Nilotinib and imatinib inhibit cytarabine cellular uptake: Implications for combination therapy
Introduction
Chronic myeloid leukemia (CML) is characterized by a reciprocal translocation between chromosomes 9 and 22. This genetic modification leads to the generation of the bcr-abl fusion gene that encodes a chimeric protein with constitutive tyrosine kinase activity [1]. Imatinib (Gleevec) is a very efficient inhibitor of the Bcr-Abl fusion protein that has been used for a decade to treat CML patients and acute lymphoblastic leukemia (ALL) patients that harbor a very similar chromosome translocation. An efficient and sustained response to imatinib is obtained in patients with CML in chronic phase but its efficacy is generally transient in late stages CML (accelerated and blast crisis phases) [2], [3], [4]. Moreover, imatinib is not an optimal treatment for 30–35% of the patients in chronic phase; some patients have to discontinue treatment due to side effects, and others lose or do not achieve a complete cytogenetic response, which significantly increases the risk of disease progression [2]. Thus, new therapeutic approaches that increase the cytogenetic response would be beneficial to CML patients.
Nilotinib (Tasigna) is a rationally designed Bcr-Abl inhibitor with greater potency and specificity than imatinib [1]. Furthermore, it is active against several Bcr-Abl mutants that are resistant to imatinib [1]. Nilotinib efficacy was first demonstrated in patients who failed imatinib therapy but its superiority over imatinib has been also confirmed in newly diagnosed CML patients [1], [5], [6], [7].
Treatments that combine imatinib with other chemotherapeutic agents are also actively investigated in CML and ALL patients. The combination of imatinib with the nucleoside analog cytarabine was an obvious regimen to test in the clinic since cytarabine combined to interferon alpha was the previous standard of care for CML before the imatinib era [8], and that several in vitro studies reported synergistic activities with imatinib and cytarabine [9], [10], [11]. A similar combination treatment could be envisaged for nilotinib that is also able to synergize with cytarabine [12].
Nucleosides use specific transporters to enter into cells, with the equilibrative nucleoside transporter 1 (ENT1) being the main cytarabine transporter; its low abundance in acute myeloid leukemia blasts correlates with a lack of response to cytarabine treatment [13]. Few years ago it was reported that some p38 MAPK inhibitors could block nucleoside transport, and that this inhibition occurs in a p38 MAPK-independent manner [14]. This finding was later extended to other types of kinase inhibitors that have different protein targets. Imatinib was tested and it was among the most efficient to block nucleoside transport; it inhibited uridine and thymidine transport by almost 70% at a 10 μM concentration [15]. In another study, it was also reported that imatinib would inhibit the uptake of the nucleosidic analog fludarabine with a similar order of magnitude [16].
We then hypothesized that a combination treatment with cytarabine and imatinib or nilotinib would not be optimal for the treatment of CML if both drugs were used simultaneously. Thus, we investigated to which extent imatinib and nilotinib would inhibit the cellular uptake of the natural nucleoside thymidine, and the nucleoside analog cytarabine in CML cells.
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Cell lines and cell culture
K562 (CCL-243) and MEG-01 (CRL-2021) cell lines were obtained from ATCC. Both cell lines were cultured in RPMI-1640 (Wisent, St-Bruno, Canada) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2.
Uptake assays of [3H]-thymidine and [3H]-cytarabine
K562 or MEG-01 cells (4 × 105 cells per sample) were incubated at 37 °C for 15 min with 5 μl imatinib or nilotinib at different concentrations, in 0.5 ml of transport buffer (20 mM Tris/HCl, 3 mM K2HPO4, 1 mM MgCl2·6H2O, 2 mM CaCl2, 5
Imatinib inhibits thymidine and cytarabine uptake in CML cells
The effect of imatinib on nucleoside transport was first examined with thymidine. At a 10-μM concentration, imatinib was able to inhibit the entry of radiolabeled thymidine in K562 cells by 76%. Imatinib was still efficient and blocked by 43% the entry of thymidine at 1 μM, but its effect on thymidine transport at 0.1 μM was not significant (Fig. 1a). With the MEG-01 CML cell line, thymidine entry was inhibited by 72 and 52% with 10 and 1 μM imatinib, respectively. As with K562 cells, the effect
Discussion
At a common regimen of 400 mg daily, imatinib reaches a mean peak concentration of 4.6 μM and a mean serum trough concentration of 1.5 μM [1]. We report that 1 μM imatinib inhibits cytarabine by 23% in K562 cells, and by 25% in MEG-01 cells (Fig. 1c and d). These results are in line with those reported previously with uridine in K562 cells and fludarabine with human T-lymphocytes [15], [16]. Results from a French clinical trial and from the HOVON-51 study that investigate the upfront combination
Conflict of interest statement
The authors declare no conflict of interest in connection with this work.
Acknowledgements
Contributions. JBS, KG and MC performed experiments, and POdeL and MC designed the study and wrote the manuscript.
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