In Vitro Activity of a Novel Small
Molecule Cyclin Dependent Kinase
Inhibitor, CYC202 (seliciclib or R-
Roscovitine), in Multiple Myeloma
Noopur Raje MD
Dana-Farber Cancer Institute
Jerome Lipper Myeloma Center
Boston
IMW 2005
嵩yclins are over-expressed
in MM cells.
嵯ysregulation of Cyclin D1-
3 occurs in all MM making
them susceptible to
proliferative stimuli
愁C classification based on
IgH Translocations and
cyclin D expression
Hideshima et al, Blood 2004
Mechanisms of Drug Resistance in MM
嵯efects in apoptotic
signaling
慈ver-expression of
MDR genes
嵩ytokines: IL-6,IGF-
1
嵩AM-DR
Hideshima et al, Blood 2004
Novel Therapies targeting MM cell and BM microenvironment
Hideshima et al, Blood 2004
CDK Inhibitors:
Cyclacel, CYC202, R-Roscovitine,
Seliciclib
感urine analog
嵩ompetes with ATP for its binding site on CDKs
愚ore specific for cdk2 inhibition and action on cyclin
A and E
廈as shown activity against 19 human cancer cell
lines
廈as shown activity in a xenograft model
慈ngoing phase I and phase II studies in breast, lung,
and B cell hematologic cancers
T ime course of effect of Cyclacel on MM1.S cells by MT T
140
24 hrs
48hrs
120
72hrs
l 100
ro
ontc 80
of
ge
60
ta
IC
n
50
erc 40
pe
20
0
0
1
10
25
50
100
Cyclacel dose (micromola r)
Cyclacel induces cytotoxicity in MM cells within 24 hours
Cyclacel induces cytotoxicity in MM cells sensitive
and resistant to conventional chemotherapy and
steroids.
SENSITIVE CELL LINES
RESISTANT CELL LINES
MM1S
MM1.R
120
120
OPM2
LR5
100
RPMI
100
DOX40
l
U266
l
ro
ro
nt
80
nt
80
o
o
C
C
of
60
IC
of
60
e
50
IC
g
ge
50
ta
ta
n
40
n
e
40
e
rc
rc
e
e
P
20
P
20
0
0
0
10
15
202550
100
0
1015
20
2550
100
CYCLACEL (micromolar)
CYCLACEL (micromolar)
The IC
ranges between 15 and 25 micromolar for most
50
cell lines.
Cyclacel is cytotoxic to patient MM cells.
Effect of Cyclacel on MM pt Cells
140
120
l 100
ro
ont
C
80
Pt #1
of
ge
60
Pt#2
tane 40
rceP
20
0
0
5
10
15
20
25
50
Cycla ce l Dose (micromola r)
Cyclacel induces apoptosis in MM cells in a time and dose dependent
manner as evidenced by PARP cleavage and Caspase Cleavage.
A
B
0
6
12
18 hrs
0 hrs
2 hrs
6 hrs
Cleaved PARP
Cleaved Caspase 8
C
+ ZVAD-Fmk
C
25
50 25
50然ol
Caspase 3
12 hrs
18 hrs
24 hrs
Cleaved Caspase 3
+ ZVAD-Fmk
C
25
50 25
50然ol
Caspase 8
Cleaved Caspase 8
Cyclacel down-regulates Mcl-1 and pSTAT3 in a
time and dose dependent manner.
Cyclacel 25 然
0
2
4
6
8hrs
pSTAT3
STAT3
MCL1
Tubulin
+ZVAD
Cyc(然)
0
25
50
0
25
50
Caspase 8
Cleaved
caspase 8
MCL1
Tubulin
Down-regulation of Mcl-1 is independent of
caspase cleavage
Down-regulation of Mcl-1 was seen in resistant cell lines as
well.
Dox-40
LR5
Seliciclib (然)
0
25
50
0
25
50
Mcl-1
Tubulin
Cyclacel overcomes the protective effect of
BMSCs on MM cells
Effect of Cyclacel by T [H]T dR Uptake in co-culture system
35000
BMSCs
30000
MM Cells
MM + Stroma
25000
20000
m
cp 15000
10000
5000
0
0
1015
202550
Cyclace l (micromolar)
MM1.S
MM1.S +
stromal cells
O 2
然
然
然
然
然
然
ddH
0
25
50
0
25
50
MCL1
+BMSCs
Cyc (然)
0
25
50
0
25
50
pSTAT3
GAPDH
STAT3
MCL1
80
70
60
els)x 50(Pi
40
rea
ra 30
o
teri 20
In 10
0
Cyclacel [然]
Cyclacel down-regulates both MCL1 and pSTAT3 in the
presence of BMSCs.
Cyclacel down-regulates Mcl-1 even in the
presence of IL-6
cela
Control
IL-6
IL-6+Cycl
MCL1
Tubulin
Effect of Cyclacel on adhesion mediated IL-6
700
IL-6
600
Cyclacel inhibits adhesion
500
400
l
mediated IL-6 production
/m
pg 300
by BMSCs.
200
100
0
0
0
5
101520
2550
Cycla cel Dose (micromolar)
MM.1S
MM.1S + stromal cells
O 2
然
然
然
然
然
然
ddH
0
25
50
0
25
50
Cyclacel downregulates IL-6
transcription and protein
IL-6
expression in MM cells even in
the presence of BMSCs
GAPDH
MM 1.S + BMSCs
IL-6
160
140
ls)e 120
pSTAT3
ix
(P 100
rea
80
aro 60
teri
40
STAT3
In
20
0
Cyclacel [然]
MCL1
IL-6 ELISA
Cyc 25 然
-
+
-
+
35
MM1.s with BMSCs
MM1.S
IL-6 Ab
-
-
+
+
30
25
l)
20
/mg
(p 15
-6
MCL1 downregulation by
IL
10
5
cyclacel is only in part
0
025
50
related to blockade of IL6
Cyclacel Dose (micrograms)
Cyclace l in combination with Ve lcade (1nM )
120
Cyclacel
*
Cyclacel + Velcade (1nM)
100
*
l
* CI <1
ro
80
ntoc
of
*
60
egtane 40
rc
pe
20
0
0
10152025
Cyclacel Dose (micromolar)
Cyclacel in combination with Doxorubicin (88nM)
120
Cyclacel
Combination of
Cyclacel + Doxorubicin (88nM)
*
100
*
* CI <1
l
Cyclacel with Velcade
tron 80
oc
and Doxorubicin
of
60
geta
results in synergism
ne 40
rc
pe
20
0
0
1015
2025
Cyclacel Dose (micromolar)
CONCLUSIONS:
嵩yclacel is cytotoxic to MM cells which are sensitive and
resistant to chemotherapy as well as patient MM cells.
嵩yclacel causes apoptosis in MM cells in a time and dose
dependent manner.
嵩yclacel can overcome the protective effects of cytokines
and BMSCs and therefore overcome drug resistance.
嵩yclacel causes apoptosis of MM cells in part by down-
regulation of Mcl-1 and pSTAT3.
嵯own regulation of Mcl-1 is in part related to inhibition of
IL-6
嵩ombinations with Velcade and Dox are synergistic and
should be tested in future clinical trials.
Acknowledgements:
Ken Anderson
Teru Hideshima
Dharminder Chauhan
Shaji Kumar
Paul Richardson
Kenji Ishitsuka
Nikhil Munshi
Yasui Hiroshi
Norihiko Shiraishi
Simon Green
Emma Voinescu
Cyclacel inhibits cyclins differentially in a time
dependent manner
0
2
4
6
8hrs
Cyclin A
Cyclin B1
Cyclin D3
Cyclin H
Cyclacel overcomes the protective effects of IL-6, VEGF
and IGF-1 on MM cells
Cyclacel overcomes the protective effect of cytokines on MM cell
proliferation
(3[H]T dR Uptake)
Control
0
15
10000
25
9000
50
8000
100
7000
6000
m
5000
cp
4000
3000
2000
1000
0
MM1.S
IL-6
IGF-1
VEGF
Cyclacel down-regulates genes involved in transcription and
regulation of transcription of the pol II promoter