Lyn, a tyrosine kinase closely linked to the differentiation status of primary acute myeloid leukemia blasts, associates with negative regulation of all-trans retinoic acid (ATRA) and dihydroxyvitamin D3 (VD3)-induced HL-60 cells differentiation

Abstract

Background: Lyn, an import member of Src family kinases (SFKs), is supposed to be implicated in acute myeloid leukemia (AML) pathogenesis and development by participation in AML differentiation, yet the details still remain incompletely understood. The expression status of Lyn and its correlation with multiple clinical parameters including cell differentiation degree, different cytogenetic risk classification, and the activity of myeloperoxidase (MPO) were thus investigated. To address the mechanisms underlying the involvement of Lyn in differentiation induction, the effects of dasatinib, an inhibitor for SFKs including Lyn, on the alterations of all-trans retinoic acid (ATRA)- or dihydroxyvitamin D3 (VD3)-induced differentiation, and c-Myc protein expression were investigated.

Methods: Primary AML blasts were obtained from 31 newly diagnosed AML patients with different French-American-British (FAB) subtypes. The expression of phosphorylated and total Lyn, c-Myc, and CD11b, CD11c and CD15 was analyzed by flow cytometry. The activation of Akt and Erk known to be involved in the regulation of c-Myc expression was investigated using western blotting.

Results: Significant higher expression levels of total Lyn were observed in AML patients with favorable cytogenetics, higher MPO activity and FAB M2 subtype. A clear positive correlation between the expression levels of Lyn and differentiation status of primary AML blasts was observed. Dasatinib inhibited the expression of phosphorylated Lyn, and further enhanced the differentiation-inducing activity of ATRA and VD3 in HL-60 cells. Augmented downregulation of c-Myc protein expression was observed in the combination treatment with ATRA, VD3 and dasatinib compared to treatment with each reagent alone in HL-60 cells. The suppression of the activation of Akt and Erk was also observed concomitantly.

Conclusions: The expression level of total Lyn is closely linked to the differentiation status of AML blasts. The enhancement of differentiation-inducing activity of ATRA/VD3 by dasatinib suggested that Lyn was associated in the negative regulation of ATRA/VD3-induced HL-60 cells differentiation. The enhancement probably was attributed to the downregulation of c-Myc implicated with the suppression of the activation of Akt and Erk. These results provide novel insights into a possible combinational therapeutic approach by targeting Lyn for AML patients, and offer new possibilities for the combination therapy with VD3 and dasatinib.

Keywords: Acute myeloid leukemia; All-trans retinoic acid; Dihydroxyvitamin D3; Lyn; c-Myc; dasatinib.

Fig. 1

Representative flow cytometry histograms of Lyn expression and a positive correlation between the expression levels of total and phosphorylated Lyn in primary AML blast cells from patients. The expression levels of Lyn in primary AML cells were analyzed and presented as histograms plotted by flow cytometry as described in “Methods” section. a Histograms show the high (patients #9 and #25) and low (patients #7 and #21) Lyn expression level in respective primary AML blast cells of individual patients. b A strong correlation between the expression levels of total and phosphorylated Lyn (p-Lyn). MFI mean fluorescence intensity

Fig. 2

Correlation between the expression level of Lyn and clinical laboratory parameters. The expression levels of Lyn in primary AML cells obtained from 31 patients with different subtypes of AML were analyzed with flow cytometry. Multiple clinical parameters of individual patients were collected in Nihon University Itabashi Hospital, and the assessment of the expression level of CD15 was entrusted to Bio Medical Laboratories, Inc. The correlation between the expression levels of Lyn and different cytogenetic risk groups (a), different AML subtypes (b), MPO activity (c), the expression level of CD15 (d), patients’ age (e) and WBC numbers (f) were shown respectively

Fig. 3

Inhibition of phosphorylation of Lyn and enhancement of differentiation-inducing activity of both ATRA and VD3 by dasatinib in HL-60 cells. a After treatment with 300 nM dasatinib for 30 min, the expression levels of phosphorylation of Lyn were evaluated by flow cytometry as described in “Methods” section. b–d After treatment with 1 μM ATRA or 100 nM VD3 in the presence or absence of 300 nM dasatinib for 72 h, morphological changes and the expression profiles of CD11b and CD11c were evaluated by Wright-Giemsa staining, and flow cytometry, respectively, as described in “Methods” section. b Representative photomicrographs are shown from three independent experiments. c Flow cytometry profiles of CD11b and CD11c. d Percent of CD11b and CD11c positive cells were calculated, respectively, based on flow cytometry profiles shown in (c). Experiments were independently repeated at least three times and results are shown as mean ± SD). p-Lyn dasatinib+, the expression level of phosphorylated Lyn in the presence of dasatinib; p-Lyn dasatinib−, the expression level of phosphorylated Lyn in the presence of dasatinib. *p < 0.05 vs. control; ^# p < 0.05 vs. ATRA; ^† p < 0.05 vs. VD3

Fig. 4

Potentiation of ATRA- or VD3-induced c-Myc downregulation by dasatinib in HL-60 cells. After treatment with 1 μM ATRA, 100 nM VD3, alone or in combination with 300 nM dasatinib, for 72 h, the alteration of c-Myc expression in HL-60 cells was examined by intracytoplasmic staining using flow cytometry as described in “Methods” section. The relative expression level of c-Myc is shown as the mean fluorescence intensity (MFI). a Representative histogram rofiles are shown. b The relative expression level of c-Myc was calculated based on flow cytometry profiles shown in (a). Six independent experiments were carried out and results are shown as mean ± SD. *p < 0.05 vs. control; ^# p < 0.05 vs. ATRA; ^† p < 0.05 vs. VD3

Fig. 5

Expression profiles of phosphorylated and total Akt and Erk in HL-60 cells treated with ATRA and VD3, each alone or in combination with dasatinib. After treatment with 1 μM ATRA and 100 nM VD3, each alone or in combination with 300 nM dasatinib for 24 h, the alterations of the expression level of phosphorylated and total Akt and Erk proteins were evaluated by western blotting method as described in “Methods” section