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The sequence of application of methotrexate and histone deacetylase inhibitors determines either a synergistic or an antagonistic response in childhood acute lymphoblastic leukemia cells

Leukemia volume 25, pages 359–361 (2011)Cite this article

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We have acknowledged with great attention the recent article of Leclerc et al.1 reporting a synergistic cytotoxicity of methotrexate (MTX) and histone deacetylase inhibitors (HDACis) combination treatment in childhood acute lymphoblastic leukemia (ALL) cell lines. Both the group of Leclerc1 and ours2 have suggested that the introduction of HDACis into current ALL therapy regimes may provide a promising alternative. Leclerc et al.1 have observed synergism after sequential treatment with MTX followed by the HDACi suberoylanilide hydroxamic acid (SAHA). However, when we changed the sequence of application by treating cells primarily with different HDACis, such as SAHA and valproic acid (VPA), and afterwards with MTX or combined MTX and HDACi concomitantly, a strong antagonistic effect was observed.

Furthermore, we analyzed the effect of concomitant treatment with MTX and/or VPA on Nalm6 cell cycle distributions during a 72-h time course (Figure 2b). Treatment with MTX alone resulted in an accumulation of cells in G0/1 and S phase at 24 h, followed by a potent shift towards the sub-G1 peak, indicating DNA fragmentation and apoptosis at subsequent time points. VPA induced an accumulation of cells mainly in G0/1 phase and a nearly complete loss of S phase after 24 h of treatment. After 48 h VPA-treated cells entered again into S phase, indicating a resumption of proliferative activity, which resulted in a cell cycle distribution comparable to that of the untreated control at 72 h. The apoptotic sub-G1 peak in VPA-exposed cells was moderately increased compared with untreated controls. Concomitant treatment with MTX and VPA produced cell cycle distributions largely resembling those of VPA alone, confirming again an antagonistic interaction. MTX as an S-phase selective agent might be especially prone to treatment sequence-specific drug interactions. For example, exposure of L5178Y murine leukemia cells to MTX followed by asparaginase produces synergistic effects, whereas reversal of the treatment sequence results in antagonism.6 It was shown that asparaginase pre-treatment inhibited intracellular accumulation of MTX polyglutamates, which have a central role in mediating MTX cytotoxicity. Furthermore, it has been observed that asparaginase induced a temporary inhibition of leukemia cell proliferation, as shown by a reversible loss of S-phase cells in cell cycle distributions. Interestingly, the extent of inhibition and recovery of MTX polyglutamate accumulation in the asparaginase pre-treated cells largely paralleled the inhibition and recovery of DNA synthesis.6 Further studies supported the assumption that cellular accumulation of MTX polyglutamates, and thereby MTX cytotoxicity, is directly related to the proliferation rate of tumor cells.7 With regard to our experiments, it seems possible that the temporary loss of S-phase cells in cultures treated with VPA or the combination of VPA and MTX might contribute to the sequence-specific antagonism of MTX and HDACi in a similar way as was shown for MTX and asparaginase.

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Acknowledgements

This work was supported by a grant of Berliner Krebsgesellschaft e.v.

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  1. Department of Pediatric Oncology/Hematology, Charité - Universitätsmedizin Berlin, Berlin, Germany

    L Bastian, H G Einsiedel, G Henze, K Seeger & S Shalapour

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Bastian, L., Einsiedel, H., Henze, G. et al. The sequence of application of methotrexate and histone deacetylase inhibitors determines either a synergistic or an antagonistic response in childhood acute lymphoblastic leukemia cells. Leukemia 25, 359–361 (2011). https://doi.org/10.1038/leu.2010.259

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