Post on 26-Mar-2015
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