Phenotypic and Functional Alterations of Bone Marrow Mesenchymal Stem and Progenitor Cells in Chronic Myeloid Leukemia
Authors: Dolinska Monika , Klang Johannis , Xiao Pingnan , Durgaryan Andranik , Sandhow Lakshmi , Johansson Anne-Sofie , Kondo Makoto , Deneberg Stefan , Ungerstedt Johanna , Le Blanc Katarina , Stenke Leif , Ekblom Marja , Stromberg Ulla , Mustjoki Satu , Lehmann Soren , Qian Hong 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a myeloproliferative stem cell neoplasm characterized by the presence of the BCR-ABL1 fusion gene. Although current treatment with tyrosine kinase inhibitors (TKI) has dramatically improved the prognosis of CML, these inhibitors do not eradicate leukemic stem cells (LSC) in most patients with the risk of recurrence of leukemia if TKI are stopped. In vitro studies have suggested that this might be attributable to protection of bone marrow (BM) stromal cells, such as osteoblasts, adipocytes, endothelial and mesenchymal stem cells (MSCs). However, how different BM stromal cells contribute to the persistence of LSC remains largely unknown. To investigate this issue we have compared freshly isolated BM stromal cell subsets including MSCs from newly diagnosed CML patients (n=10) with that from age-matched healthy donors (n=12). Distinct from the previous studies on culture-selected BM stromal cells, the naive stromal cells isolated by multi-color fluorescence activated cell sorting (FACS) were phenotypically, molecularly and functionally characterized in the present study.
We observed: 1) Similar to the immunophenotype of normal MSCs (CD45-CD235a-CD31-CD44-, most of which were CD271+CD146+CD106+) (Qian et al., JBC, 2012), the CML MSCs, estimated by colony forming unit-fibroblast (CFU-F), were also enriched in the CD45-CD235a-CD31-CD44- cell fraction. 2) The frequency of CFU-Fs was significantly increased in CML BM compared to that in the age-matched healthy controls (p=0.005). 3) A decreased osteogenic, but enhanced adipogenic differentiation potential of CML MSC was revealed in multilineage differentiation assay. This suggests a skewed differentiation potential of the CML MSCs towards adipocytes, possibly related to an altered stromal cell composition in the patients; 4) An increased proportion of CD31+ endothelial cells was seen in CML BM stroma compared to controls (p=0.023) by FACS. 5) An upregulation of the adhesion receptor integrin α4/CD49D was seen in the CD44- MSCs from CML patients (p=0.0087). Conversely, a downregulation of transcripts of Angiopoietin 1, CXCL12, KIT ligand and LAMA4 in the patient MSCs was detected by Quantitative-PCR, indicating an altered hematopoiesis-supportive function of CML MSCs. 6) Importantly, no BCR-ABL fusion were found in the freshly sorted MSCs and mature stromal cells using Fluorescence In Situ Hybridization analysis, suggesting that these MSCs were not part of the leukemic clone. Taken together, our data provide evidence for phenotypic and functional alterations of BM mesenchymal cells in CML patients. The functional relationship between the stromal cell alterations and the growth of LSC as well as the underlying molecular mechanisms are currently under investigation.

Disclosures
Mustjoki: Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding.