Cereblon expression is required for the antimyeloma
activity of Lenalidomide and Pomalidomide
Yuan Xiao Zhu, Esteban Braggio, Chang-Xin Shi, Laura A Bruins, Jessica
E Schmidt, Rafael Fonseca, P. Leif Bergsagel, Chad C. Bjorklund, Robert
Orlowski, and A. Keith Stewart
Scottsdale, Arizona
Rochester, Minnesota
Jacksonville, Florida
Immunomodulatory drugs (IMiDs)
O
O
O O
O
H
H
O
H
N
N
N
N
O
N
O
N
O
O
NH
O
NH
2
2
Thalidomide
Lenalidomide
Pomalidomide
1
Identification of a Primary Target of
Thalidomide Teratogenicity
Takumi Ito, Hideki Ando, Takayuki Suzuki, Toshihiko
Ogura, Kentaro Hotta, Yoshimasa Imamura, Yuki
Yamaguchi, Hiroshi Handa
Half a century ago, thalidomide was found to be
teratogenic, causing multiple birth defects.......Here,
we identified cereblon (CRBN) as a thalidomide
binding protein.
CRBN forms an E3 ubiquitin ligase complex with
damaged DNA binding protein 1 (DDB1) and Cul4A
that is important for limb outgrowth.
Thalidomide initiates its teratogenic effects by
Science 12 March 2010
binding to CRBN and inhibiting the associated
ubiquitin ligase activity.
Vol. 327, pp1345-1350
CRBN knockdown inhibited LPS induced TNF
secretion and impaired IMiDs mediated inhibition
of TNF production from differentiated U937 cells
3500
3000
l)
2500
m
Control
g/p 2000
(
LPS (1ug/ml)
LPS + Len (50 uM)
lpha
1500
a
LPS + Pom (500 nM)
F
TN
1000
LPS + Tha ( 50uM )
500
0
NT
CRBN shRNA
2
Some lenalidomide resistant MM cell lines have low
CRBN expression
MM1.S
OPM2
OCI/MY5
JJN3
KMS11
OPM1
MM1.S
OPM2
KMS11
JJN3
OCI/MY5 OPM1 SKMM2 KMS12PE MM1.S res
CRBN
-actin
Overexpression of CRBN in OPM1 cell that has low CRBN
expression increased its sensitivity to lenalidomide
MTT (day 6 after treatment with lenalidomide)
l)
1.4
tron 1.2
cog
1
ru
OPM1
d
0.8
o
OPM1/CRBN w t
n
0.6
OPM1/CRBN mut
100%
0.4
(y 0.2
ilitbia 0
V
0
3.1 6.2 12.5 25
50
Lenalidom ide uM
3
CRBN knockdown resulted in cytotoxicity in MM cells
1. 5
o
NT
tde
#1
liz
)
a
rol
1. 0
#1 0
rm
#1 3
o
ont
(n
cTN 0.5
ilitybiaV
0. 0
KMS18
MM1.S
H929
OPM2
JJN3
OPM2 / NT d5
OPM2 / CRBN shRNA d5
PI
81%
35%
Annexin VFITC
Lenalidomide treatment and CRBN knockdown
reduce interferon regulatory 4 expression in MM cells
1 2 3 4
1 2 3 4
IRF4
IRF4
actin
actin
1. H929 control 48h
1. OPM2 NT 72h
2. H929 lenalidomide 48h
2. OPM2 CRBN shRNA 72h
3. H929 control 72h
3. H929 NT 48h
4. H929 lenalidomide 72h
4. H929 CRBN shRNA 48h
4
Small percentage of MM cells survived from
CRBN silencing
GFP%
Parental cells NT CRBN shRNA
OPM2
H929
KMS18
CRBN knockdown confers
Lenalidomide and Pomalidomide resistance
OPM2 MTT (day6 post treatment)
l) 1.2
trono 1
cdtea 0.8
trenu
OPM2 NT
o 0.6
t
OPM2 #13
de
liz 0.4
a
rmo( 0.2
yitilb 0
Via
0
5uM
10uM
20uM
