Liat Josefsberg Ben-Yehoshua, Michal Samookh & Arnon Nagler

1. Heterogenic Expression of Cyclin E in Multiple Myeloma Liat Josefsberg Ben-Yehoshua, Michal Samookh & Arnon Nagler Department of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel

2. Multiple Myeloma MM A malignancy of antibody-secreting B cells A neoplasm of fully differentiated plasma-cells Low proliferative rate A multi-step process - immortalization Extended duration transformation Upon transformation plasma cells accumulate within the bone marrow (BM) chemo-sensitive drug resistance resistance to apoptosis microenvironment-growth factor independence Monoclonal Gammopathy of Uncertain Significance

3. Multiple Myeloma Genetic Heterogeneity MM Affects disease initiation, progression, and therapeutic response. Karyotypic instability is seen at the earliest stage of the disease and increases with disease progression. A major challenge in the treatment of MM

4. MM Cyclin D Overexpression Cdk G1-S phase Translocations (11q13 & 6p21). Biallelic expression. Trisomies. Insertion of regulatory elements, 3'  Ig enhancer (U266). Transcription regulation. No lymphoid tumors G1- arrest Bergsagel et al 2005 The biological significance remains unknown

5. Cyclins Regulate the Cell Cycle cyclin CDK Cyclin is a regultory subunit of CyclinDependent Kinase regulatory catalytic G1-S transition Genomic instability

6. CDK Inhibition a desired therapeutic target overview First generation Pan-CDK inhibitors No improved clinical outcomes Non-specific drug targets, high toxic side effects & limited efficacy. Second generation Increased specificity & potency. PD-0332991 Pfizer: CDKs 4, 6, SNS-032 Bristol-Myers Squibb/Sunesis:CDKs 1, 2, 4, 7, and 9.  TG02 Tragara Pharmaceuticals: CDK 1, 2, 7 and 9, JAK2 and FLT3.

7. Seliciclib a Potent & Specific CDK Inhibitor CDK2/CCNE CDK2/CCNA CDK7/CCNH (TFIIH) CDK9/CCNT G1-S transition phosphorylates RNA polymerase II Cyclacel

8. The Human Kinome - a dendogram of kinases overview Tyrosine kinase-like kinases Tyrosine kinase MAP kinases CDKs MAP kinases GSK CDK-like Casein Kinases PKA PKG PKC S6 kinases Ca2+/calmodulin-dependent protein kinases

9. Kinomescan High Throughput Kinase Inhibition Assay Seliciclib Flavopiridol Karaman et al Nat. Biotechnology 2008

10. Study Design 7 human Multiple Myeloma lines (hMMCLs) were examined Expression profile of CCND/CCNE Sensitivity to CDK inhibition Mechanism of apoptosis Effect on adhesion

11. Heterogenic Expression of Cyclin D Western blot NCI H929 ARH77 U266 CAG MM RPMI 8226 ARP1 37 KDa CCND2 25 50 β-Actin 37

12. ** * p<0.05 ** p<0.01 Heterogenic Expression of Cyclin E western blot qRT-PCR CCNE1 50 KDa 37 50 37 CDK2 NCI H929 RPMI 8226 CAG ARP1 U266 ARH77 10 9 ** 8 ** Relative levels of CCNE1 transcript 2 -ΔΔCt 7 6 5 4 3 * 2 1

13. hMMCLsSensitivty to Seliciclib survival MTT assay Sensitive Resistant NCI H929 ARP ARH CAG U266 RPMI 8226 1 1 1 Cell survival (percentage of control) 0.5 0.5 0.5 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 seliciclib (µM) seliciclib (µM) seliciclib (µM)

14. control seliciclib control seliciclib G1 G1 G1 G1 S S S S Apo Apo Apo Apo G2 G2 G2 G2 Seliciclib Induces Apoptosis cell cycle (propidium iodide) NCI H929 ARH 77 control seliciclib 100 100 90 90 ** p<0.01 80 80 Fraction of cells (%) Fraction of cells (%) ** 70 70 60 60 50 50 40 40 30 30 ** 20 20 10 10 0 0 G1 S G2 apoptosis G1 S G2 apoptosis

15. Selicilcib Induces Apoptosis annexin V 2.81% 2.41% 1.33% 2.18% 24.68% 15.68% 0.44% 80.78% control DMSO Seliciclib 5 hours 50 µM Seliciclib 16 hours 50 µM NCI H929 92.29% 2.49% 93.5% 3.35% 27.48% 32.18% 3.61% 14.17% 3.52% 4.46% 3.65% 4.68% 3.33% 3.57% 4.35% 4.95% PI PI Annexin V Annexin V 91.54% 0.48% 91.39% 0.28% 92.75% 0.35% 89.93% 0.77% ARH77 120 NCI H929 100 80 Fraction of cells (%) 60 40

16. 0 4 8 0 4 8 0 4 8 0 25 50 100 0 25 50 100 0 25 50 100 SeliciclibDownregulates MCL1 western blot ARP1 MM RPMI 8226 ARH77 CAG NCI H929 U266 37 KDa MCL1 37 CDK2 25 RPMI 8226 CAG NCI H929 RPMI 8226 CAG NCI H929 MCL1 37 KDa 37 CDK2 25 seliciclib (µM) Time (hours)

17. Adhesion propery of hMMCLs Stroma Cells VLA-4 α4 β1 Fibronectin MM MM

18. 1.2 1 0.8 Cell adhesion (O.D) 0.6 0.4 0.2 0 ARH77 ARP1 CAG MM NCI H929 RPMI 8226 U266 0.6 ctrl 0.5 Non-specific ab Fibronectin 4alpha block BSA 0.4 Cell adhesion (O.D) 0.3 0.2 0.1 0 ARH77 ARP1 CAG MM NCI H929 RPMI 8226 U266 adhesion assay hMMCLs Adhere to FN via VLA-4

19. 1.2 1.0 FN 0.8 BSA 0.6 0.4 0.2 0 0 1 2 3 0 20 40 60 80 100 survival MTT assay Cell Adhesion Mediated Drug Resistance CAM-DR NCI H929 1.2 MM 1.0 Cell survival Fold over control Cell survival Fold over control 0.8 0.6 0.4 0.2 0 selicicilib (µM) doxorubicin (µM)

20. ARH77 ARP1 NCI H929 RPMI 8226 adhesion assay Seliciclib Inhibits Adhesion 1.2 1.0 0.8 Cell-adhesion fold treated / untreated cells 0.6 0.4 0.2 0 0 20 40 60 80 100 selicicilib (µM)

21. Summary Heterogenic expression of CCND/E in several hMMCLs. Seliciclib a CDK inhibitor is a potent inducer of apoptosis. The apoptosis is mediated by downregulation of MCL1 Selicilib results in reduced adhesion to fibronectin

22. An additional advantage of seliciclib, which is currently in phase I/II clinical trials, may be the prevention of adhesion-mediated- drug resistance P MCL1 seliciclib Induces apoptosis. Evades CAM-DR by preventing adhesion. cyclin CDK PEST163 MCL1 MCL1

23. Thanks Arnon Nagler Michal Samookh Doron Ginsburg Bar-Ilan UNiversity Ronen Alon Weizmann Institute of Science