Enhancing Neutrophil Differentiation - A Novel Role for the Death-Associated Protein Kinase 2 (DAPK2).

DAPK2 is a 42-kDa Ca²⁺/Calmodulin-regulated serine/threonine kinase involved in apoptosis. In gene expression profiles derived from in vitro differentiated myeloid leukemic NB4 cells treated with all-trans retinoid acid (ATRA), we found that DAPK2 was decisively induced during differentiation towards neutrophils. DAPK1, a close relative of DAPK2, is inactivated in a number of hematopoietic malignancies (AML, lymphoma, myeloma), and it may play a role during normal and leukemic myeloid cell differentiation. We therefore investigated DAPK2 for its possible role in both normal and leukemic myelopoiesis. Real time quantitative RT-PCR (RQ-PCR) and Western blot analysis of DAPK2 gene expression in primary myeloid cells revealed significantly higher DAPK2 expression in granulocytes (G; n=9) compared with monocytes/macrophages (M; n=8) and CD34⁺ progenitor cells (CD34⁺; n=6) (Δ, p< 0.001; figure, left panel). Moreover, significantly increased DAPK2 mRNA levels were also seen when cord blood CD34⁺ progenitor cells were induced to differentiate towards neutrophils with human recombinant G-CSF (hrG-CSF). In addition, ATRA-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, showed significantly higher DAPK2 mRNA expression paralleled by DAPK2 protein induction. However, during differentiation of CD34⁺ cells (with hrM-CSF) and U937 cells (with PMA) towards monocytes/macrophages, DAPK2 mRNA levels remained low. DAPK2 expression in primary leukemic cells revealed significantly lower DAPK2 expression levels in AML blasts (AML; n=100) than in samples from chronic myeloid leukemia patients in chronic phase (CML-CP; n=9) (ΔΔ, p< 0.001; figure, right panel).

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Stable lentiviral-mediated expression of wild-type DAPK2 enhanced ATRA-induced granulocytic differentiation of NB4 cells as shown by morphology and by increased CD11b expression. Furthermore, upregulation of mRNA levels of key regulator genes for terminal differentiation, such as C/EBPe, the G-CSF receptor and the secondary granule protein lactoferrin, was also enhanced. Expression of a kinase-inactive DAPK2 mutant did not show these effects, a finding consistent with a role of DAPK2 in granulopoiesis.

Conclusion: we demonstrate for the first time, that DAPK2 expression levels correlate with the degree of granulocytic differentiation, and that DAPK2 upregulation is restricted to granulopoiesis. Furthermore lentiviral-mediated DAPK2 expression enhances granulocytic differentiation. The finding that DAPK2 expression is low in AML and high in CML-CP patients suggests that suppressed DAPK2 expression may contribute to the differentiation block in AML.