MEDITERRANEAN SCHOOL OF ONCOLOGY Advanced Courses and SeminarsLLC e MM oggi: un paradigma per nuovi “standard” nelle neoplasie

ematologiche

Orvieto (Terni), 20-21 novembre 2009

Sopravvivenza e Resistenza all’Apoptosi Sopravvivenza e Resistenza all’Apoptosi delle Cellule LLC-Bdelle Cellule LLC-B

Stefano CoaccioliEmanuela Rosati

Clinica Medica, Terni - Immunologia, Perugia Università degli Studi di Perugia

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Chronic Lymphocytic Leukemia Kipps TJ Curr Opin Haematol 2000; 7:223-34

CLL come disordine eterogeneo

• anormalità cromosomiche 6, 11, 12, 13• anormalità citogenetiche espressione di geni per Ig

mutati vs non-mutati• fenotipi Ag di superficie CD5 (CD19, CD23, CD20)

• marker prognostici dubling time < 12mo, 2mg, sCD23, wbc > 30x109/L, Tk

• terapia:– CHT di combinazione fludarabine - mafosfamide– nuove molecole cladribine– molecole “biologiche” rituximab, CAMPATH-1H, lym-1– altri agenti terapeutici GC, melarsoprol, depsipeptide,

bryostatin1, lactacystin– trapianto cellule staminali– terapia genica

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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– l’attività costitutiva della via PI3K nelle cellule CLL-B quando coltivate in vitro con fibroblasti: prevenzione dell’attivazione della caspasi e dell’apoptosi

• conclusione: la via PI3K sembra rivestire un ruolo chiave nella crescita e nella sopravvivenza delle cellule CLL-B

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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

l’apoptosi spontanea di B-CLL cells in vitro correla con la downregulation di bcl-xL che è prevenuta dalla co-coltura con Ltk-

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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

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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

l’attività NF-kB dipendente da PI3K, nelle cellule B-CLL induce l’espressione delle proteine XIAP e FLIPL

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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

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apoptosi % di B cellule dopo 48 hdi coltura

control MEMstromal cell contactno stromal cell contact

control MEMno stromal cell contactstroma CM

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

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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 microambientesu espressione di bcl-2

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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

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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

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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

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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

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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

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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

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Notch Signaling: Cell Fate Control and Signal Integration in

Development Artavanis-Tsakonas S et al. Science 1999; 284:770-6

• elementi del segnale Notch

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Notch Signaling: Cell Fate Control and Signal Integration in

Development Artavanis-Tsakonas S et al. Science 1999; 284:770-6

• interazioni

inter-cellulari

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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

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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

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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

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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 expressionlymphoma absent weak strong EB-virus ALK-protein

B-CLL 3 5 - not d’ed not d’edmantle cell 5 4 - not d’ed not d’edfollicle center 2 8 - not d’ed not d’edmarginal zone B-cell 3 5 - not d’ed not d’eddiffuse large B-cell - 10 - not d’ed not d’edsporadic Burkitt - 8 - - not d’edclassical HD - - 25 14 -ALCL - - 12 - 9

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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 cellsc) ALCLd) undetectablee) Jagged 1 in HRS cells f) cHDg) Jagged 1 in ALCL cells

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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

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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

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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

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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 Notch2lCPEST

were incubated with bortezomib

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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 l’espressione e l’attivazione dei recettori per Notch nelle cellule B-CLL, ed il ruolo del segnale Notch nel sostenere la sopravvivenza di questa popolazione cellulare

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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 all’apoptosi dopo 24 h di coltura ex vivo

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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

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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 dell’espressione di Notch1, Notch2 e Hes1 nelle

cellule B-CLL coltivate ex vivo

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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

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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

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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 * sull’attività del NF-kB, sull’espressione della proteina c-IAP e sulla attivazione della caspasi

* -secretase inhibitor I

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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

rappresentazione schematica del segnale Notch con le potenziali strategie per la sua manipolazione farmacologica

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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, Perugia Emanuela ROSATIPierfrancesco MARCONI

MSO Lorenzo FALCHIMarta GUBBIOTTI

Oncoematologia, Terni Anna Marina LIBERATI

Clinica Medica, Terni Adolfo PUXEDDU

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