1. MEDITERRANEAN SCHOOL OF ONCOLOGY Advanced Courses and Seminars LLC e MM oggi: un paradigma per nuovi standard nelle neoplasie ematologiche Orvieto (Terni), 20-21 novembre 2009 Sopravvivenza e Resistenza allApoptosi delle Cellule LLC-B Stefano Coaccioli Emanuela Rosati Clinica Medica, Terni - Immunologia, Perugia Università degli Studi di Perugia __________________________________________________________________

2. Chronic Lymphocytic Leukemia Kipps TJ Curr Opin Haematol 2000; 7:223-34 CLL come disordine eterogeneo anormalità cromosomiche6, 11, 12, 13 anormalità citogenetiche espressione di geni per Ig mutati vs non-mutati fenotipi Ag di superficieCD5 (CD19, CD23, CD20) marker prognosticidubling time 30x10 9 /L, Tk terapia: – CHT di combinazionefludarabine - mafosfamide – nuove molecolecladribine – molecole biologicherituximab, CAMPATH-1H, lym-1 – altri agenti terapeuticiGC, melarsoprol, depsipeptide, bryostatin1, lactacystin – trapianto cellule staminali – terapia genica ______________________________________________________________________________________________________________________

3. A Sustained Activation of PI3K/NF-kB Pathway is Critical for the Survival of CLL-B Cells Cunì S et al. Leukemia 2004; 18:1391-1400 incremento di B-linfociti maturi CD5 + in presenza di una bassa proporzione di cellule proliferanti: deficit di apoptosi CD5 + long-lived in vivo vs apoptosi spontanea in vitro: – ruolo del microambiente in vivo: apoptosi spontanea associata a perdita di attività NF-kB – lattività costitutiva della via PI3K nelle cellule CLL-B quando coltivate in vitro con fibroblasti: prevenzione dellattivazione della caspasi e dellapoptosi conclusione: la via PI3K sembra rivestire un ruolo chiave nella crescita e nella sopravvivenza delle cellule CLL-B ______________________________________________________________________________________________________________________

4. A Sustained Activation of PI3K/NF-kB Pathway is Critical for the Survival of CLL-B Cells Cunì S et al. Leukemia 2004; 18:1391-1400 lapoptosi spontanea di B-CLL cells in vitro correla con la downregulation di bcl-x L che è prevenuta dalla co-coltura con Ltk - ______________________________________________________________________________________________________________________

5. A Sustained Activation of PI3K/NF-kB Pathway is Critical for the Survival of CLL-B Cells Cunì S et al. Leukemia 2004; 18:1391-1400 il crosslink con CD40 induce la sintesi di DNA nelle cellule B-CLL ______________________________________________________________________________________________________________________

6. A Sustained Activation of PI3K/NF-kB Pathway is Critical for the Survival of CLL-B Cells Cunì S et al. Leukemia 2004; 18:1391-1400 lattività NF-kB dipendente da PI3K, nelle cellule B-CLL induce lespressione delle proteine XIAP e FLIP L ______________________________________________________________________________________________________________________

7. CLL-B Cells but not Normal B Cells are Rescued from Apoptosis by Contact with Normal Bone Marrow Stromal Cells Lagneaux L et al. Blood 1998; 91(7):2387-96 ruolo del microambiente su apoptosi ______________________________________________________________________________________________________________________ apoptosi % di B cellule dopo 48 h di coltura control MEM stromal cell contact no stromal cell contact control MEM no stromal cell contact stroma CM

8. CLL-B Cells but not Normal B Cells are Rescued from Apoptosis by Contact with Normal Bone Marrow Stromal Cells Lagneaux L et al. Blood 1998; 91(7):2387-96 ruolo del microambiente su adesione ______________________________________________________________________________________________________________________

9. CLL-B Cells but not Normal B Cells are Rescued from Apoptosis by Contact with Normal Bone Marrow Stromal Cells Lagneaux L et al. Blood 1998; 91(7):2387-96 ruolo del microambiente su espressione di bcl-2 ______________________________________________________________________________________________________________________

