ALBERT

Description: The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential. In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated. Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential. Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche. In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures: No relevant conflicts of interest to declare.

Description: 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. Disclosures No relevant conflicts of interest to declare.