Interferon-alpha targets JAK2V617F-positive hematopoietic progenitor cells and acts through the p38 MAPK pathway

Abstract

Objective: Interferon-alpha (IFNalpha) therapy leads to hematological remissions and a reduction of the JAK2V617F allele burden in patients with polycythemia vera (PV). In this study, the cellular target by which IFNalpha affects hematopoiesis in PV patients was evaluated.

Materials and methods: CD34(+) cells were isolated from normal bone marrow and the peripheral blood of patients with PV and were treated in vitro with each of the three commercially available forms of IFNalpha: IFNalpha 2b, pegylated IFNalpha 2a (Peg-IFNalpha 2a), and pegylated IFNalpha 2b (Peg-IFNalpha 2b).

Results: Each form of IFNalpha was equally potent in suppressing hematopoietic colony formation by normal CD34(+) cells, but Peg-IFNalpha 2a and IFNalpha 2b were more effective than Peg-IFNalpha 2b in inhibiting burst-forming unit erythroid-derived colony formation by PV CD34(+) cells. In addition, exposure of PV CD34(+) cells to equal doses of Peg-IFNalpha 2a and IFNalpha 2b resulted in a 38% to 40% reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells (HPC), while equivalent doses of Peg-IFNalpha 2b did not reduce the number of malignant HPC. Further studies explored the mechanism by which IFNalpha induced PV HPC growth inhibition. Treatment of Peg-IFNalpha 2a increased the rate of apoptosis of PV CD34(+) cells and the phosphorylation/activation of p38 mitogen-activated protein kinase in PV CD34(+) cells, while the p38-specific inhibitor SB203580 reversed the growth inhibition and apoptosis induced by Peg-IFNalpha 2a.

Conclusion: These data suggest that low doses of IFNalpha selectively and directly suppress PV JAK2V617F HPC and that these agents act through the p38 mitogen-activated protein kinase pathway.

Figure 1. Comparison of the effects of increasing concentrations of IFNα 2b, Peg-IFNα 2b and Peg-IFNα 2a on hematopoietic colony formation by normal bone marrow and polycythemia vera peripheral blood CD34⁺ cells

Hematopoietic colony numbers were enumerated after 14 days of incubation. A, CFU-GM derived colony formation assayed from normal BM CD34⁺ cells in the presence of varying concentrations of the three preparations of IFNα; B, BFU-E derived colony formation assayed by normal BM CD34⁺ cells in the presence of varying concentrations of the three preparations of IFNα; C CFU-GM derived colony formation assayed from CD34⁺ cells from PV patients in the presence of varying concentrations of the three preparations of IFNα; D, BFU-E derived colony formation assayed from CD34⁺ cells from PV patients in the presence of varying concentrations of the three preparations of IFNα;

Figure 2. Effects of the addition of equal doses of the three forms of IFNα on the number of JAK2V617F positive hematopoietic colonies assayed in vitro from CD34⁺ cells from patients with PV

CD34⁺ cells were incubated with either 200U/ml or 500U/ml of IFNα for 14 days and the numbers of colonies were enumerated. Individual colonies were randomly plucked and assayed for JAK2V617F. A, Comparison of the effects of the three forms of IFNα on JAK2V617F positive hematopoietic colony formation; (*: P<0.05; **: P<0.01; ***: p<0.001). B, Effects of addition of the three forms of IFNα at 500unit/ml on hematopoietic colony size, in each panel the upper colonies are representative CFU-GM derived colonies and the lower colonies are representative BFU-E derived colonies.

Figure 3. Activation of the p38 MAPK pathway by Peg-IFNα 2a in PV CD34⁺ cells

A. Three days of treatment with Peg-IFNα 2a at a dose of 500units/ml significantly increased the number of apoptotic PV CD34⁺ cells as determined by flow cytometric analysis. B. Treatment with 500units/ml of Peg-IFNα 2a elevated the number of CD34+ cells with cleaved caspase 3 as determined by flow cytometrically. The elevation of CD34+ cells with cleaved caspase 3 was reduced by prior-incubation with SB203580 for 30 min at dose of 10 uM. C. FACS analysis also showed that the number of PV CD34+ cells with phosphorylated form of p38 were increased after treatment with 500units/ml of Peg-IFNα 2a, this increase was not observed with prior-incubation with SB203580 followed by Peg-IFNα 2a treatment. D. Western blotting demonstrated that the phospho-p38 level in PV CD34⁺ cells from two individual patients (PV1 and PV2) was increased after treatment with Peg-IFNα 2a, but the effect was not observed with prior-treatment with SB203580. Similarly, Peg-IFNα 2a inhibited both CFU-GM (E.) and BFU-E (F.) derived colony formation by PV CD34⁺ cells, this inhibitory activity was blocked by prior-treatment with SB203580. The colonies were enumerated after 14 days of incubation. (*: p<0.05; **: p<0.01).