A novel Bim-BH3-derived Bcl-XL inhibitor: Biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity

Raffaella Ponassi, Barbara Biasotti, Valeria Tomati, Silvia Bruno, Alessandro Poggi, Davide Malacarne, Guido Cimoli, Annalisa Salis, Sarah Pozzi, Maurizio Miglino, Gianluca Damonte, Pietro Cozzini, Francesca Spyraki, Barbara Campanini, Luca Bagnasco, Nicoletta Castagnino, Lorenzo Tortolina, Anna Mumot, Francesco Frassoni, Antonio DagaMichele Cilli, Federica Piccardi, Ilaria Monfardini, Miriam Perugini, Gabriele Zoppoli, Cristina D'Arrigo, Raffaele Pesenti, Silvio Parodi

Research output: Contribution to journalArticle

Abstract

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-XL, 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-XL, 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.

Original languageEnglish
Pages (from-to)3211-3224
Number of pages14
JournalCell Cycle
Volume7
Issue number20
Publication statusPublished - Oct 15 2008

Keywords

  • Apoptosis
  • Bim-BH3
  • Leukaemia
  • Multi-parameter flow cytometry
  • Targeted therapy

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Developmental Biology

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    Ponassi, R., Biasotti, B., Tomati, V., Bruno, S., Poggi, A., Malacarne, D., Cimoli, G., Salis, A., Pozzi, S., Miglino, M., Damonte, G., Cozzini, P., Spyraki, F., Campanini, B., Bagnasco, L., Castagnino, N., Tortolina, L., Mumot, A., Frassoni, F., ... Parodi, S. (2008). A novel Bim-BH3-derived Bcl-XL inhibitor: Biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity. Cell Cycle, 7(20), 3211-3224.