Ihab Abd El-Rahman Hematology Department, Hadassah University Hospital

1. The Role of Livin in Tumorigenicityand it’s Expression in Normal Hematopoiesis and in Hematological Malignancies Ihab Abd El-Rahman Hematology Department, Hadassah University Hospital

2. Apoptosis=Programmed Cell Death Death receptors FAS/CD95 TNF family Mitochondria Fadd Tradd Cytochrome C IAP IAP IAP Caspase8 Caspase9 Caspase3 Caspase 6 Caspase 7 Apoptosis Cell membrane BCL2 P53 NFkB

3. BIR RING Livin The Family of Inhibitor of Apoptosis Proteins (IAP) Baculoviruses Mammals CiIAP 239 a.a OpIAP 268 a.a ASFIAP 223 a.a XIAP 497 a.a cIAP-1 604 a.a cIAP-2 618 a.a NIAP 1403 a.a BRUCE 4845 a.a ILP-2 Survivin 142 a.a BIR RING Baculoviral IAP Repeat Really Interesting New Gene, Zn-Finger

4. LIVIN LIVIN Caspase 8 Caspase 9 Caspase 3 Caspase 6 Caspase 7 LIVIN Livin Blocks Apoptosis Through its Ability to Bind and Inhibit Specific Caspases

5. Caspases Cleave Livin 20mM 20mM 200mM 200mM - - - - ZVAD-FMK - + + - + + + + Staurosporine Livin α Livin β cleavage fragments Livin a Livin b tLivin a tLivin b Nachmias et al. Cancer Research, 2003

6. tLivin Acts in a Paradoxical Fashion as aPro-Apoptotic Factor that Causes More Cell Death 30 25 20 % Apoptosis 15 10 5 0 tLivin α tLivin b Empty vector Livin α Livin β Nachmias et al. Cancer Research, 2003

7. Livin - Regulator of Apoptosis Protein Major apoptotic signal Livin Cleaved Livin Confirming the kill Pro-apoptotic activity Feeling Good Transient death stimuli Caspase 9 Caspase 9 Caspase 9 Cytochrome C Caspase 3, 7 Caspase 3, 7

8. Cleavage of Livin will Induce Apoptosis in Cancer Cell • Livin-cleavage promoting agent • Genetically engineered pro apoptotic subunit 

9. Animal Model for Livin Hodgkin’s Disease model L428, a Hodgkin’s Disease cell line - Does not express the Livin protein - Poorly tumorigenic in NOD/SCID mice

10. Hodgkin’s Disease Model Livin G418 Cloning Transfection Selection L428 cells Measuring tumor volume Subcutaneous injection NOD/SCID

11. 100 100 80 80 60 Livin β 60 40 40 20 20 TNF-a Etoposide BIR RING 0 0 pLXSN pLXSN - Livin pLXSN Livin - pLXSN pLXSN Livin - pLXSN - Livin 100 80 % Apoptosis Anti-Fas Staurosporine 60 40 100 20 0 80 1 2 3 60 40 20 0 Vector Livin a Livin b Vector Livin a Livin b Vector Livin a Livin b pLXSN pLXSN - Livin pLXSN - Livin Livin has Two Splicing Variants with Different Anti-Apoptotic Effects Livin α 18 aa BIR RING Ashhab et al. FEBS Lett, 2001

12. 0.6 87 % ) * 3 0.5 * P = 4 E - 05 0.4 47 % 40 % Tumor volume (cm 0.3 0.2 13 % 6 % 0 % 0.1 0.0 EV EV Livinα Livinα Livinβ Livinβ 11 13 Time post injection (Days) Livinα Tumor development EV Livinβ P = 0 . 006 Livin in Hodgkin’s Disease cells Tumor initiation How can we explain the different effects of Livin α and Livin β on tumor development? 100% 80% cm 60% mice with tumors 3 . 0 40% > 20% % 0% EV EV Livinα Livinα Livinβ Livinβ 11 13 Time post injection (Days)

13. Livin α 12D 17D 44D Livin b Livin β tLivin β 3D 8D 1 2 1 2 1 2 1 2 1 2 Cleavage of Livin in Tumors Hodgkin’s Disease tumors Livin a Melanoma tumors tLivin EV Livinα tLivin α Livinβ

14. EV Livin β Livin βD→A Livin βRING Abrogation of The Pro-Apoptotic Effect of Livin p=0.004 LivinβRING p=5.11E-06 LivinβD→A EV Livinβ CCD camera, 26 days post injection

15. The Paradoxical Activity of Livin Livin tLivin Anti-apoptotic activity Pro-apoptotic activity

16. Childhood ALL Livin=1\2 Livin=0 Livin=3+ Choi et al. Blood2007 Low Expression of Livin Improves Prognosis 114 Melanoma patients

17. What Can be Learned from The Animal Model and The Clinical Studies • Livin is a regulator of apoptosis rather than an inhibitor of apoptosis • Livin promotes tumor development • tLivin induces apoptosis and inhibits tumor development

18. SCREENING FOR LIVIN EXPRESSION IN NORMAL HEMATOPOIESIS AND HEMATOLOGICAL MALIGNANCIES BY IMMUNOHISTOCHEMISTRY

19. Normal Bone Marrow immature myeloid precursors megakaryocytes

20. Anti-BCL-2 Anti-Livin germinalcenter germinalcenter mantle zone mantle zone mantle zone Normal Lymph Node Anti-CD20 germinalcenter