Negative regulatory effects of Mnk kinases in the generation of chemotherapy-induced antileukemic responses

Mnk kinases are downstream effectors of mitogen-activated protein kinase pathways and mediate phosphorylation of the eukaryotic initiation factor (eIF4E), a protein that plays a key role in the regulation of mRNA translation and is up-regulated in acute myeloid leukemia (AML). We determined the effects of chemotherapy (cytarabine) on the activation status of Mnk in AML cells and its role in the generation of antileukemic responses. A variety of experimental approaches were used, including immunoblotting, apoptosis assays, small interfering RNA (siRNA)-mediated knockdown of proteins, and clonogenic hematopoietic progenitor assays in methylcellulose. Cytarabine induced phosphorylation/activation of Mnk and Mnk-mediated phosphorylation of eIF4E on Ser209, as evidenced by studies involving pharmacological inhibition of Mnk or experiments using cells with targeted disruption of Mnk1 and Mnk2 genes. To assess the functional relevance of cytarabine-inducible engagement of Mnk/eIF4E pathway, the effects of pharmacological inhibition of Mnk on cytarabine-mediated suppression of primitive leukemic progenitors [leukemic colony forming unit (CFU-L)] were examined. Concomitant treatment of cells with a pharmacological inhibitor of Mnk or siRNA-mediated knockdown of Mnk1/2 strongly enhanced the suppressive effects of low cytarabine concentrations on CFU-L. It is noteworthy that the mammalian target of rapamycin (mTOR) inhibitor rapamycin also induced phosphorylation of eIF4E in a Mnk-dependent manner, whereas inhibition strongly enhanced its antileukemic effects. These data demonstrate that Mnk kinases are activated in a negative-feedback regulatory manner in response to chemotherapy and impair the generation of antileukemic responses. They also identify this pathway as a novel target for the design of new approaches to enhance the antileukemic effects of chemotherapy or mTOR inhibitors in AML.