LLC e MM oggi: un paradigma per nuovi standard nelle neoplasie ematologiche Mediterranean School of Oncology Orvieto, Palazzo Coelli Novembre Novembre 2009 Ruolo di bendamustina: un nuovo standard? Annamaria Rauco UO Oncologia Medica Ospedale S.Camillo de Lellis, Rieti Lorenzo Falchi SC Oncoematologia con Autotrapianto, Az. Osp. S.Maria Terni Università degli studi di Perugia

BENDAMUSTINE HISTORY

BENZIMIDAZOLE RING ALKYLATING GROUP BUTYRIC ACID SIDE CHAIN BENDAMUSTINE CHEMICAL STRUCTURE

BENDAMUSTINE CICLOFOSFAMIDE CHLORAMBUCIL MELPHALAN BENDAMUSTINE CHEMICAL STRUCTURE

DNA Alkylation Cross-links DNA single and double stands inhibiting DNA replication, repair and transcription Significantly more double strand breaks when compared to cyclophosphamide and carmustine Double strand breaks more durable when compared to cyclophosphamide and carmustine Extent and durability of effect results in mitotic catastrophe BENDAMUSTINE ALKYLATING ACTIVITY

BENDAMUSTINE UNIQUELY REGULATES APOPTOSIS PATHWAYS COMPARED WITH OTHER ALKYLATORS p21,wip1,NOXA,DR5/KILLER,BTG2 PROAPOPTOTICI p53-RESPONSE PRESENTANO p53-RESPONSE ELEMENTS ELEMENTS NELLE LORO REGIONI PROMOTER E SONO DEFINITI p53-DIPENDENTI MICROARRAYS GENI ATTIVATI DALLA BENDAMUSTINA BENDAMUSTINE APOPTOSIS PATHWAYS

Q-PCR validation was used to confirm the effects of bendamustine on p21 and NOXA both genes were induced in SU-DHL-1 cells after 8 h of exposure to bendamutine genes were also induced by equitoxic concentration of phosphoramide mustard and chlorambucil but to a much lower extent BENDAMUSTINE APOPTOSIS PATHWAYS

immunoblotting analysis, using antibodies that specifically recognize Ser 15 - phosphorilated p53 shows that bendamustine led to an 8-fold up-regulation of Ser 15 - phosphorilated p53 in SU-DHL-1 cells when testing the proapoptotic mitochondrial protein Bax, Bendamustine, but not chloramucil or phosphoramide caused an appreciable increase in the protein expression of Bax BENDAMUSTINE APOPTOSIS PATHWAYS

the DNA repair enzyme APE is an apurinic/apyrimidinic endonuclease that plays a critical role in the base excision repair pathway APE is inhibited by methoxyamine, a drug that specifically binds to abasic sites in DNA and reduces APE activity by 300-fold The cytotoxic activities of bendamustine and phosphoramide were assessed The IC 50 of bendamustine was reduced 6-fold with methoxyamine addition The IC 50 of phosphoramide mustard did not change with methoxyamine addition BENDAMUSTINE UNIQUELY INDUCES A BASE EXCISION REPAIR PATHWAY RESPONSE BENDAMUSTINE DNA REPAIR PATHWAYS

DNA repair enzyme O 6 -alkylguanine-DNA alkyltransferase is an important DNA-repair protein that protects cells from the toxic effects of DNA alkylators The activity of bendamustine and phosphoramide was examined in presence of an alkylguanyl transferase inhibitor, O 6 -benzylguanine The cytotoxicity of bendamustine was not enhanced by the addition of O 6 -benzylguanine OTHER ALKYLATORS BUT NOT BENDAMUSTINE BUT NOT BENDAMUSTINE INDUCE AN ALKYLTRANSFERASE MECHANISM OF DNA REPAIR BENDAMUSTINE DNA REPAIR PATHWAYS

Bendamustine inhibits mitotic checkpoints and induces mitotic catastrophe Treatment with bendamustine resulted in a 60-80% down-regulation of the in a 60-80% down-regulation of the mRNA expression of all three genes Phosphoramide mustard or chlorambucil had more modest inhibitory effects on these genes transcript BENDAMUSTINE MITOTIC CATASTROPHE

To determine whether bendamustine can cause mitotic catastrophe it was tested in cell lines with deficiencies in apoptotic pathways Increased incidence of chromatin condensation and multinucleation/micronucleation, hallmarks of mitotic catastrophe, in both cell lines Micronucleation compared with only 6% in DMSO control cells with only 6% in DMSO control cells BENDAMUSTINE MITOTIC CATASTROPHE

CONCLUSIONI - Meccanismo dazione unico - Attivazione apoptosi p53-dipendente - Alti livelli di attivazione p53 e dei geni p53 dipendenti - Inibizione numerosi checkpoint mitotici - Danno esteso al DNA/innesco catastrofe mitotica ATTIVITÀ NEI PAZIENTI RESISTENTI AGLI ALCHILANTI TRADIZIONALI