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.
Ponassi
Raffaella
R
Department of Oncology, University of Genoa, Genoa, Italy.
Biasotti
Barbara
B
Tomati
Valeria
V
Bruno
Silvia
S
Poggi
Alessandro
A
Malacarne
Davide
D
Cimoli
Guido
G
Salis
Annalisa
A
Pozzi
Sarah
S
Miglino
Maurizio
M
Damonte
Gianluca
G
Cozzini
Pietro
P
Spyrakis
Francesca
F
Spyraki
Francesca
F
Campanini
Barbara
B
Bagnasco
Luca
L
Castagnino
Nicoletta
N
Tortolina
Lorenzo
L
Mumot
Anna
A
Frassoni
Francesco
F
Daga
Antonio
A
Cilli
Michele
M
Piccardi
Federica
F
Monfardini
Ilaria
I
Perugini
Miriam
M
Zoppoli
Gabriele
G
D'Arrigo
Cristina
C
Pesenti
Raffaele
R
Parodi
Silvio
S
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