Hematopoiesis in Chronic Phase CML emerges from Hematopoietic Stem Cells with a Transcriptional Phenotype Resembling Normal Myeloid Progenitor Cells.

Introduction: Composition of cell subsets within the CD34+ cell population is markedly altered in chronic phase (CP) CML. Specifically, megakaryocyte-erythrocyte progenitors (MEP) show a proportional increase and the percentage of granulocyte-macrophage progenitors (GMP) is significantly lower in comparison to healthy bone marrow. To understand the molecular basis for this, we have used microarray technology to analyze transcriptional differences between distinct subsets from normal and CP CML bone marrow.

Methods: We examined highly purified HSCs, CMPs, GMPs and MEPs from patients with chronic phase CML and healthy volunteers by FACS for subset analyses and applied high-speed cell-sorting to obtain the distinct CD34+ subsets. Then, we performed gene expression analyses (HU-133A 2.0) of each separate subset. Further assays included quantitative RT-PCR, FISH, adhesion and migration assays and transient transfection experiments.

Results: Subset analyses showed a significant proportional expansion of CMPs and MEPs and a decrease of HSCs in chronic phase CML. When comparing the concentration of each subset by means of absolute cell counts, HSC counts were similar whereas counts for other subsets were significantly higher in CML ranging between 2.8 and 7.7-fold. When looking at the gene expression data, it was surprising to see that the CML HSC has a transcriptional profile, which is similar to CML progenitors and healthy CMP as determined by Euclidian Distance Analysis. In contrast, for normal individuals the cluster tree clearly resembled the current model for hierachical development with the HSC sitting at the top of the hierarchy, and the more differentiated subsets of MEP and GMP at the bottom. Given that differences between the determined progenitors were minor, we focused on the characterisation of the CML HSC. We found 614 genes differentially expressed including downregulation of genes encoding for adhesion molecules, transcription factors and regulators of stem cell fate as well as upregulation of proliferation-associated genes in CP CML. Impaired adhesive and migratory capacity and a novel role of the nuclear receptors NR4A1 and NR4A3 for the transcriptional regulation of c-Jun and JunB was functionally corroborated in CML HSC.

Conclusion: Based on these data we propose a loss of quiescence of CML HSC upon detachment from the niche leading to expansion of myeloid progenitors as determined by quantitative analysis.