imids mechanism of action and future applications

IMiDs Mechanism of Action and Future Applications

A. Keith Stewart Mayo Clinic in Arizona

Scottsdale, Arizona Rochester, Minnesota Jacksonville, Florida

IMiD Structures

PotencySide effects Potency

• 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

Identification of a Primary Target of Thalidomide Teratogenicity

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

0

0.2

0.4

0.6

0.8

1

1.2

Via

bilit

y (%

con

trol

)

Lenalidomide

MM1.S Res

MM1.S

MM1.S MM1.S res

CRBN

b-actin

Zhu YX, et al. Blood. 2011;118:4771-9.

Lenalidomide Resistant MM Cells Lack Cereblon

<0.81 <0.9 >0.910 15 36

0

5

10

15

20

25

30

35

N =

CRBN expression as a percentage of the mean levels in all

MM

0%

19%

33%

Gene Expression Levels of Cereblon Predict Response Rate to Pomalidomide

Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28

0 12 24 36 48 600

20

40

60

80

100

Lowest 25%Top 25%

Months

% A

live

Gene Expression Levels of Cereblon Predict Overall Survival of Pomalidomide Treated Patients

0 12 24 36 48 600

20

40

60

80

100

Lowest 25%

Months

% A

live

P=0.01 P=0.005

9.1 vs 27 months

Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

CRBN Binding Proteins Altered by Lenalidomide

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

CRBN Binding Proteins Altered by Lenalidomide

Zhu et al. Blood. 2014 Jun 9. [Epub ahead of print]

CRBN Binding Proteins Altered by Lenalidomide

IKZF1/IRF4/Myc Degradation After Lenalidomide

IKZF3

IKZF1

IRF4

MYC

b-actin

Len - + - + - + - +

Time 3h 6h 24h 48h

Degradation of Ikaros by Cereblon Binding Small Molecules

1 2 3 4 5 6 7 8 9 10 110

200

400

600

800

1000

1200

1400

1600

1800

ThalLenPom

Proteasome Inhibitors block IKAROS degradation by Lenalidomide

0 1 2 3 4 5 6 7 80

200

400

600

800

1000

1200

1400

1600

1800

0

Len (2uM)

Bortezomib Concentration (nM)

0 2.5 5 10 15 20 25 30 35 400

200

400

600

800

1000

1200

1400

1600

1800

2000

CarfilzomibCar+Len 2uM

Carfilzomib concentration: nM

8226

KMS12PE

EJM My5

SKMM

2JJN

3FR

4H112

OPM2

U266

KMS18

H929

MM

1.S

MM

1.R

KMS11

XG10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Len 2uM 5hrs

Sensitivity of MM to Lenalidomide is Correlated with IKAROS Degradation Efficiency

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

IZKF1 L208R substitution and codon deletion mutation

XG1 MM1.S KMS11 H929 JJN3 EJM OCI-MY5 FR4 SKMM

IZKF1

IZKF3

b-actin

Len Sensitive Len Resistant

CRBN

IRF4

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]

Tumor, 702 X, 171 mutation reads (24%)

Germline, 462 X, 0 mutation reads

IKZF3 point mutation (p.Gly157Arg)• exonic, missense, damaging

77 gene panel IKZF3 (Aiolis) Mutation: Patient Progressing on Pomalidomide and Dexamethasone

• 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

Summary

Thank you