GLUCOSE AVAILABILITY IN HYPOXIA REGULATES THE SELECTION OF CML PROGENITOR SUBSETS WITH DIFFERENT RESISTANCE TO IMATINIB-MESYLATE

Introduction. We previously showed that clonal Chronic Myeloid Leukaemia (CML) cell lines are highly heterogeneous populations, where incubation under severe hypoxia (0.3% O2) selects hypoxia-resistant BCR/Abl-independent Leukaemia Stem Cells (LSC) and suppresses clonogenic progenitors (CFC). Accordingly, hypoxia-selected LSC exhibited complete “primary” resistance to imatinib-mesylate (IM), a feature highly relevant to the estabilishment of Minimal Residual Disease. The refractoriness of CML stem/progenitor cells (LSC/LPC) to IM was explored in relation to their resistance to hypoxia or its combination with glucose shortage, as a basis to design therapeutical approaches targeted to the long-term eradication of leukaemia. Methods. Hypoxic (0.3% O2) CML cultures were established with different cell densities and glucose concentrations and treated or not with IM. BCR/Abl expression was assessed by western blotting. BCR/abl transcription was determined by RT-PCR. The maintenance of stem cell potential in hypoxic primary cultures (LC1) was assessed by the Culture-Repopulating Ability (CRA) assay on the basis of the capacity of LC1 cells to repopulate secondary liquid cultures (LC2) incubated in normoxia. Results. By varying cell density and glucose concentration in culture, it was possible to identify hypoxia-resistant LSC/LPC subsets endowed with different kinetic properties and IM-resistance. Glucose availability in hypoxia appeared to regulate the balance between the LSC and LPC phenotypes. BCR/Ablprotein was suppressed in hypoxia following a kinetic paralleling that of glucose concentration decrease in culture medium, so that hypoxiaresistant cells maintained BCR/Abl-protein expression until glucose was available. LPC surviving merely hypoxic conditions were immediately recruitable to clonal expansion upon transfer to growth-permissive secondary cultures in normoxia. Such a property, due to the prompt rescue of BCR/Abl signaling, as confirmed using an inducible BCR/Abl expression system, was paralleled by LPC sensitivity to IM. LSC selected under hypoxia/ischemia were instead capable of delayed clonal expansion only and refractory to IM. IM-resistant LSC were also selected in hypoxia/ischemia from primary BCR/Abl-positive leukemia cells. These LSC are suitable to home in vivo within hypoxic stem cell niches and to represent the CML cell subset responsible for MRD and relapses of disease. Conclusions. We demonstrated that different combinations of hypoxia and glucose shortage differently modulate BCR/Abl expression and result in the selection of of hypoxia-resistant, BCR/Abl-protein–positive LPC sensitive to IM, as well as of ischemia-selected, BCR/Abl-protein– negative LSC refractory to IM.