10. Involvement of Notch1 Signaling in Bone Marrow Stroma-Mediated de novo Resistance of Myeloma and Other Malignant Lymphoid Cell Nefedova Y et al. Blood 2004; 103:3503-10 the bone marrow microenvironment plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies interaction with bone marrow stroma activated Notch signaling in tumor cells activation of only Notch-1, but not Notch-2, resulted in protection of tumor cell from melphalan- and mitoxantrone- induced apoptosis overexpression of Notch-1 in Notch-1 - U266 myeloma cells up regulated p21 and resulted in protection from drug- induced apoptosis ______________________________________________________________________________________________________________________

11. Recent Insight into the Role of Notch Signaling in Tumorigenesis Leong KG et al. Blood 2006; 107:2223-33 the mammalian Notch receptor and ligand families ______________________________________________________________________________________________________________________

12. Recent Insight into the Role of Notch Signaling in Tumorigenesis Leong KG et al. Blood 2006; 107:2223-33 ligand-mediated activation of Notch signaling ______________________________________________________________________________________________________________________

13. Recent Insight into the Role of Notch Signaling in Tumorigenesis Leong KG et al. Blood 2006; 107:2223-33 oncogenic Notch signaling pathways in hematologic malignancies ______________________________________________________________________________________________________________________

14. Recent Insight into the Role of Notch Signaling in Tumorigenesis Leong KG et al. Blood 2006; 107:2223-33 oncogenic Notch signaling pathways in solid tumors ______________________________________________________________________________________________________________________

15. Recent Insight into the Role of Notch Signaling in Tumorigenesis Leong KG et al. Blood 2006; 107:2223-33 tumor-suppressive Notch signaling pathways in solid tumors ______________________________________________________________________________________________________________________

16. Notch Signaling: Cell Fate Control and Signal Integration in Development Artavanis-Tsakonas S et al. Science 1999; 284:770-6 elementi del segnale Notch ______________________________________________________________________________________________________________________

17. Notch Signaling: Cell Fate Control and Signal Integration in Development Artavanis-Tsakonas S et al. Science 1999; 284:770-6 interazioni inter-cellulari ______________________________________________________________________________________________________________________

18. Arbiter of Differentiation and Death: Notch Signaling Meets Apoptosis Miele L et al. J Cell Physiol 1999; 181:393-409 il segnale Notch come arbitro del destino cellulare ______________________________________________________________________________________________________________________

19. An Invitation to T and More: Notch Signaling in Lymphopoiesis Allman D et al. Cell 2002; 109:S1-S11 impact of Notch activity on lymphopoiesis ______________________________________________________________________________________________________________________

20. An Invitation to T and More: Notch Signaling in Lymphopoiesis Allman D et al. Cell 2002; 109:S1-S11 Notch signaling may modulate positive selection by inhibiting TCR signaling in a dose-dependent fashion ______________________________________________________________________________________________________________________

21. Activated Notch1 Signaling Promotes Tumor Cell Proliferation and Survival in Hodgkin and Anaplastic Large Cell Lymphoma Jund F et al. Blood 2002; 99:3398-3403 Notch expression in human malignant B- and T-cell lymphoma entities Notch1 expression lymphomaabsent weak strong EB-virusALK-protein B-CLL 3 5-not ded not ded mantle cell 5 4-not ded not ded follicle center 2 8-not ded not ded marginal zone B-cell 3 5-not ded not ded diffuse large B-cell -10-not ded not ded sporadic Burkitt - 8- - not ded classical HD - - 25 14 - ALCL - - 12 - 9 ______________________________________________________________________________________________________________________

22. Activated Notch1 Signaling Promotes Tumor Cell Proliferation and Survival in Hodgkin and Anaplastic Large Cell Lymphoma Jund F et al. Blood 2002; 99:3398-3403 a) HRS cells b) HRS cells c) ALCL d) undetectable e) Jagged 1 in HRS cells f) cHD g) Jagged 1 in ALCL cells ______________________________________________________________________________________________________________________

