ETV6/ARG Oncoprotein Induces Autonomous Cell Growth By Enhancing c-Myc Expression through STAT5 Activation in the Acute Promyelocytic Leukemia Cell Line HT93A

Background: A fusion gene comprising ETS-variant gene 6 (ETV6) and ABL- related gene (ARG) has been identified in some patients with acute leukemia. Although the ETV6/ARG fusion gene is considered to contribute to the leukemogenesis, the detailed underlying mechanism is still unknown. Here, we investigated the role of ETV6/ARG fusion genes by exposing the HT93A cell line, which was established from a patient with relapsed acute promyelocytic leukemia carrying ETV6/ARG fusion gene, to the tyrosine kinase inhibitor, nilotinib.

Material and Methods: HT93A cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum with or without nilotinib ± 50 ng/ml of granulocyte colony-stimulating factor (G-CSF). The cells were intracellularly stained anti-phosphorylated ARG (Y429), anti-phosphorylated STAT3 (Y705), anti-phosphorylated STAT5 (Y694), anti-p21/Waf1, anti-p27/Kip1, and anti-c-Myc polyclonal antibodies. Thereafter, protein expression and the degree of phosphorylation were evaluated with a flow cytometer. Cell growth was evaluated by trypan blue exclusion test, cell cycle analysis, and by intracellular staining with propidium iodide followed by flow cytometric analysis. Experiments were independently repeated at least thrice. Results shown are the mean ± standard deviation (SD) values. Data were analyzed using Student’s t-test, and p < 0.05 was considered statistically significant.

Results: ARG was predominantly activated in HT93A cells and nilotinib treatment inhibited ARG phosphorylation. HT93A cells were sensitive to nilotinib, which inhibited cell growth in a dose-dependent manner for 96 h of incubation. Nilotinib treatment for 48 h significantly increased the probability of cells in the G0/G1 phase and decreased that in the S+G2/M phase. Further, nilotinib alone inhibited the expression of both phosphorylated STAT3 and STAT5. Moreover, nilotinib downregulated the expression levels of p21/Waf1 and c-Myc. However, p27/Kip expression level did not alter. Because the activation of STAT3 and/or STAT5 was assumed as the major pathway induced by ETV6/ARG oncoprotein, HT93A cells were coincubated with 50 ng/mL G-CSF in the presence or absence of nilotinib. After stimulation with G-CSF for 60 min, STAT5 was significantly phosphorylated in both nilotinib-treated and untreated cells; the expression level of phosphorylated STAT5 returned to the control level when treated with nilotinib and G-CSF together. In contrast, STAT3 phosphorylation was unaltered by G-CSF stimulation in nilotinib-treated and untreated cells. G-CSF alone had an insignificant effect on cell growth. The downregulation of the expression levels of p21/Waf1 and c-Myc by nilotinib was abolished by the combination of G-CSF. G-CSF had slight effect on HT93A cell cycle distribution; results of the combination therapy with nilotinib and G-CSF returned the cell cycle distribution to the control level.

Conclusions: We demonstrated that ETV6/ARG inhibition by nilotinib abrogates autonomous cell growth via the downregulation of c-Myc through the inactivation of STAT5 but not of STAT3. Our data suggest that ETV6/ARG oncoprotein has a contributory effect for the autonomous cell growth and is a potential target in the treatment of leukemia.