40uM
80uM
120uM
5
CRBN knockdown confers complete
Lenalidomide and Pomalidomide resistance
OPM2 MTT (day6 post treatment)
l) 1.2
trono 1
cdtea 0.8
trenu
OPM2 NT
o 0.6
t
OPM2 #13
de
liz 0.4
a
KMS18 MTT (6 post treatment)
rmo( 0.2
yit
1.2
il
l)
b
0
tro
Via
1
n
0
5uM
10uM
20uM
40uM
80uM
120uM
ocd 0.8
tea
KMS18 NT
tre 0.6
n
KMS18 #13
u% 0.4
(yitil 0.2
biaV 0
0
5uM
10uM 20uM 40uM 80uM 120uM
CRBN knockdown confers complete
Lenalidomide and Pomalidomide resistance
OPM2 MTT (day6 post treatment)
l) 1.2
tron 1
co
ed
0.8
treatnu
OPM2 NT
o 0.6
t
OPM2 #13
ediz 0.4
al
rm
KMS18 MTT (6 post treatment)
(o 0.2
ility
1.2
iab
0
V
l)
0
5uM
10uM
20uM
40uM
80uM
120uM
otr 1
nocd 0.8teae
KMS18 NT
tr 0.6
n
KMS18 #13
0.6
u 0.4
(%
0.5
ility 0.2
b
0.4
iaV
OPM1/ NT
0
0.3
0
5uM
10uM 20uM 40uM 80uM 120uM
OPM1/ CRBN
0.2
#13
0.1
0
0 nM M M M M M M
0n
100 200n 300n 400n 60 800n 1000n
Pomalidomide
6
CRBN silencing inhibits IMiDs but not other active agents
1.2
1
0.8
.
OPM2 /NT
D 0.6
O.
OPM2/CRBN#13 r
0.4
0.2
0
02.5
33.5
44.5
5
Bortezom ib nM
CRBN silencing inhibits IMiDs but not other active agents
1.2
1
0.8
.
OPM2 /NT
D 0.6
O.
OPM2/CRBN#13 r
0.4
0.2
0
02.5 33.5 44.5 5
Bortezom ib nM
1.2
1
0.8
.
OPM1/NT
D
0.6
O.
OPM1/CRBN#13 r
0.4
0.2
0
0
0.1
1
10
100
De x uM
7
CRBN deletion inhibits IMiDs but not other active agents
1.2
1
0.8
.
OPM2 /NT
D
0.6
O.
OPM2/CRBN#13 r
0.4
0.2
0
0
2.5
3
3.5
4
4.5
5
Bortezom ib nM
1.2
1
0.8
.
OPM1/NT
D 0.6
O.
OPM1/CRBN#13 r
0.4
0.2
0
00.1
1
10
100
1.2
Dex uM
1
0.8
.D
OPM1/NT
0.6
O. 0.4
OPM1/CRBN#13 r
0.2
0
02.5
5
10
20
Mel uM
Impairment of response to Lenalidomide after
CRBN knockdown (gene expression profile)
Lenalidomide 48h
636 30
637 150
OPM2 / non targeting
OPM2/CRBN kd
8
Lenalidomide Resistant Cell Line from Dr. Orlowski
RT-PCR (CRBN)
l)
tro 1.4
noc 1.2
T
No 1
tde 0.8
liza
rm 0.6
on(T 0.4
Calt 0.2
eda 0
lte
MM1S
MM1S Res
D
MM1.S
MM1.S res
CRBN
actin
Genomic abnormalities affecting CRBN confer MM1.S
cell resistance to IMiDs
4N: 88
CRBN: 2 copies
CRBN
-3p12-pter
Tetraploidization
-10
+X
CRBN
CRBN
44 chromosomes
88 chromosomes
CRBN: 1 copy
CRBN: 1 copy
Chr3
Chr3
MM1.S res (provided by Dr.Orlowski)
MM1.S
9
Low CRBN expression is common in lenolidomide
refractory or exposed samples
Q-PCR
nosis) 3.5
diag
3
at
2.5
sample
the
2
to
diagnosis
ed 1.5
relapse
1
(normaliz
ession 0.5
Expr
0
12345
6789
CRBN
Summary
1. We demonstrate here that CRBN is essential for IMiDs activity in MM.
2. Preliminary data support that low levels of CRBN may predict poor drug
response.
3. Our data suggest that CRBN is a critical molecule but not unique source
of IMiDs resistance.
Acknowledgement
MD Anderson Cancer
Center
Myeloma Research Labs
Scottsdale, AZ
Dr. Orlowski's Lab
10