Evaluation of Extrinsic and Intrinsic Cues Involved in BCR-ABL-Induced Leukemogenesis

Intrinsic and extrinsic signals together contribute to determine self renewal, quiescence or the specific metabolic status of leukemic stem cells (LSC) in BCR-ABL mediated chronic myeloid leukemia (CML). Our previous studies have shown that expression of BCR-ABL together with the polycomb repression complex 1 member BMI1 in human CD34+ cells is sufficient to induce a serially transplantable lymphoid leukemia in vivo while a myeloid phenotype was never observed. Yet in vitro, both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in CML patients. Since NSG models are typically lymphoid biased due to the absence of species-specific myeloid growth factors, we hypothesized that extrinsic factors might dictate lineage fate. Using a “humanized” NSG mouse model in which scaffolds seeded with human mesenchymal stromal cells were implanted we observed that, in contrast to the murine niche, BCR-ABL overexpression alone was sufficient to induce a serially transplantable leukemia of both the lymphoid and myeloid lineage. Using myeloid blast-crisis CML patient cells, engraftment was also observed whereby the immature blast-like phenotype was predominantly maintained in the humanized scaffold niche, and to a much lesser extent in the murine niche. This distinction could also be demonstrated functionally by using in vitro long-term self-renewing cultures. Blast cells retrieved from the human scaffold niche could readily be established while no long-term cultures could be initiated from cells retrieved from the murine bone marrow niche. Genome-wide transcriptome analyses of leukemic cells retrieved from the mouse BM niche and from the human scaffold niche revealed striking differences in gene expression imposed on BCR-ABL+ cells by these different environments. For example, endogenous BMI1 levels were significantly higher in BCR-ABL cells retrieved from human scaffold niche as compared to murine BM harvested cells suggesting that BMI1 might still be required as additional factor to prevent oncogene-induced senescence.

Apart from epigenetic modifiers, we hypothesized that the hypoxic microenvironment might play an important role in maintaining CML LSCs and studied that in detail. Hypoxia inducible factor 1α (HIF1) and HIF2 act as transcription factors that are stabilized under hypoxic conditions. HIF1 has been characterized as an important factor that controls cellular metabolism while the role of HIF2 is still less clear. Earlier we identified HIF2 as downstream target of STAT5 and observed elevated glucose uptake in STAT5 activated HSCs. Several genes associated with glucose metabolism were upregulated by STAT5 in an HIF2 dependent manner, including SLC2A1 and GYS2. Here, we investigated metabolic changes in BCR-ABL expressing human stem/progenitor cells and focused on the role on HIF1 and HIF2. Genome-wide transcriptome analyses were performed on human CB CD34+ cells transduced with BCR-ABL as well as on BCR-ABL-positive CML and B-ALL patient samples. GSEA analyses indicated that these transcriptome changes were strongly enriched for STAT5 and MYC signatures as well as for hypoxia, embryonic stem cell and glucose metabolism gene signatures which included upregulation of e.g. SLC2A3, SLC2A1 and HIF1 and HIF2. These data suggest that BCR-ABL imposes hypoxic signaling under normoxic conditions. Moreover, downregulation of HIF1 and HIF2 using a shRNA approach impaired proliferation and reduced progenitor frequencies of BCR-ABL+ cells.

Next we studied metabolic changes in BCR-ABL+ cells using NMR spectroscopy. We observed striking differences in uptake and secretion of metabolites when BCR-ABL CB CD34+ cells were compared to normal CB CD34+ cells under normoxia and hypoxia. As expected, BCR-ABL cells exhibited enhanced glycolysis as determined by an increased production and secretion of lactate under both normoxic and hypoxic conditions. Interestingly, glutamine levels were strongly enhanced in BCR-ABL+ cells, in a HIF1/2-dependent manner, possibly via enhanced glutamine import or glutamine production via upregulation/activation of Glutamine Synthase. Our current hypothesis is that BCR-ABL+ cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells, and studies focus on whether targeting this pathway might provide alternative means to eradicate LSCs.