Synergistic Activity of Nilotinib and Established Chemotherapeutic Agents in Imatinib-Sensitive and -Resistant BCR-ABL-Positive Leukemia Cells.

Resistance towards the tyrosine kinase inhibitor (TK) imatinib frequently emerges in advanced stages of chronic myelogenous leukemia (CML) and complicates disease treatment. Nilotinib (AMN107) represents a novel BCR-ABL TK inhibitor with reported antiproliferative activity against imatinib-resistant cells in vitro. Since combination chemotherapy is a strategy to increase the antileukemic efficacy, we were interested whether combinations of nilotinib and other established chemotherapeutic agents such as mafosfamide, busulfan, treosulfan, daunorubicin or mitoxantrone display synergistic activity in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. Tetrazolium-based MTT assays (48 h incubation) revealed as expected that substantially higher concentrations of single agent nilotinib are required for growth inhibition of the imatinib-resistant K562-R (IC50=19.76±4.94 μM) and LAMA84-R (IC50=0.14±0.03 μM) cell lines as compared to the imatinib-sensitive counterparts (IC50=0.10±0.03 μM for K562 and IC50=0.03±0.01 μM for LAMA84, respectively). Combination treatment of both imatinib-sensitive LAMA84 and -resistant LAMA84-R cells resulted in variable antiproliferative effects. Combination index (CI) values calculated according to the median-effect method of Chou and Talalay denoted mostly additive (CI=1) and antagonistic (CI>1) effects. In both lines a clear synergistic activity with CI<1 could be observed only for the combination nilotinib plus mitoxantrone. By comparison in the imatinib-sensitive K562 and -resistant K562-R lines all applied combinations showed improved antileukemic efficacy with CI values predominantly lower than 1 indicating synergism between nilotinib and most of the substances investigated (see Table I, n=3, results are given as mean CI values at different growth inhibition levels (fraction affected, Fa)).

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In BaF3 cells expressing imatinib-resistant mutants of BCR-ABL (E255K and T315I) IC50-values of single agent nilotinib were 0.37±0.04 μM and 23.58±1.92 μM, respectively. Importantly, combination treatment of imatinib-resistant BaF3 cells with both nilotinib plus daunorubicin and nilotinib plus mitoxantrone led to enhanced antiproliferative effects with synergism (CI<1) at high growth inhibition levels even in the T315I resistant mutant cell line (see Table II, n=3).

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The results demonstrate the ability of established chemotherapeutic agents to enhance the antileukemic efficacy of nilotinib and indicate the potential of the applied combinations for further study in the clinical setting.