ERK5-INHIBITION AS A NOVEL APPROACH TO TARGET CML STEM CELLS

Introduction. Evidences obtained in our laboratory indicated that the leukaemia stem cell (LSC) potential of chronic myeloid leukaemia (CML) cell populations is resistant to, and selected in, severe hypoxia (0.1-1% oxygen). The Extracellular signal-Regulated Kinase 5 (ERK5) is a member of the mitogen-activated protein kinase family involved in the control of cell survival and proliferation and in the pathogenesis of different types of cancer, including CML. The main target of this study was to address the effects of ERK5 inhibition on the maintenance of hypoxiaselected LSC of CML by the Culture-Repopulating Ability (CRA) assay. Methods. The K562 and KCL-22 human stabilized CML cell lines, where ERK5 is constitutively activated, were used both for all the experiments. Cells were incubated in hypoxic (~0.1% O2) or normoxic (used as control) primary cultures (LC1) in the absence or the presence of MEK5 or ERK5 inhibitors. Cells were harvested at different times and analysed. Cell survival, cycling, proliferation and apoptosis were assessed by counting Trypan blue-negative cells or flow cytometry using propidium iodide (PI) and anti-annexin-V antiboidies. At day 7 of incubation, LC1 cells were transferred to non-selective normoxic secondary cultures (LC2) of CRA assays in the absence of inhibitors, to evaluate LC2 repopulation. Results. Hypoxia prevents the cell number increase which occurred in normoxia and determined early and massive apoptosis, as well as cell cycle arrest of surviving cells. Consistently, the AKT protein, an important pro-survival signal, was cleaved in hypoxia. Hypoxia decreased the intensity and duration of ERK1/2, p38 and JNK phosphorylation/ activation occurring in normoxia, without altering the expression of these proteins. On the other hand, hypoxia suppressed ERK5 constitutive activation and protein expression, unchanged in normoxia, but not ERK5 mRNA. The effects of the inhibition of the MEK5>ERK5 pathway were then determined. We tested several MEK5>ERK5 pathway inhibitors and identified one specific ERK5 inhibitor that completely suppressed LSC maintenance in hypoxia. In LC1, this inhibition resulted in a significant increase of the percentage of cells in G0/G1 and in a modest reduction of apoptosis, pointing to a cytostatic, rather than cytotoxic, effect of ERK5 inhibition. Conclusions. The maintenance of leukaemia stem cells of CML is impaired by ERK5 inhibition, which therefore emerges as a potential novel strategy for CML therapy.