LLC e MM oggi:LLC e MM oggi:un paradigma per nuovi standard un paradigma per nuovi standard

nelle neoplasie ematologichenelle neoplasie ematologiche

Mediterranean School of OncologyMediterranean School of OncologyOrvieto, Palazzo CoelliOrvieto, Palazzo Coelli 20-22 Novembre 200920-22 Novembre 2009

Ruolo di bendamustina: un nuovo standard?Ruolo di bendamustina: un nuovo standard?

Annamaria RaucoAnnamaria RaucoUO Oncologia Medica Ospedale S.Camillo de Lellis, RietiUO Oncologia Medica Ospedale S.Camillo de Lellis, Rieti

Lorenzo FalchiLorenzo FalchiSC Oncoematologia con Autotrapianto, Az. Osp. S.Maria TerniSC Oncoematologia con Autotrapianto, Az. Osp. S.Maria Terni

Università degli studi di PerugiaUniversità degli studi di Perugia

BENDAMUSTINE HISTORYBENDAMUSTINE HISTORY

BENZIMIDAZOLE RINGBENZIMIDAZOLE RING

ALKYLATINGALKYLATING GROUPGROUP BUTYRIC ACID SIDE CHAINBUTYRIC ACID SIDE CHAIN

BENDAMUSTINE CHEMICAL STRUCTUREBENDAMUSTINE CHEMICAL STRUCTURE

BENDAMUSTINE

CICLOFOSFAMIDE

CHLORAMBUCIL

MELPHALAN

BENDAMUSTINE CHEMICAL STRUCTUREBENDAMUSTINE 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 ACTIVITYBENDAMUSTINE ALKYLATING ACTIVITY

BENDAMUSTINE UNIQUELY REGULATES APOPTOSIS PATHWAYSBENDAMUSTINE UNIQUELY REGULATES APOPTOSIS PATHWAYSCOMPARED WITH OTHER ALKYLATORSCOMPARED WITH OTHER ALKYLATORS

p21,wip1,NOXA,DR5/KILLER,BTG2PROAPOPTOTICI

PRESENTANO p53-RESPONSEp53-RESPONSEELEMENTS ELEMENTS NELLE LORO REGIONI

PROMOTER E SONO DEFINITIp53-DIPENDENTI

MICROARRAYSGENI ATTIVATI DALLA

BENDAMUSTINA

BENDAMUSTINE APOPTOSIS PATHWAYSBENDAMUSTINE 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 cellsafter 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 PATHWAYSBENDAMUSTINE APOPTOSIS PATHWAYS

immunoblotting analysis, using antibodiesthat specifically recognize Ser15 - phosphorilated p53

shows that bendamustine led to an 8-foldup-regulation of

Ser15 - phosphorilated p53in 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 PATHWAYSBENDAMUSTINE APOPTOSIS PATHWAYS

the DNA repair enzyme APE is an apurinic/apyrimidinic endonuclease that playsthe DNA repair enzyme APE is an apurinic/apyrimidinic endonuclease that playsa critical role in the base excision repair pathwaya critical role in the base excision repair pathway

APE is inhibited by methoxyamine, a drug that specifically binds to abasic sites in APE is inhibited by methoxyamine, a drug that specifically binds to abasic sites in DNA and reduces APE activity by 300-foldDNA and reduces APE activity by 300-fold

The cytotoxic activities of bendamustineand phosphoramide were assessed

The IC50 of bendamustine was reduced6-fold with methoxyamine addition

The IC50 of phosphoramide mustard didnot change with methoxyamine addition

BENDAMUSTINE UNIQUELY INDUCESBENDAMUSTINE UNIQUELY INDUCESA BASE EXCISION REPAIR PATHWAYA BASE EXCISION REPAIR PATHWAY

RESPONSERESPONSE

BENDAMUSTINE DNA REPAIR PATHWAYSBENDAMUSTINE DNA REPAIR PATHWAYS

DNA repair enzyme O6-alkylguanine-DNAalkyltransferase is an important DNA-repair

protein that protects cells from the toxic effectsof DNA alkylators

The activity of bendamustine and phosphoramidewas examined in presence of an alkylguanyl

transferase inhibitor, O6-benzylguanine

The cytotoxicity of bendamustine was not enhanced by the addition of O6 -benzylguanine

OTHER ALKYLATORSOTHER ALKYLATORS BUT NOT BENDAMUSTINEBUT NOT BENDAMUSTINE

INDUCE AN ALKYLTRANSFERASE MECHANISM INDUCE AN ALKYLTRANSFERASE MECHANISM OF DNA REPAIROF DNA REPAIR

BENDAMUSTINE DNA REPAIR PATHWAYSBENDAMUSTINE DNA REPAIR PATHWAYS

Bendamustine inhibits mitotic checkpoints and Bendamustine inhibits mitotic checkpoints and induces mitotic catastropheinduces mitotic catastrophe

Treatment with bendamustine resultedTreatment with bendamustine resulted in a 60-80% down-regulation of thein a 60-80% down-regulation of themRNA expression of all three genesmRNA expression of all three genes

Phosphoramide mustard or chlorambucilPhosphoramide mustard or chlorambucilhad more modest inhibitory effects had more modest inhibitory effects

on these genes transcripton these genes transcript

BENDAMUSTINE MITOTIC CATASTROPHEBENDAMUSTINE 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 Increased incidence of chromatin condensation and multinucleation/micronucleation, multinucleation/micronucleation, hallmarks of mitotic catastrophe,hallmarks of mitotic catastrophe,

in both cell linesin both cell lines

Micronucleation comparedMicronucleation compared with only 6% in DMSO control cellswith only 6% in DMSO control cells

BENDAMUSTINE MITOTIC CATASTROPHEBENDAMUSTINE MITOTIC CATASTROPHE

CONCLUSIONICONCLUSIONI- Meccanismo d’azione 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 ATTIVITÀ NEI PAZIENTI RESISTENTI AGLI ALCHILANTI TRADIZIONALITRADIZIONALI