tub
Tub (µM)
C
2.5
C 2.5 5
Ac-Lys
IB:HDAC6
HDAC6
IB:Hsp90
Hsp90
HDAC6 siRNA
Tub C 2.5 5 (µM, 6h)
c 100 200 nM
p-Akt
HDAC6
Akt
p-Akt
Akt
Actin

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17AAG Inhibits Tubacin-Induced Akt Phosphorylation
MM.1S
RPMI
Tub
AAG
Tb
Tub
AAG
C
2.5 5 0.5
1 T/AAG
C
2.5 5 0.5
1 T/AAG
Ac-a-tubulin
Ac-a-tubulin
HDAC6
HDAC6
Hsp90
Hsp90
p-Akt
p-Akt
Akt
17AAG does
t
no alter T b
u acin function

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Tubacin Enhances 17AAG-Induced Cytotoxicity
Tubacin + 17AAG
140
48h MTT
120
100
Tubacin
lo
0M
80
0 uM
2.5 uM
contr
60
%
5 uM
40
20
0
0
125
250
500
0
125
250
500
MM.1S
RPMI
17AAG (nM)

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HDAC Inhibitors in Clinical Trials
Compound
Clinical trial
Short-chain fatty acid
Butylate
I, II
Va
V lporoic
a
acid
II
I, III
AN-9
I, II
Hydroxamate
SAHA
I, IIII, III
PXD101
I
LAQ824
I
LBH589
I
Pyroxamide
I
Benzamide
MS-275
I, II
CI-
CI 994
I, IIII, III
Cyclic peptide
Depsipeptide
I, II
Others
MGCD-0103
I

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LBH589 Downregulates STAT3 Phosphorylation
INA6
MM1S
U266
INA6
C
1
2
4
6
8 h
p-STAT3
LBH
0 50
50 100 0
50
50 100 0
50
50 100 nM
STAT3
p-STAT3
p-Akt
STAT3
Akt
p-
p ERK
p-ERK
GAPDH
Ac-H3
H3
GAPDH
LBH 50nM
50nM

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LBH589 Inhibits Phosphorylation of Jak2/STAT3 Induced
by IL-6 or BMSC Co-Culture
cont
IL-6
SC #1
SC #2
LBH
-
+
-
+
-+
-+
p-JAK2
JAK2
p-STAT3
STA
ST T
A 3
T
MM.1S

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17AAG Enhances LBH589-Induced Cytotoxicity
120
U266
100
24h MTT 17AAG
80
0
60
125
control
250
40
%
500
40
%
20
0
0
12 5
.
25
50
LBH589 (nM)

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LBH589 Activates NF-B Associated with Increased p65 Acetylation
MM1S
LBH
0 25 50 nM
Cyto
Nuc.
LBH
+
-+
-
Ac-p65
p65
p84

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IKK Inhibitors Enhances LBH589-Induced Cytotoxicity
LBH+MLN120B (24h MTT)
120
100
80
MM.1S
60
INA6
control%
RPMI
40
20
0
LBH589
0 nM
12.5 nM
0 nM
12.5 nM
MLN(-)
MLN(10 µM)

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PI3K/AKT pathway
Receptor tyrosine kinase
P
PDK1
P
AKT
PIP3
P110
P
PIP3
IRS
P85
Ser473
P
P Thr308
PI3K
PTEN
ROS
p70s6k
p
mTOR
AKT
eNOS
P
P
MDM2
FKHR
GSK3

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Akt inhibitor Perifosine induces cytotoxicity
in MM cell lines
M.W. = 462
120
24h MTT
Perifosine(5 uM) 0 0.5 1
2 4 6 (h)
100
MM1S
p-Akt
p
MM1R
80
Akt
U266
60
INA-6
p-PDK1
RPMI
control
p-FKHRL1
% 40
LR5
p-GSK3 /

/
20
Dox40
tubulin
0
0
1.5 3
6 12.5 25 50 100
Perifosine (µM)
Hideshima et al. Blood 2006

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Perifosine induces cytotoxicity in patient cells
Sample # 1
2
3 4
5
6
7 8
9 10 11 12 13 14 15 16 17 18
p-Akt
Akt
GAPDH
Pat MM Cells
120
100
l
80
o
60
contr
40
%
20
0
0
5
10
20
Perifosine (µM)

