A novel Bim-BH3-derived Bcl-XL inhibitor: biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity.
BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-X(L), involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-X(L), we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.
Department of Oncology, University of Genoa, Genoa, Italy.
Raffaella
R
Ponassi
Barbara
B
Biasotti
Valeria
V
Tomati
Silvia
S
Bruno
Alessandro
A
Poggi
Davide
D
Malacarne
Guido
G
Cimoli
Annalisa
A
Salis
Sarah
S
Pozzi
Maurizio
M
Miglino
Gianluca
G
Damonte
Pietro
P
Cozzini
Francesca
F
Spyrakis
Francesca
F
Spyraki
Barbara
B
Campanini
Luca
L
Bagnasco
Nicoletta
N
Castagnino
Lorenzo
L
Tortolina
Anna
A
Mumot
Francesco
F
Frassoni
Antonio
A
Daga
Michele
M
Cilli
Federica
F
Piccardi
Ilaria
I
Monfardini
Miriam
M
Perugini
Gabriele
G
Zoppoli
Cristina
C
D'Arrigo
Raffaele
R
Pesenti
Silvio
S
Parodi
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
C533898:072RB peptide; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D008565:Membrane Proteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein