Hypoxia Selects Bortezomib-Resistant Stem Cells of Chronic Myeloid Leukemia

We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML) cells and selects stem cells where BCR/Abl(protein) 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

Effects of BZ on hypoxia-selected K562 cells.

<p>Cells from day-7 hypoxic LC1 treated with BZ at day 6 as indicated were transferred into normoxic LC2 (3×10⁴ viable cells/ml) and trypan blue-negative cells counted at the indicated times of incubation in LC2. Values represent means±S.E.M. of data from 3 independent experiments.</p

Effects of BZ on hypoxia-resistant K562 cell subsets.

<p>Cells treated as indicated in hypoxic or normoxic LC1 (established at 3×10⁴ viable cells/ml) were transferred at day 2 (<b>A</b>) or day 7 (<b>B</b>) into normoxic LC2 (3×10⁴ viable cells/ml) and trypan blue-negative cells counted at the indicated times of incubation in LC2. Values represent means±S.E.M. of data from 3 independent experiments.</p

Induction of apoptosis by BZ in hypoxia.

<p>(<b>A</b>) Total cell lysates in Laemmli buffer were subjected to immuno-blotting with the indicated antibodies. Anti-H4 was used to verify equalization of protein loading. One representative experiment out of 3 is shown. (<b>B</b>) Percentages cells undergone “early” (annexin-V+/PI-) or “late” (annexin-V+/PI+) apoptosis, as determined by flow-cytometry. Values are means±S.E.M. of 3 independent experiments.</p

Effects of BZ on BCR/Abl protein expression in hypoxia.

<p>Total cell lysates in Laemmli buffer were subjected to immuno-blotting with an anti-Abl antibody. Anti-vinculin and anti-ERK1/2 antibodies were used to verify equalization of protein loading. One representative experiment out of 3 is shown.</p

Effects of Bortezomib (BZ) on K562 cell bulk.

<p>Cultures were treated or not with a single dose of 0.5 nM BZ from time 0 to day 7 or from day 1 to day 7 and trypan blue-negative cells counted at the indicated times. Values represent means±S.E.M. of data from 3 independent experiments. (<b>A</b>): hypoxia (∼0.1% O₂); (<b>B</b>): normoxia (21% O₂).</p