Energy shortage regulates BCR-Abl expression in Chronic Myeloid Leukemia cells via transcriptional/post-transcriptional control

Low oxygen tension is a critical aspect of the metabolic milieu of stem cell niches. It has been hypothesized that low oxygen tensions in stem cell niches in vivo offer a selective advantage to the maintenance of Hematopoietic Stem Cells (HSC), being HSC, but not less immature progenitors, well suited to stand low oxygen tensions. It was indeed shown in our laboratory that resistance to severe hypoxia in vitro defines hierarchical levels within hematopoietic populations. The cancer stem cell (CSC) hypothesis postulates that tumor cells are hierarchically organized with respect to tumor growth initiation and maintenance. The source of CSC is not entirely clear, and may differ depending on the specific disease. Some experimental results are consistent with the idea that CSC can derive from normal stem cells that have undergone oncogenic transformation. In contrast, it is possible that malignant non-stem progenitor cells acquire the capacity of self-renewal. In addition, tumors have an elevated rate of glucose uptake and consumption through glycolysis, that offers a growth advantage to cancer cells under a hypoxic environment. Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder driven by the BCR-Abl oncogenic fusion protein with constitutive kinase activity, which drives autonomous cell survival and growth. It was also shown in our laboratory that stabilized and cloned leukemia cell lines are highly heterogeneous, to comprise cells with a hypoxia-adapted “stem” phenotype as well as others with a hypoxia-sensitive “progenitor” phenotype, suggesting that these phenotypes are not genetically “frozen”, but flexibly, and reversibly, expressed. Thus, leukemia cell lines emerged as suitable models to assess independently the sensitivity to chemoterapy of the “stem” or the “progenitor” leukemia cell phenotypes to chemotherapy, provided an experimental system capable to select the one from the other (such as incubation in hypoxia or in glucose starvation for different times) is established. BCR-Abl protein suppression emerged as a crucial feature of CML cells adaptation to hypoxia. Furthermore, in hypoxia, while surviving leukemia cells are generally growth-arrested, Leukemia Stem Cells (LSC) are in large part cycling, indicating that cycling in hypoxia is a specific property of LSC. Thus, hypoxia-selected LSC, while remaining genetically leukemic, are phenotypically independent from BCR-Abl signaling. The suppression of BCR-Abl, molecular target of the current CML therapy, combined with the capacity of cycling within the hypoxic niches, makes of hypoxia-adapted LSC the most likely candidate to sustain treatment-insensitive Minimal Residual Disease (MRD) of CML. The main target of this study was to characterize molecular mechanisms regulating BCR-Abl expression in blast crisis CML cell lines (K562 and KCL22). We found that hypoxia strongly inhibits the overall growth of CML cell populations and suppresses both the expression and phosphorylation of BCR-Abl protein and transcription of BCR-abl mRNA. These pointed to a dual action of hypoxia as far as BCR-Abl expression in concerned, transcriptional and/or post-transcriptional. To test whether hypoxia per se is capable of driving the selection of CML cells, or it additional environmental/metabolic factors are required, such as glucose availability, we maintained cells in normoxia in the absence or the presence of glucose for 14 days and compared the data with those obtained in hypoxia: glucose shortage reduced cell survival and downregulated BCR-Abl protein but, unlike hypoxia, did not affect BCR-abl mRNA levels. The effect of environmental/metabolic factors on LSC were determined using the Culture Repopulating Ability (CRA) assay, where the time-dependent selection of LSC in hypoxic/glucose-starved cultures is estimated following cell transfer to secondary standard cultures where the expansion of population is allowed. Results allowed us to confirm our hypothesis that energy shortage (hypoxia or glucose deprivation) represents the condition selecting LSC cells, BCR-Abl-negative.