Abstract
The t(6,9)(p23,q34) translocation occurs in 1-5% of adult patients with acute myeloid leukemia (AML). It is associated with a poor prognosis and defines a high risk group of AML in the WHO classification. The t(6;9) is in most of the cases the only cytogenetic aberration at diagnosis. The hallmark of t(6;9)-positive AML is the DEK/CAN fusion protein. DEK/CAN is a leukemogenic oncogene, but little is known about the molecular mechanism of DEK/CAN-induced leukemogenesis. The 165 kDa DEK/CAN phosphoprotein is encoded by a single transcript of 5.5Kb. The DEK portion of the DEK/CAN contains all the major functional domains of DEK mediating DNA-binding and multimerization. DEK increases life span of primary cells in culture by inhibiting cellular senescence and apoptosis. Post-translational modifications of DEK, mainly phosphorylation, influence the activity of DEK; unphosphorylated DEK has a higher affinity for DNA than the phosphorylated form, which in turn has a higher ability for multimerization. The main kinases that phosphorylate DEK are Glycogen synthase kinase 3 β (GSK3β) and Casein kinase 2 (CK2). The respective phosphorylation sites are conserved in the DEK portion of DEK/CAN. However, little is known about the role of phosphorylation for the biological functions of DEK/CAN. Therefore we generated several mutants of DEK and DEK/CAN by point-mutating the putative GSK3β-sites (ΔP1) from S to A and by deleting the CK2 sites in addition to these mutations (ΔP2). The reduction of S/T phosphorylation was confirmed by a ProQ staining and affinity chromatography on lysates of 293T cells expressing DEK, DEK/CAN and the respective ΔP1 or ΔP2 mutants. Further biological and biochemical consequences of these mutations for DEK and DEK/CAN were investigated in murine factor dependent 32D progenitor cells and in primary murine Sca1+/lin- hematopoietic stem cells (HSC), retrovirally or lentivirally transduced with DEK, DEK/CAN and/or their phosphorylation mutants ΔP1 or ΔP2, respectively.
Here we report that the loss of the GSK3β- and CK2-phosphorylation sites did not interfere with the subnuclear localization of either DEK or DEK/CAN as revealed either by subnuclear fractionation experiments or by co-localization with native DEK/CAN in confocal laser scan microscopy assays on 32D cells co-expressing DEK/CAN and ΔP1-DEK/CAN or ΔP2-DEK/CAN. In contrast, the destruction of GSK3β-phosphorylation sites not only led to a loss of apoptosis inhibition by DEK and DEK/CAN upon factor withdrawal in 32D cells, but also abolished the increased self renewal potential of DEK/CAN-positive HSC. In fact DEK/CAN-positive HSCs significantly increased colony numbers in colony forming units spleen-day 12 (CFU-S12) assays as compared to empty vector controls, whereas ΔP1-DEK/CAN and ΔP2-DEK/CAN did not have any effect.
In summary, our results suggest an important role of the GSK3β-phosphorylation for the DEK/CAN-induced leukemogenesis, which establishes the GSK3β-activity as a molecular target for therapeutic intervention in t(6;9)-positive AML.
Disclosures
No relevant conflicts of interest to declare.
Exploiting an Asp-Glu “switch” in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia