Inhibition of Human Plasmacytoma Cell
Growth by a Novel JAK Kinase Inhibitor
Renate Burger, Steven Le Gouill, Yu-Tzu Tai, Reshma Shringarpure,
Klaus Podar, Laurence Catley, Pierfrancesco Tassone, Teru
Hideshima, James D. Rodgers, Jordan S. Fridman, and
Kenneth C. Anderson
Jerome Lipper Multiple Myeloma Center / Dana-Farber Cancer Institute
Incyte Corp., Wilmington, DE
10th IMW, Sydney, Australia, 2005: Experimental Agents
Inhibition of IL-6 Signaling by INCB20 in MuItiple Myeloma
IL-6
INCB20
gp130
gp130
gp80
Jak1
Ras
SHP-2 Grb2
Sos
Jak2
Raf-1
Tyk2
STAT1
PI3K
MEK1/2
STAT3
p42/p44 MAPK
AKT
Survival
Drug resistance
Cell cycle progression
Proliferation
INCB000020 (INCB20) - Profile
M.W. ~400 - soluble in DMSO - stable at room temperature
inhibits all JAK family members (JAK1, JAK2, JAK3, TYK2):
IC
1 nm (enzyme assays)
50
selectivity for JAKs over other kinases 100- to 1,000-fold higher
(>80 tested)
INCB20 inhibits IL-6 dependent growth of INA-6 cells
ek
175000
upta
150000
e
) 125000
in
100000
id
cpm
75000
m
(
50000
y
25000
th
0
H]3
6 (ng/ml)
[
0
.001
.01
.1
1
10
IL-6
[3H] thymidine uptake*
MTS assay*
l)
120
100
tro
100
80
trol)
80
con
60
IC
con
60
50
of
40
of
40
(%
20
%
20
~ 0.5 然
(
0
0
OD
cpm
0.1
0.5
1
5
0.1
0.5
1
5
INCB20 (然)
INCB20 (然)
*) 72 h, IL-6 10 ng/ml
INCB20 induces apoptosis in INA-6 cells
in the presence of IL-6
INCB20 (然)
no
DMSO
0.25
0.5
1
2
treatment
11.1%
10.8%
12.7%
14.2%
32.6%
74.2%
events
Apo 2.7
48 h, IL-6 10 ng/ml
INCB20 inhibits IL-6 induced STAT3 phosphorylation
in INA-6 cells
IL-6
+
INCB20 (然)*
IL-6
INCB20
Co
0
.03
.1
.3
1
P-STAT3
gp130
gp80
gp130
STAT3
Ras
SHP-2
Jak
P-ERK1/2
ERK1
*) o.n. starvation
STAT3
INCB20 2 h
ERK1/2
15 min IL-6 (10 ng/ml)
INA-6 cell growth is enhanced in the presence of BMSC
and inhibited by INCB20
250000
48 h
200000
BMSC alone
uptake
150000
INA-6 alone
INA-6 with BMSC
(cpm)
100000
50000
H]thymidine3[
0
00.1
0.5
1
INCB20 (然)
+ BMSC
INCB20 (然)*
Co
0
.1
.3
1
*) o.n. starvation
P-STAT3
24 h on BMSC
INCB20 2 h
STAT3
INCB20 inhibits growth of patient MM cells
IL-6
BMSC
)
)
cpm
cpm
(
(
30000
20000
25000
15000
20000
uptake
uptake
15000
10000
10000
5000
5000
0
0
H]thymidine
0
0.2
1
5
H]thymidine
0
1
5
3 [
3 [
INCB20 (然)
INCB20 (然)
medium alone
MM cells alone
IL-6 (50 ng/ml)
MM cells with BMSC
Effect of INCB20 on human MM cell line growth
RPMI8226
120
LP-1
100
L363
uptake
OCI-My7
80
MM1.S
control)
60
NCI-H929
of
thymidine
40
U266
thymidine
(%
H]3
20
Dox40
[
INA-6 + IL-6
0
0.1
0.5
1
5
INCB20 (然)
Mechanisms of IL-6 mediated Dex resistance in MM
IL-6
INCB20
gp130
gp130
gp80
Dexamethasone
Jak1
SHP-2
Jak2
Tyk2
Mitochondria
STAT3
RAFTK-P
PI3K
Smac
Bcl-2 family
AKT
proteins
Caspase-9, -3
Survival
Apoptosis
Drug resistance
INCB20 overcomes IL-6 mediated protection against
dexamethasone in sensitive MM1.S cells
w/o IL-6
+ IL-6
)
50000
cpm(
40000
30000
uptake
20000
10000
H]thymidine
0
3 [
-
Dex INCB Dex/
-
Dex INCB Dex/
INCB
INCB
Dex 1 然
INCB20 5 然
IL-6 50 ng/ml
72 h
INCB20 inhibits IL-6 induced signaling pathways
in MM1.S cells
IL-6 + INCB20 (然)*
Co
-
D 1.25 2.5
5
10
20
Phospho-SHP-2
Phospho-AKT
Phospho-STAT3
STAT3
Phospho-ERK1/2
ERK1/2
*) o.n. starvation
INCB20 2 h
Tubulin
15 min IL-6 (50 ng/ml)
Summary & Conclusions
INCB20 inhibits growth of myeloma cell lines and
patient tumor cells, alone and in the presence of
IL-6 and bone marrow stromal cells.
INCB20 specifically abrogates IL-6 induced
signaling pathways and inhibits IL-6 mediated cell
growth, survival, and drug resistance.
Disruption of JAK activity and thereby blocking
cytokine mediated effects may be a valuable
therapeutic approach in multiple myeloma.
Acknowledgments
Jerome Lipper Multiple Myeloma
Incyte Corp., Wilmington, DE
Center, DFCI, Boston, MA
Jordan S. Fridman
Laurence Catley
Kris Vaddi
Dharminder Chauhan
James D. Rodgers
Teru Hideshima
Claire Neilan
Steven Le Gouill
Paola Neri
Klaus Podar
University Clinic Schleswig-Holstein
Reshma Shringarpure
Campus Kiel, Germany
Yu-Tzu Tai
Martin Gramatzki
Pierfrancesco Tassone
Kenneth C. Anderson
Supported by
National Institutes of Health, The Multiple Myeloma Research Foundation,
The Fund to Cure Myeloma, Doris Duke Distinguished Clinical Research Scientist
Award (K.C. Anderson)