Understanding the cellular targets of IMiDs ®

and the relevance to clinical efficacy

Dr. A. Keith Stewart

Scottsdale, Arizona Rochester, Minnesota Jacksonville, Florida

Molecular Structure of Thalidomide, Lenalidomide and Pomalidomide

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 binding to CRBN and inhibiting the associated ubiquitin ligase activity.

Identification of a Primary Target of Thalidomide Teratogenicity

Ito T, et al. Science. 2010;327:1345-50.

Cereblon Levels Are Highest in Myeloma, Leukemias and Neuroblastoma

Schuster SR, et al. Blood 2012;120:[abstract 194].

MM1.S MM1.S res

CRBN

β-actin

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

Lenalidomide Resistant Myeloma Cells Lack Cereblon

Lenalidomide Pomalidomide

Control

Cereblon knockdown

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

Cereblon Knockdown Confers CompleteLenalidomide and Pomalidomide Resistance

CR

BN

Ex

pre

ssio

n (

no

rma

lize

d t

o t

he

sa

mp

le a

t d

iag

no

sis)

Low Cereblon Expression is Common inLenalidomide Refractory Patients

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

Truncating Mutation of Cereblon in Drug Resistant pa tient

Egan J, et al. Br J Haematol. 2013:[E-pub ahead of print].

Pomalidomide: Measurable Parameter From Baseline (Serum, Urine, FLC)

N = 331

Lacy MQ, et al. Blood. 2012;120:[abstract 201].

N =

CRBN % of mean MM

0%

19%

33%

Gene Expression Levels of Cereblon Predict Response to Pomalidomide

Schuster SR, et al. Blood 2012;120:[abstract 194].

Gene Expression Levels of Cereblon Predict PFSof Pomalidomide Treated Patients

P=0.0001

3 months versus 17 months

Schuster SR, et al. Blood 2012;120:[abstract 194].

P=0.01 P=0.005

9.1 months versus 27 months

Gene Expression Levels of Cereblon Predict OSof Pomalidomide Treated Patients

Schuster SR, et al. Blood 2012;120:[abstract 194].

Cereblon Expression in HOVON -65/GMMG-HD4

Broyl A, et al. Blood. 2013;121:624-7.

48

1.0

0.8

0.6

0.4

0.2

0.00 12 24 36

Pro

gres

sion

free

sur

viva

l

Months

p = 0.009

> median< median

42 31 13 430 19 4 0

A

1.0

0.8

0.6

0.4

0.2

0.00 12 24 36

Pro

gres

sion

free

sur

viva

l

Months

p = 0.18

> median< median

38 19 3 239 33 10 4

C

48

1.0

0.8

0.6

0.4

0.2

0.00 12 24 36

Ove

rall

surv

ival

Months

p = 0.81

> median< median

42 35 15 847 43 25 10

D

48

–2

60

1.0

0.8

0.6

0.4

0.2

0.00 12 24 36

Ove

rall

surv

ival

Months

p = 0.13

> median< median

48 38 18 940 31 16 6

B

48

11

60

A-B: thal-treated, C-D: bort-treated

Zhu YX, et al. Leuk Lymphoma. 2013;54:683-7.

NN

O

O

O

O

S

Immuno-modulation

Tumoricidal

•↑Signaling (Rho, IL2)•↓TNFα•↑F-Actin/capping•↑Antigen Presentation

•↓Cell Cycle/Prolif •↑Apoptosis•↑Tumor Suppr•↓Oncogenes•↓Migration/invasion•↓Angiogenesis

•↕Progenitor cell expansion/fate•↕Hb

Differentiationand side effects

SIMiD

S

Ubiquitin E3 ligase

ProteasomeSubstrates ?

Lopez-Girona A, et al. Leukemia. 2012;26:2326-35. Carter S, et al. Nat Cell Biol. 2007;9:428-35.

Xu Y, et al. Blood. 2009;114:338-45. Noonan K. Clin Cancer Res. 2012;18:1426-34.

Chauhan D. Blood. 2010;115:834-45.

IMiDs® Bind to a Cereblon Mediated E3 Ligase Resulting in Pleiotropic Clinically Relevant Effect s

Cereblon k/dLenalidomide 48h

ControlLenalidomide 48h

150

30 636

637 136

27

Impairment of Response to Lenalidomide After Cereblon Knockdown (Gene Expression Profile)

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

ββββ-actin

IRF4

1 2 3 4

1. OPM2 NT 72h2. OPM2 CRBN shRNA 72h3. H929 NT 48h4. H929 CRBN shRNA 48h

ββββ-actin

IRF4

1 2 3 4

1. H929 control 48h2. H929 lenalidomide 48h3. H929 control 72h4. H929 lenalidomide 72h

Lenalidomide Treatment and Cereblon Knockdown Reduce IRF -4 Expression in MM Cells

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

KMS11/vector

KMS11/pIRF4

Control lenalidomide 30 uM d7

Viable cells Dead cells

Overexpression of IRF4 Protects MM cells From Lenalidomide Induced Cytoxicity

Zhu YX, et al. Blood. 2012;120 [abstract 1807].

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

Pathway Analysis of Genes Regulated by IMiD-Cereblon

IMiDs Synergize With Dexamethasone to Overcome Resistance

H929 D1 1053 1054D

MS

OLe

nP

omD

exLe

n-D

exP

om-D

exD

MS

OLe

nP

omD

exLe

n-D

exP

om-D

exD

MS

OLe

nP

omD

exLe

n-D

exP

om-D

exD

MS

OLe

nP

omD

exLe

n-D

exP

om-D

ex

C-Myc

IRF4

Actin

Len sensitive Len resistant

Celgene data on file. Courtesy of Dr. R. Chopra

CRBN

ββββ-actin

MM1.S OPM2 KMS11 JJN3 OCIMY5 OPM1 SKMM2 KMS12PE MM1.S res

Cereblon is Not the Only Story in Drug Resistance

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

Do Lenalidomide and Pomalidomide Have Different Substrate Specificities After Binding

the Cereblon E3 Ligase Complex?

Directbinding

POMS

Ubiquitin E3 ligase

Directbinding

LEN

S

Ubiquitin E3 ligase

A

B D

E

C

F

Lenalidomide Proteome

H

BD

EA

F

G

Pomalidomide Proteome

Celgene on file. Courtesy of Dr. R. Chopra

Assay Development and Clinical Role of Cereblon Measurement

What we need• Gold standard reagents and methods

• Reproducibility in multiple laboratories for confirmation of results

• Large clinical trial sample sets

Current Status• Lack of standardization of reagents and assays

• Uncertainty over what to measureo RNA (RNA seq, GEP)o Protein (immunohistochemistry, flow cytometry, western blot)o Splice variants

Cereblon: IMiDs Next Steps

1.What are the substrates that link Cereblon to MM cell death

2.How does Cereblon regulate immune activity of IMiD s

3.What are the relevant differences between IMiDs

4.Extend clinical data into prospective studies

5.Determine value (or not) as a clinical biomarker

6.New drugs targeting the Cereblon - IRF4 pathway