Bortezomib Specifically Targets a Subset of Hypoxia-Selected CML Stem Cells

Background: We previously demonstrated that severe hypoxia selects LSC from CML. We found that BCR/Abl protein is suppressed in hypoxia-selected CML cells and that these cells resulted refractory to Imatinib-mesylate (IM). Methods: Cells of the stabilized CML line K562 were used. BCR/Abl protein expression was determined by SDS-PAGE/western blotting. The maintenance of stem cell potential in hypoxic (0.3% O2) 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: The addition of BZ at time-zero (t0) of incubation in hypoxia markedly reduced the number of viable cells, whereas that at day 1 of hypoxia was completely ineffective. In cells treated with BZ at day 1, BCR/Abl suppression was delayed at least of 1 day. The CRA assay was carried out with cells recovered from hypoxic LC1 at days 2, 3 or 7. Cells recovered at day 2 started to repopulate LC2 immediately, due to the maintenance of BCR/Abl, to peak at day 10. LC2 repopulation by cells from day-3 and from day-7 LC1 was delayed, to peak at day 21. BZ (added at t0 or at day 1) did not alter either day-2 or day-3 LC2 kinetics but significantly reduced day-7 LC2 repopulation. Conclusions: Incubation of K562 cells in hypoxia for 1 day protects cells bulk from the toxic effects of BZ. The CRA assay defined a CML-LSC subsets hypoxia selected at day 7, BCR/Abl- typically refractory to IM but sensitive to BZ.