Abstract
We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML) cells and selects stem cells where BCR/Ablprotein is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ) on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1), by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2) and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening.
Highlights
We previously demonstrated that severe hypoxia inhibits growth of leukemia cell populations and selects cells exhibiting properties of leukemia stem cells (LSC) [1,2,3]
We proposed that LSC responsible for minimal residual disease (MRD) of Chronic Myeloid Leukemia (CML) home in vivo within severely hypoxic areas of bone marrow [3] where normal hematopoietic stem cells physiologically reside [4,5,6,7]
This study showed, in primary as well as K562 CML cells, differences with respect to response to BZ between cell bulk and a cell subset containing LSC selected following prolonged incubation in hypoxia
Summary
We previously demonstrated that severe hypoxia inhibits growth of leukemia cell populations and selects cells exhibiting properties of leukemia stem cells (LSC) [1,2,3]. Incubation of Chronic Myeloid Leukemia (CML) cells in hypoxia, including K562 as well as the corresponding primary cells (from blast-crisis patients), results in the complete suppression of BCR/Abl protein, but not mRNA, so that hypoxia-selected CML stem cells, while remaining genotypically leukemic, are phenotypically independent of BCR/Abl signaling and thereby refractory to the treatment with Imatinibmesylate (IM) [1,3] This is very well in keeping with the notions that IM, despite its impressive efficacy as first-line therapy for patients with chronic phase CML, induces a state of minimal residual disease (MRD), rather than cure, and that LSC are the source of MRD. The study reported here addressed the effects of BZ on K562 as well as primary CML cells in severe hypoxia, to assess the sensitivity to BZ of hypoxia-selected LSC independently of, and in comparison with, that of unselected cells
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