Lenalidomide & Pomalidomide
Inhibit Expression
Expression of
of
IRF4/MUM1
Antonia López Girona
Disclosures
Employ
pyee of Celgene
g
Corporation
p
2
Lenalidomide Mechanisms Defined
In Context Of Disease State
3
Lenalidomide Mechanisms Defined
In Context Of Disease State
ˇ
Identify EARLY factors/pathways required for anti-proliferative function
of lenalidomide
Provide scientific rational for combined
combined used of lenalidomide with
complementary therapeutics
Provide potential biomarker associated with MM sensitivity to IMiDs
4
Lenalidomide induces cell cycle arrest in the
M l
u ti
lti l
p e Myeloma
l
ce lll liline NCI-H929
Lenalidomide (M)
Cell cycle
cy
profile
profile (2 days
day )
DMSO 01
0.1
03
0.3
1
3
10
100
ppRB(S807/811)
80
ppRB(S608)
60
G2/M
S
pRB total
fcellso
G0/G1
40
%
SubG1
p27
20
c-Myc
0
DMSO
0.1
0.3
1
3
10
Actin
Lenalidomide (M)
(Average of three experiments for each condition)
5
Gene Expression Profile is Linked to IMiD
Function in H929
Len or Pom
2
4
8
24
Time (hours)
Immune Response
reased
Interferon signaling
Inc
(30) DNA
Replication
ed
E2F binding site
genes
Decreas
(11) Response to
DNA damage
6
Gene Expression Profile is Linked to IMiD
Function in H929
Len or Pom
2
4
8
24
Time (hours)
M01
DMSO
Lena
DMSO
Lena
Poma
900
700
5.0
40
500
4.0
e
xpression
enes
3.0
2.5
g
e
2.0
1.5
92
1.2
1.0
300
gene
0.9
0.8
0.7
0.6
IRF4
0.5
04
0.4
100
0.3
0.2
0.1
0.0
0
DMSO
Lena
Poma
7
Lenalidomide Inhibits IRF4 Expression in
aD
a ose
Dose-dependent manner
manner in
in H929
H929 cells
Gi
Gene expression
Lenalidomide (M)
100
DMSO 0.1
0.3
1
3
10
)H
IRF4
75
GAPD
Actin
to
mRNA
50
ed
IRF4
25
(normaliz
0 0
0.1
0.3
1
3
10
Lenalidomide (M)
8
IRF4 is required for cell cycle
progression in NCI-
NCI H929 cells
60
Cell cycle (4 days)
50
Mock Tr
T ansfection
100
G2/M
Control siRNA
S
G0/G1
40
IRF4 siRNA
SubG1
80
fcells 30
o
%
60
20
cells%
80 % Dead
10
40
0
SubG1
G0/G1
S
G2/M
20
M C
IRF4si
0
M
Control
siIRF4
3
3
1
2
3 days
(sp)
IRF4
ppRB
IRF4 siRNA mimics lenalidomide
Total pRb
effect on replication markers
p27
9
IRF4 overexpression rescues pomalidomide
effects on cell cycle regulators
H929 cells, transfected with IRF4- or GFP-CMV-based expression
plasmids for
for 24 hr, were treated with DMSO (-)o
) r
or 1 M CC4047 (+)
(+)
for 24 hr
Ctrl
IRF4
P
lid
oma
omide (1 M)
-
+
-
+
0
4
8
16
24 hr
IRF4
IRF4
ppRB (S807/11)
p27
ppRB (S608)
ppRb
pRB Total
Total pRb
p27
p
Actin
Actin
10
Combination of IRF4 siRNA and Pomalidomide
Enhanced Anti-proliferative
p
effect of IMiD
H929 cells were transfected with control (Ctr) or IRF4 siRNAs and
treated after with DMSO or Pomalidomide 1 M for 1 and 2 days
y
Ctr siRNA
IRF4 siRNA
24
48
24
48
hr
Cl
Ce lll
l
cyc e (2
(2 d
)
ays
-
+ - + - + -
+
70
IRF-4
60
Ctrlsi
s DMSO
SO
RB
pp
S807/811
S807/81
50
Ctrlsi Poma
