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

Lenalidomide Resistant MM Cells Lack Cereblon MM1.S MM1.S res CRBN b-actin Zhu YX, et al. Blood. 2011;118:4771-9.

CRBN expression as a percentage of the mean levels in all MM Gene Expression Levels of Cereblon Predict Response Rate to Pomalidomide 33% 19% CRBN expression as a percentage of the mean levels in all MM 0% N = Schuster SR, et al. Leuk Res. Jan 2014; 38(1):23-28

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

IKZF1/IRF4/Myc Degradation After Lenalidomide Time 3h 6h 24h 48h Len - + - + - + - + IKZF1 IKZF3 IRF4 MYC b-actin

Degradation of Ikaros by Cereblon Binding Small Molecules

Proteasome Inhibitors block IKAROS degradation by Lenalidomide

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

IZKF1 L208R substitution and codon deletion mutation CRBN/IZKF1/IRF4 Expression and Drug Resistance XG1 MM1.S KMS11 H929 JJN3 EJM OCI-MY5 FR4 SKMM IZKF1 IZKF3 b-actin Len Sensitive Len Resistant CRBN IRF4 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%) IKZF3 point mutation (p.Gly157Arg) exonic, missense, damaging Germline, 462 X, 0 mutation reads

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

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