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. 2016 Nov;5(11):3223-3234.
doi: 10.1002/cam4.925. Epub 2016 Oct 10.

Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes

Noriyoshi Iriyama  1 , Yoshihiro Hatta  1 , Masami Takei  1
Affiliations

Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes

Noriyoshi Iriyama et al. Cancer Med. 2016 Nov.

Abstract

It has been shown that an increase in cytotoxic lymphocyte counts in the peripheral blood occurs rapidly after taking dasatinib, but the underlying mechanism is not yet elucidated. To investigate the influence of dasatinib on signal transduction pathways, we investigated the changes in JAK-STAT, mitogen-activated protein kinase (MAPK), and AKT in cytotoxic lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), before and after dasatinib treatment in chronic myeloid leukemia patients. Among a total of 30 patients, 18 were treated with dasatinib, nine with imatinib, and three with nilotinib. At constitutive levels, the expression of phosphorylated proteins, pSTAT1, pSTAT3, and pERK in NK cells and pSTAT3 in CTLs, was significantly higher in dasatinib-treated patients. Among the patients evaluated, only dasatinib-treated patients showed inhibition of multiple signaling pathways after taking a tyrosine kinase inhibitor. The magnitude of pERK and pAKT inhibition was closely associated with an increase in NK cells and CTLs, respectively, after taking a tyrosine kinase inhibitor. Those responses were more evident in patients with cytomegalovirus IgG positivity. In this study, we demonstrated for the first time, the influence of dasatinib on cell events in cytotoxic lymphocytes in vivo and explained the possible underlying mechanism that results in lymphocyte mobilization after dasatinib treatment.

Keywords: AKT; ERK; cytotoxic lymphocyte; dasatinib; mobilization.

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Figures

Figure 1
Figure 1
Flow cytometric analysis of each lymphocyte subset and expression of phosphorylated proteins. Representative data are shown. Lymphocyte fractions are classified according to surface antibodies including CD3, CD8, and CD56. Natural killer (NK) cells were defined as the CD3‐CD56+ immunophenotype and cytotoxic T lymphocytes (CTLs) were CD3+ CD8+ (A). Cells were costained with phospho‐specific antibodies, including antibodies targeting pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT, and expression levels of the isotype control and phosphorylated proteins in NK cells are presented (B). The values for the isotype control and each phosphorylated protein are shown as the median fluorescence intensity (MFI).
Figure 2
Figure 2
Changes in the number of lymphocytes, natural killer (NK) cells, and cytotoxic T lymphocytes (CTL) are shown. The values were compared before and after treatment (1 h for dasatinib and 2 h for imatinib or nilotinib) or according to treatment (A). The relative changes in the number of lymphocytes, NK cells, and CTLs are also shown and are expressed as log2 (fold change [FC]) (B).
Figure 3
Figure 3
Constitutive levels of phosphorylated proteins including pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT in natural killer (NK) cells (A) and cytotoxic T lymphocytes (CTLs) (B) grouped according to treatment (dasatinib [n = 18] or other TKI [n = 12]) are shown. The values for phosphorylated proteins in each fraction are shown as the median fluorescence intensity (MFI).
Figure 4
Figure 4
Heat map analysis showing changes in the values in natural killer (NK) cell (A) and cytotoxic T lymphocyte (CTL) (B) fractions (n = 28). Changes in the median fluorescence intensity (MFI) values of phosphorylated proteins in each fraction were compared before and after treatment (1 h for dasatinib [patient no. 1–15, 17, 18] and 2 h for imatinib or nilotinib [patient no. 19–29]) and expressed as log2 (fold change [FC]).
Figure 5
Figure 5
Correlations between changes in cell number and changes in the expression levels of each phosphorylated protein in natural killer (NK) cell and cytotoxic T lymphocyte (CTL) fractions (n = 28). Values are shown as log2 (fold change [FC]).
Figure 6
Figure 6
Representative histograms showing expression changes of pERK (A) and pAKT (B) in the natural killer (NK) cell fraction.
Figure 7
Figure 7
Direct effect of dasatinib on cytotoxic lymphocytes. Mononuclear cells obtained from peripheral blood before taking dasatinib were incubated with dimethylsulfoxide (DMSO) vehicle, 100 nmol/L or 300 nmol/L dasatinib, 2 μmol/L imatinib, or 2 μmol/L nilotinib, for 1 h, fixed with 1.5% paraformaldehyde, and permeabilized with 90% methanol, followed by surface and intracellular staining. The staining was evaluated by flow cytometry. Changes in the expression of phosphorylated proteins, including pERK and pAKT, were evaluated in five samples derived from patients who responded to dasatinib. Levels of each phosphorylated protein were evaluated as the median fluorescence intensity (MFI), and the ratio compared to the control was shown as log2 (fold change [FC]). Results are shown as the mean ± standard error of the mean. *P < 0.05
Figure 8
Figure 8
Influence of prior history of cytomegalovirus (CMV) infection on the change of signal transduction pathways. CMV IgG status was examined in patients receiving dasatinib treatment, with positivity in 13 and negativity in five. Eleven patients were treated with other tyrosine kinase inhibitors (TKIs). Changes of pERK and pAKT expression in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) according to CMV IgG status are shown as log2 (fold change [FC]).
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