Abstract
ObjectiveThe aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models.MethodsThe potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model.ResultsIbrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model.ConclusionsTaken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.
Highlights
Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase member of the Tec kinase family, plays a significant role in B-cell development
The Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity in primary B-chronic lymphocytic leukemia (B-CLL) samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib
Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X
Summary
Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase member of the Tec kinase family, plays a significant role in B-cell development. BTK is known to be important for B cell migration and homing and is activated upon chemokine binding to CXCR4 and CXCR5 through direct interaction with the chemokine receptor G protein subunits [2, 3] For all these reasons, BTK represents an unique therapeutic target in B-cell malignancies [1]. The present indication is that there are very few complete responses with Ibrutinib (2% in previously treated CLL, 13% in treatment of naïve CLL) and when treatment is interrupted disease progression is rapidly seen. It is intensely debated the search of combination therapy to improve responses, with the aim of attaining a minimal residual disease (MRD) negative response, without significant toxicity for the patients. Another evidence suggesting the importance of searching new therapeutic combinations with Ibrutinib is the emergence of resistance to Ibrutinib monotherapy [10,11,12,13]
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