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Perifosine Inhibits MM cell growth associated with
inhibition of Akt phosphorylation in
in vivo
vivo
1.2
)
control
3
1.0
daily
1200
weekly
.8
al
e(mm
la
p=0.04
iv
m
04
vr viv
m
u
800
.6
Sm Sur
volu
Cu
Weekly
.4
Cum
Control
250mg/kg (n=7)
400
mor
Log rank
(n=6)
Tu
.2
Daily 36mg/kg
p=0.01
p0.01
(n=7)
0
0.0
1
6
13
19
25
32
10
20
30
40
50
60
70
80
90
Days
DAYS
Days
control
Perifosine-treated
p-Akt
Akt
Hideshima et al. Blood 2006

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Nat Reviews Immunol 2007 7:191-201

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P110 Expression in Primary MM Cells and MM Cell Lines
Patients
1 2345 67 8910
M1S
PM1
PM2
5
PMI8226
OX40
R
M1R
R
266
A6
929
B
P110
P1
10
M
O
O
R
D
L
M
N
M
U
I
H
L
GAPDH
P110
11 12 13 14 15 16 17 18
P110
P110
GAPDH
P110
19 20 21 22 23 24
P110
P110
-Tubulin
GAPDH

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P110
P1
10
ifi
spec c i hibit
n
or CAL101 (C li
a t
s
)
oga
PI3K inhibitor ( Currently Clinical Trial )
Profiler of CAL101
CAL101 (Calistoga)
XL147 (Exelixis)
PI3K/p110
110 (
M)
200
XL765 (Exelixis)
PI3K/P110 (M)
16
GDC-0941 (Genentech)
PI3K/P110 (M)
0.13
PI3K/P11
PI3K/P1 0 (M)
61
BEZ235 (Novartis)
PX866 (ProlX Pharmaceuticals )

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CAL101 I hibit
n
s Akt
Akt
d
an it
its D
t
owns ream M l
o
l
ecu es
CAL101 1.0M treatment
LB
INA-6
Time (h)
Time (h)
0 15
1.5 3
6
0 15
1.5 3
6
AKT
P-AKT (ser473)
P-AKT (Thr308)
P-FKHRL
-tubulin

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CAL101 Inhibits MM Cell Growth
U266
B 120
A
48h
H929
A
48h
H
120
100
LR5
100
80
LB
80
60
Patient1
INA-6
60
40
Patient2
OPM1
40
20
Patien
e t3
20
RPMI8226
0
0
OPM2
0
0.31 0.625 1.25 2.5
5.0 10.0 20.0
0
0.625 1.25 2.5
5.0 10.0 20.0
Drug concentration (M)
Drug concentration (M)
C
120
100
80
60
Hl
Hea th
lthy donor1
40
Healthy donor2
20
Healthy donor3
Healthy donor4
0
0
0.625 1.25 2.5
5.0 10.0 20.0
Drug concentrat
concentra ion (M)

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CAL120 (CAL101 Analog) Inhibits MM Cell Growth in Vivo
e
1600
Control vehicle
1400
volum
Survival proportions
Control vehicle
)3 1200
1.1
10mg/kg
1.0
1000
10mg/kg
al 0.9
30mg/kg
Tumor
(mm
T
800
viv 0.8
0.7
600
sur 0.6
30mg/kg
Mean
400
0.5
0.4
200
0.3
0
Fraction 0.2
1
3
5
8
10 12 15 17 19 22 24 26
0.1
26
00
0.0
0 1020
304050
60
Days from starting treatment
Days from starting treatment
Control
30mg/kg
Vehicle
Control
Treated
Control
vehicle
with
30mg/kg
H&E
P-PDK-1
P-AKT
ACTIN
P-AKT
Detail will be presented by Dr. H. Ikeda

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Conclusions
1. HDAC inhibitors target not only histones, but also other
molecules mediating signaling cascades in MM cells.
2. Small molecule inhibitors targeting those signaling cascades
could augment HDAC inhibitors-induced cytotoxicity.
3. I h
n ibit
hibitors f
o PI3K/Akt
PI3K/Akt path
thway demonstrate signifi
ificant anti
ti-
MM activities.

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Acknowledgement
This study
study is supported by
by National
National Institutes
Institutes of
of Health
Health
Grant Specialized Programs of Research Excellence
(SPORE) IP50 CA10070-01, PO-1 78378 and RO-1 CA
50947; the Multiple
Multiple Myeloma
Myeloma Research
Research Foundation; the
Multiple Myeloma Research Consortium and the Cure
for Myeloma Research Fund.

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