Transcript Hepatitis C in Liver Transplantation

IMiDs Mechanism of Action
and Future Applications
Scottsdale, Arizona
Rochester, Minnesota
Jacksonville, Florida
A. Keith Stewart
Mayo Clinic in Arizona
IMiD Structures
Side effects
Potency
Potency
Identification of a Primary Target
of Thalidomide Teratogenicity
• 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 binding to CRBN
and inhibiting the associated ubiquitin ligase activity
Takumi Ito, Hideki Ando, Takayuki Suzuki, Toshihiko Ogura, Kentaro Hotta, Yoshimasa Imamura,
Yuki Yamaguchi, Hiroshi Handa. Science. 2010;327:1345-50.
Cereblon
• Cereblon on chromosome 3
was first described as
associated with human intelligence
(Cerebral protein with Lon protease)
• Functions in the brain as an ionic channel regulator
• Highly conserved from plants to humans,
broadly expressed
• Regulates AMP kinase in insulin resistance, obesity
• Forms an E3ligase complex with DDB1, Cul4A,
Roc1
Cereblon Levels are Highest in MM,
Leukemias, and Neuroblastoma
Viability (% control)
Lenalidomide Resistant MM Cells Lack Cereblon
1.2
1
0.8
0.6
0.4
MM1.S Res
0.2
MM1.S
0
Lenalidomide
MM1.S MM1.S res
CRBN
b-actin
Zhu YX, et al. Blood. 2011;118:4771-9.
Gene Expression Levels of Cereblon Predict
Response Rate to Pomalidomide
35
33%
30
25
19%
20
15
10
5
0%
0
<0.81
N = 10
<0.9
>0.9
15
36
CRBN
expression as
a percentage
of the mean
levels in all
MM
Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28
Gene Expression Levels of Cereblon Predict
Overall Survival of Pomalidomide Treated Patients
100
100
P=0.01
80
P=0.005
80
60
% Alive
% Alive
60
40
40
Lowest
25%
20
Lowest
25%
Top 25%
20
0
0
0
12
24
36
Months
48
60
0
12
24
36
48
60
Months
9.1 vs 27 months
Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28
CRBN Binding Proteins Altered by Lenalidomide
Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]
CRBN Binding Proteins Altered by Lenalidomide
Top Lenalidomide Regulated Putative CRBN Interacting Proteins
Experiment 1*
Experiment 2*
Experiment 3*
Regulation
Proteins
-Len
+Len
-Len
+Len
-Len
+Len
GNL2
2
0
2
0
2
0
Down
STUB1
2
0
2
0
2
0
Down
IKZF1
5
0
6
0
3
0
Down
IKZF3
5
0
7
0
10
0
Down
ANKRD12
2
1
4
0
6
0
Down
KPNA2
15
2
16
9
36
19
Down
CUL4A
30
66
34
45
31
58
Up
CUL4B
18
43
9
45
44
130
Up
DDB1
112
179
82
261
122
271
Up
SQSTM1
11
17
7
17
12
21
Up
*The number in each column represents the number of assigned spectra for that
protein
Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]
CRBN Binding Proteins Altered by Lenalidomide
Top Lenalidomide Regulated Putative CRBN Interacting Proteins
Experiment 1*
Experiment 2*
Experiment 3*
Proteins
Regulation
-Len
+Len
-Len
+Len
-Len
+Len
GNL2
2
0
2
0
2
0
Down
STUB1
2
0
2
0
2
0
Down
IKZF1
5
0
6
0
3
0
Down
IKZF3
5
0
7
0
10
0
Down
ANKRD12
2
1
4
0
6
0
Down
KPNA2
15
2
16
9
36
19
Down
CUL4A
30
66
34
45
31
58
Up
CUL4B
18
43
9
45
44
130
Up
DDB1
112
179
82
261
122
271
Up
SQSTM1
11
17
7
17
12
21
Up
*The number in each column represents the number of assigned spectra for that
protein
Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]
IKZF1/IRF4/Myc Degradation After Lenalidomide
Time
Len
IKZF1
IKZF3
IRF4
MYC
b-actin
3h
-
6h
+
-
24h
+
-
48h
+
-
+
Degradation of Ikaros by Cereblon Binding Small Molecules
1800
1600
1400
1200
Thal
Len
Pom
1000
800
600
400
200
0
1
2
3
4
5
6
7
8
9
10
11
Proteasome Inhibitors block IKAROS degradation by
Lenalidomide
1800
2000
1600
1800
1400
1600
1400
1200
1000
0
1200
1000
800
Len (2uM)
Carfilzomib
800
Car+Len 2uM
600
600
400
400
200
200
0
0
1
2
3
4
5
6
7
8
Bortezomib Concentration (nM)
0
0 2.5 5 10 15 20 25 30 35 40
Carfilzomib concentration: nM
Sensitivity of MM to Lenalidomide is Correlated
with IKAROS Degradation Efficiency
0.9
0.8
0.7
0.6
0.5
0.4
Len 2uM 5hrs
0.3
0.2
0.1
0
Most resistant cell lines
Most Sensitive cell lines
• MM cell lines with adenoviral vector expressing IKAROS 1 - luciferase
fusion gene. Luciferase activity was measured and normalized to cells
treated with DMSO
CRBN/IZKF1/IRF4 Expression and Drug Resistance
Len Sensitive
XG1
MM1.S
KMS11
H929
Len Resistant
JJN3
EJM
OCI-MY5
FR4
SKMM
IZKF1
IZKF3
CRBN
IRF4
b-actin
IZKF1 L208R substitution and
codon deletion mutation
t(6;14) IgH-IRF4 translocation
Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]
Survival after Pomalidomide/Dexamethasone
Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]
Ikaros Expression and Pomalidomide/Dexamethasone
Overall survival 7.3 vs 27.2 months
p=0.004
Lowest Quartile
0/11 Patients Responded
Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]
77 gene panel IKZF3 (Aiolis) Mutation: Patient
Progressing on Pomalidomide and Dexamethasone
Tumor, 702 X, 171 mutation
reads (24%)
Germline, 462 X, 0 mutation
reads
IKZF3 point mutation
(p.Gly157Arg)
• exonic, missense, damaging
Summary
• Cereblon is the IMiD target and accumulates with
IMiD binding
• Ikaros is rapidly degraded in presence of IMiD and
blocked by bortezomib or carfilzomib in vitro
• Low Cereblon and Ikaros levels may predict
response and survival
• Resistance can be explained in many cases by
disruption of this pathway
Thank you