Glucose Availability in Hypoxia Regulates the Selection of CML Progenitor Subsets with Different Resistance to Imatinib-Mesylate

Background: Incubation under severe hypoxia of CML cell lines selects hypoxiaresistant BCR/Abl-independent Leukaemia Stem Cells (LSC) and suppresses clonogenic progenitors (LPC). Hypoxia-selected LSC exhibited “primary” resistance to imatinibmesylate (IM), a feature highly relevant to the estabilishment of Minimal Residual Disease. Methods: Hypoxic (0.3% O2) CML cultures established with different cell densities and glucose concentrations were treated or not with IM. BCR/Abl expression was assessed by western blotting and BCR/abl transcription by RT-PCR. Stem cell potential was determined by the Culture-Repopulating Ability (CRA) assay. Results: The refractoriness of CML LSC/LPC to IM was explored in relation to their resistance to hypoxia or its combination with glucose shortage. It was possible to identify three hypoxia-resistant LSC/LPC subsets with different kinetic properties and IMresistance. Glucose availability in hypoxia appeared to regulate LSC/LPC balance; In fact, hypoxia-resistant cells maintained BCR/Ablprotein 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 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. Conclusions: This study evidenced the existence of hypoxia-resistant, BCR/Ablprotein– positive LPC sensitive to IM, as well as of ischemia-selected, BCR/Ablprotein–negative LSC refractory to IM. These LSC are suitable to home in vivo within hypoxic stem cell niches and to represent the CML cell subset responsible for MRD.