Molecular Signature of Distinct CD34+ Hematopoietic Stem and Progenitor Cell Subsets of Patients with CML in Chronic Phase.
Authors: Bruns Ingmar , Czibere Akos G. , Fischer Johannes C. , Steidl Ulrich , Pechtel Sabrina , Koch Annemarie , Brünnert Daniela , Büst Sebastian , Cadeddu Ron-Patrick , Kronenwett Ralf , Haas Rainer 
Published: October 2019

Abstract
During the last decade, chronic myeloid leukemia (CML) has been mainly characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the protooncogene BCR-ABL. This constitutively active tyrosine kinase is widely considered as the cause of the disease. Even though BCR-ABL transcripts are found in every dividing hematopoietic cell and thus, the disease is likely to originate from a primitive stem cell, the “cell of origin” is still a matter of debate. Despite the active “leukemia stem cell” discussion, very few characteristics of the “cancer stem cell” are established to date. In order to get further molecular insights into CML stem and progenitor cells, we examined CD34+ cell subsets obtained from bone marrow of 7 patients with CML in chronic phase in comparison with 5 healthy volunteers. CD34+ cells were immunomagnetically selected and high-speed cell sorting of lineage-negative, CD34+, CD38−, hematopoietic stem cells and myeloid progenitors was performed. Progenitors were further subdivided by anti-IL-3Ralpha and anti-CD45RA staining. Following RNA extraction, a two-cycle amplification procedure was used to generate cDNA for the hybridization with Affymetrix U133A2.0 arrays. After performing smoothening spline normalization, we applied the perfect match-mismatch difference model algorithm to calculate expression values (dChip). Hierarchical cluster analysis was performed using a correlation based centroid linkage algorithm. Hereby we could discriminate the HSCs, CMPs, and MEP subsets. Corroboration of RNA expression was performed by real-time RT-PCR for selected genes. Comparing the HSC subsets of CML patients with healthy controls we found 98 differentially expressed genes. 87 genes had a lower expression level in CML HSCs whereas 11 genes had a higher one. Among the downregulated genes in CML were transcriptions factors involved in myelogenesis and proliferation and several adhesion molecules associated with homing and migration of the HSCs. On the other hand, the Leptin receptor and BCR-ABL downstream targets were found to be upregulated. Within the common myeloid progenitor (CMP) compartment 37 genes were significantly differentially regulated. Twenty genes had a higher expression level in CML CMPs, 17 genes were downlegulated. Hematopoietic cell-specific cell cycle inhibitor MS4A3 was among the significantly downregulated genes whereas genes of the retinoblastoma and E2F families as well as inhibitors of the Wnt-signaling pathway were upregulated. Looking at megakaryocte-erythrocyte progenitors (MEP) in CML, key mediators of G2-M cell cycle transition were downregulated indicating a lower proliferative capacity of this subset. No transcriptional differences have been observed between granulocyte-macrophage progenitors from CML patients and healthy volunteers. Interestingly, among all other subsets myeloperoxidase (MPO) was downregulated in the CML samples and the Leptin receptor was upregulated. Our results provide novel insights into the biology of CML and potentially provide the basis for the characterization of a candidate CML stem cell.