23. Notch Signaling is a Potent Inducer of Growth Arrest and Apoptosis in a Wide Range of B-cell Malignancies Zweidler-McKay PA et al. Blood 2005; 106:3898-3906 Notch ligands and Hes1 induce Notch-mediated growth arrest / cell death in human B-cell leukemia lines ______________________________________________________________________________________________________________________

24. Notch2 is Involved in the Overexpression of CD23 in B-Cell Chronic Lymphocytic Leukemia Hubmann R et al. Blood 2002; 99:3742-47 CD23a is differently regulated in B-CLL cells compared with normal lymphocytes ______________________________________________________________________________________________________________________

25. Induction of Apoptosis by Proteasome Inhibitors in B-CLL Cells is Associated with Downregulation of CD23 and Inactivation of Notch2 Duechler M et al. Leukemia 2005; 19:260-7 cell viability, CD23 and CD25 expression of B-CLL cells during incubation with bortezomib ______________________________________________________________________________________________________________________

26. Induction of Apoptosis by Proteasome Inhibitors in B-CLL Cells is Associated with Downregulation of CD23 and Inactivation of Notch2 Duechler M et al. Leukemia 2005; 19:260-7 Notch2-transfected BL41 cells exhibit higher resistance to Pl-induced apoptosis. BL41, and stably transfected sublines expressing Notch2lC or Notch2lC PEST were incubated with bortezomib ______________________________________________________________________________________________________________________

27. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 Introduzione -1 il segnale Notch appare coinvolto in alcuni meccanismi di tumorigenesi, ma il suo ruolo preciso nella leucemia linfatica cronica B-cellulare (B-CLL) non è ancora completamente definito il nostro studio ha esaminato lespressione e lattivazione dei recettori per Notch nelle cellule B-CLL, ed il ruolo del segnale Notch nel sostenere la sopravvivenza di questa popolazione cellulare ______________________________________________________________________________________________________________________

28. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 Introduzione -2 il nostro studio ha premesso di rilevare che solamente le cellule B-CLL (e non le cellule B normali) esprimono costitutivamente le proteine Notch-1 e Notch-2, così come i due rispettivi ligandi Jagged-1 e Jagged-2 il segnale Notch è costitutivamente attivato nelle cellule B-CLL, e la sua attivazione è ulteriormente aumentata nelle stesse cellule, così da resistere spontaneamente allapoptosi dopo 24 h di coltura ex vivo ______________________________________________________________________________________________________________________

29. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 espressione di Notch-1, Notch-2, Jagged-1, Jagged-2 nelle cellule B-CLL e nelle normali cellule B ______________________________________________________________________________________________________________________

30. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 analisi dellespressione di Notch1, Notch2 e Hes1 nelle cellule B-CLL coltivate ex vivo ______________________________________________________________________________________________________________________

31. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 effetto della downregulation di Notch2 sulla sopravvivenza delle cellule B-CLL ______________________________________________________________________________________________________________________

32. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 effetto del ligando solubile Jagged.-1 sulla sopravvivenza delle cellule B-CLL ______________________________________________________________________________________________________________________

33. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 effetto del ligando solubile Jagged1 e del trattamento con GSI * sullattività del NF-kB, sullespressione della proteina c-IAP e sulla attivazione della caspasi * -secretase inhibitor I ______________________________________________________________________________________________________________________

34. Notch Signaling as a Terapeutic Target in Cancer: a New Approach to the Development of Cell Fate Modifying Agents Nickoloff BJ et al. Oncogene 2003; 22:6598-6608 rappresentazio ne schematica del segnale Notch con le potenziali strategie per la sua manipolazione farmacologica ______________________________________________________________________________________________________________________

35. Constitutively Activated Notch Signaling is Involved in Survival and Apoptosis Resistance of B-CLL Cells Rosati E et al. Blood 2009; 113:856-65 Immunologia, PerugiaEmanuela ROSATI Pierfrancesco MARCONI MSOLorenzo FALCHI Marta GUBBIOTTI Oncoematologia, TerniAnna Marina LIBERATI Clinica Medica, TerniAdolfo PUXEDDU ______________________________________________________________________________________________________________________