Understanding the cellular targets of IMiDs and the relevance to clinical efficacy Dr. A. Keith Stewart

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1 Understanding the cellular targets of IMiDs and the relevance to clinical efficacy Dr. A. Keith Stewart Scottsdale, Arizona Rochester, Minnesota Jacksonville, Florida

2 Molecular Structure of Thalidomide, Lenalidomide and Pomalidomide

3 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 binding to CRBN and inhibiting the associated ubiquitin ligase activity. Ito T, et al. Science. 2010;327:

4 Cereblon Levels Are Highest in Myeloma, Leukemias and Neuroblastoma Schuster SR, et al. Blood 2012;120:[abstract 194].

5 Lenalidomide Resistant Myeloma Cells Lack Cereblon CRBN MM1.S MM1.S res β-actin Zhu YX, et al. Blood. 2011;118:

6 Cereblon Knockdown Confers Complete Lenalidomide and Pomalidomide Resistance Lenalidomide Pomalidomide Cereblon knockdown Control Zhu YX, et al. Blood. 2011;118:

7 Low Cereblon Expression is Common in Lenalidomide Refractory Patients Zhu YX, et al. Blood. 2011;118: CRBN Expression (normalized to the sample at diagnosis)

8 Truncating Mutation of Cereblon in Drug Resistant patient Egan J, et al. Br J Haematol. 2013:[E-pub ahead of print].

9 Pomalidomide: Measurable Parameter From Baseline (Serum, Urine, FLC) N = 331 Lacy MQ, et al. Blood. 2012;120:[abstract 201].

10 Gene Expression Levels of Cereblon Predict Response to Pomalidomide 33% 19% 0% CRBN % of mean MM N = Schuster SR, et al. Blood 2012;120:[abstract 194].

11 Gene Expression Levels of Cereblon Predict PFS of Pomalidomide Treated Patients P= months versus 17 months Schuster SR, et al. Blood 2012;120:[abstract 194].

12 Gene Expression Levels of Cereblon Predict OS of Pomalidomide Treated Patients P=0.01 P= months versus 27 months Schuster SR, et al. Blood 2012;120:[abstract 194].

13 Cereblon Expression in HOVON-65/GMMG-HD4 A Progression free survival C Progression free survival > median < median Months > median < median p = Months A-B: thal-treated, C-D: bort-treated p = B Overall survival D Overall survival > median < median Months > median < median Months p = p = Broyl A, et al. Blood. 2013;121:

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

15 IMiDs Bind to a Cereblon Mediated E3 Ligase Resulting in Pleiotropic Clinically Relevant Effects IMiD Signaling (Rho, IL2) TNFα F-Actin/capping Antigen Presentation Immunomodulation S S S Substrates? Proteasome Cell Cycle/Prolif Apoptosis Tumor Suppr Oncogenes Migration/invasion Angiogenesis Tumoricidal Ubiquitin E3 ligase Progenitor cell expansion/fate Hb Differentiation and side effects Lopez-Girona A, et al. Leukemia. 2012;26: Carter S, et al. Nat Cell Biol. 2007;9: Xu Y, et al. Blood. 2009;114: Noonan K. Clin Cancer Res. 2012;18: Chauhan D. Blood. 2010;115:

16 Impairment of Response to Lenalidomide After Cereblon Knockdown (Gene Expression Profile) Control Lenalidomide 48h Cereblon k/d Lenalidomide 48h Zhu YX, et al. Blood. 2011;118:

17 Lenalidomide Treatment and Cereblon Knockdown Reduce IRF-4 Expression in MM Cells IRF4 β-actin IRF4 β-actin 1. H929 control 48h 2. H929 lenalidomide 48h 3. H929 control 72h 4. H929 lenalidomide 72h 1. OPM2 NT 72h 2. OPM2 CRBN shrna 72h 3. H929 NT 48h 4. H929 CRBN shrna 48h Zhu YX, et al. Blood. 2011;118:

18 Overexpression of IRF4 Protects MM cells From Lenalidomide Induced Cytoxicity Control lenalidomide 30uM d7 KMS11/vector Viable cells Dead cells KMS11/pIRF4 Zhu YX, et al. Blood. 2012;120 [abstract 1807].

19 Pathway Analysis of Genes Regulated by IMiD-Cereblon Zhu YX, et al. Blood. 2011;118:

20 IMiDs Synergize With Dexamethasone to Overcome Resistance H929 D DMSO Len Pom Dex Len-Dex Pom-Dex DMSO Len Pom Dex Len-Dex Pom-Dex DMSO Len Pom Dex Len-Dex Pom-Dex DMSO Len Pom Dex Len-Dex Pom-Dex C-Myc IRF4 Actin Len sensitive Len resistant Celgene data on file. Courtesy of Dr. R. Chopra

21 Cereblon is Not the Only Story in Drug Resistance MM1.S OPM2 KMS11 JJN3 OCIMY5 OPM1 SKMM2 KMS12PE MM1.S res CRBN β-actin Zhu YX, et al. Blood. 2011;118:

22 Do Lenalidomide and Pomalidomide Have Different Substrate Specificities After Binding the Cereblon E3 Ligase Complex? POM S Direct binding A B F E D Pomalidomide Proteome Ubiquitin E3 ligase G LEN S Ubiquitin E3 ligase Direct binding H B A C E D F Lenalidomide Proteome Celgene on file. Courtesy of Dr. R. Chopra

23 Assay Development and Clinical Role of Cereblon Measurement Current Status Lack of standardization of reagents and assays Uncertainty over what to measure o o o RNA (RNA seq, GEP) Protein (immunohistochemistry, flow cytometry, western blot) Splice variants What we need Gold standard reagents and methods Reproducibility in multiple laboratories for confirmation of results Large clinical trial sample sets

24 Cereblon: IMiDs Next Steps 1.What are the substrates that link Cereblon to MM cell death 2.How does Cereblon regulate immune activity of IMiDs 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