HYPOXIA SELECTS DRUG-INSENSITIVE PROGENITORS WITHIN CLONAL LEUKAEMIA CELL POPULATIONS

1. We previously showed that normal haematopoietic stem and progenitor cells exhibit different levels of resistance to severe hypoxia, which contributes to maintain stem cell features, while protecting stem cells from differentiation commitment. Leukaemia stem cells also appear to require such a protection, as differentiating agents often suppress leukaemic growth. On this basis, we undertook the characterization of the effects of hypoxia on leukaemia stem and progenitor cells. 2. The question we addressed with this study was whether clonal and apparently homogeneous myeloid leukaemia populations, such as the Friend’s murine erythroleukaemia (MEL) and K562 cell lines, comprise in fact cells endowed with different levels of hypoxia-resistance, corresponding to different levels within the progenitor/stem cell hierarchy. 3. Our experimental system, based on cell incubation in normoxia or hypoxia (0.1% oxygen), allowed to discriminate between clonogenic progenitors detectable in semisolid cultures (CFC) and hierarchically higher culture-repopulating progenitors (CRC) which correspond to short-term marrow-repopulating stem cells. 4a. We found that severe hypoxia strongly inhibited the overall growth of MEL cells and almost completely suppressed the CFC subset within the MEL cell population. Such a marked sensitivity to hypoxia of CFC when compared to the overall leukaemia cell population parallels that of normal CFC we previously showed. In hypoxia, on the other hand, a cell subset survived whose expansion power after transfer to normoxia (an indicator of CRC) was unchanged in comparison to that of cells always incubated in normoxia. 5a. Thus, the MEL cell population comprises hypoxia-resistant CRC as well as hypoxia-sensitive CFC. Such a heterogeneity is therefore typical not only of leukaemia populations progressing in vivo to a polyclonal state, but also of monoclonal populations. The hypoxia-resistant CRC subset most likely contains leukaemia stem cells which resemble stem cells believed to survive in the severely hypoxic core of fast-growing solid tumors and to be responsible for tumor progression, treatment resistance and long-term relapse of disease. 4b. The use of K562 cells enabled us to match the effects of hypoxia with those of antileukaemic drugs such as the BCR/Abl inhibitor STI-571. Hypoxia strongly inhibited the overall growth of K562 cells, and hypoxia-selected cells, after transfer to normoxia, underwent a lag phase before exponential growth was triggered. The latter phase, however, followed a kinetics very similar to that of equal numbers of cells always incubated in normoxia. CFC, when compared to the overall K562 cell population, exhibited a higher sensitivity to hypoxia and much faster kinetics of decrease in hypoxia and increase after transfer to normoxia. Cell number decrease in day-7 hypoxic K562 cell cultures was unaffected by the presence of STI571. 5b. The kinetics of cell number increase after transfer to normoxia was similar irrespective of cell treatment with STI571 in hypoxia, indicating that hypoxia-resistant CRC are STI571-insensitive. When the cell population generated in normoxia from CRC was tested for sensitivity to STI-571, its growth was markedly inhibited by STI571, indicating that the clonal expansion of hypoxia-resistant CRC results in the generation of an STI571-sensitive progeny.