ppRB S608
IRF4si DMSO
cells 40
IRF4si Poma
To
T tal
o
RB
of
RB
% 30
Bim
20
Mcl-1
10
Actin
0
- DMSO + pomalidomide (1 M)
SubG1
G0/G1
S
G2/M 11
Expression of Key Cell Cycle Regulators is Inhibited by
Pomalidomide through
through IRF4 Inhibition
Decreased genes
BCL-xL
BAG1
Negative
PTPN13
Pomalidomide
IRF4 siRNA
CSNK2A1
Regulation
CAV1
Cell Death
PPAN
40
127
CCNA2
903
CCNA1
G
CCND2
GPR125
INSIG1
SKP2
Proliferation
73
IRS2
CSF2RB
ITGA6
IL6R
Cell
79
PIK3R1
IL6ST
Communication
48
PTGS1
DDXs
RAB7L1
147
DHXs
147
SEMA4F
HNRPs
RNA
LSMs
metabolism
SFRSs
Pomalidomide
+ IRF4 siRNA
CCNE1
HRAS
CHAF1
RASA4
CHEK1
Cell Cycle
RASGRP1
MYC
GTPase signal
Control
AKAP13
CDC25A
Transduction
ARGAP1
CDK4
CHN2
IRF4
PKN2
MCM2-61
6, 0
10
ORC6L
RFC3
DNA
POLD3
Replication
POLE2
Data analyzed with GeneGo and NetBio
12
Lenalidomide and Pomalidomide Interfere with
IRF4/MYC
iti
pos ve A t
u
l
oregu t
a ory Loop
NF-B
IRF4
MYC
pathway
-1712
-407
+2123
-1024
Adapted from Shaffer et al. 2008.
IRF4 IP
Myc IP
50
7
DMSO
45
DMSO
6
Poma
Poma
ells 40
cells
40
c
Lena
Lena
5
35
/1000
/1000 30
4
25
vents
events
3
ev 20
15
2
binding
binding 10
F4
1
R
MYC
5
IR
0
0
6 hr
24 hr
6 hr
24 hr
Untr4 II
-1712
-407
-1024
IRF4
MYC
IRF4 + 2123
Untr4 II
13
Anti-proliferative Activity of Pomalidomide Correlates
ith
w
B
li
ase ne level f
o IRF4
IRF4
i
express on in MM
MM
20
P < 0.05
100
R2 = 0.50
)
Karpas-620
n
p
n
16
75
NCI-H929
NCI-H929
LP-1
IRF4
12
inhibitio
U266
of
50
LP1
(%
OPM2
h
level
th
RPMI-8226
8
KMS-12-BM
25
SKMM2
EJN
grow
JJN-3
OPM2
U266
Baseline 4
Cell
0
KMS-12-BM
JJN-3
RPMI-8226
SKMM2
0
-25
0.0001 0.001 0.01
0.1
1
10
100
0
20
40
60
80
100
% Inhibition of proliferation
Pomalidomide (M)
at 10 M pomalidomide
Zang L-H, et al., Abstract #447
14
Summary
Lenalidomide and pomalidomide inhibit IRF4 expression
pp
in H929 cells
IRF4 is required for cell cycle progression in H929 cells
IRF4 overexpression partially rescues pomalidomide effects on cell
cycle proteins
Combination of pomalidomide and IRF4 siRNA enhanced anti-
proliferative effect of IMiD
Lenalidomide and
and pomalidomide decrease
decrease binding of IRF4 and MYC
MYC to
specific DNA sites on IRF4 and MYC genes
Anti-
Anti proliferative activity of
of pomalidomide correlates
correlates with
with baseline
baseline level
level of IRF4
expression in MM
15
Acknowledgements
Celgene - San Diego
Celgene - Summit
Derek Mendy
Ling-Hua Zhang
Svetlana Gaidarova
Blake Bartlett
Bartlett
Billy Romanow
Peter Schafer
Lisa Morrison
George Muller
Weilin Xie
David Stirling
Frank Mercurio
Helen Brady
Norm Richard
Urvi Jhaveri
16
IRF4 siRNA does not affect cell cycle regulatiors in
the pomalidomide resistant
resistant cell line RPMI8226
RPMI8226
M
C
IRF4si
M
C
IRF4si
33 1
2
3
33
1
2
3
IRF4
ppRB
Total pRb
p27
NCI-H929
RPMI8226
17