Specific Bone Marrow Mesenchymal Subsets in Patients with Myelodysplastic Syndromes Harbor Molecular Perturbations That Alter the Dynamics of Competition between Pre-Leukemic Clones and Normal Cells
Authors: Kfoury Youmna , Jain Esha , Myers Jason R , Ji Fei , Sykes David B , Mercier Francois , Papazian Ani , Le Cam , Randolph Mark A , Steensma David P. , Abdel-Wahab Omar I , Calvi Laura M. , Sadreyev Ruslan I , Becker Michael W. , Scadden David T. 
Published: October 2019

Abstract
Myelodysplastic syndromes (MDS) are clonal disorders of aberrant hematopoietic stem cells. The role of the hematopoietic microenvironment (HMEV) in the initiation and maintenance of MDS remains unclear, though model systems have provided strong evidence for the potential of specific mesenchymal subsets, mostly osteoprogenitor cells, within the HMEV as a driver of the disease. Distinct behavior of various models emphasizes the importance of analyzing specific and highly defined populations rather than bulk mesenchymal cells when investigating patient-derived bone marrow (BM) samples.
We have developed a platform that allows us to prospectively isolate specific human BM mesenchymal subsets with a comparable transcriptional profile to mouse osteoprogenitor cells that have been shown to drive hematological malignancies when perturbed (Raaijmakers et al., 2010; Dong et al, 2016). Using a combination of three cell surface markers (CD271, CD146, CD106) that were previously shown to prospectively enrich for mesenchymal cells in human BM, we isolated and performed transcriptional and functional analysis of four distinct subsets within the non-hematopoietic (CD45, CD235), non-endothelial (CD31) compartment of the human BM. The four populations are labeled 1A (CD271+CD106-), 1B (CD271+CD106+), 2A (CD271+CD146+CD106-), and 2B (CD271+CD146+CD106+) (Figure 1A). Comparative analysis of differentially expressed gene sets based on RNA-seq within the human and mouse mesenchymal populations identified population 2B to be the most similar to osterix (OSX)-labeled mouse osteoprogenitor cells, whereas population 1A was more similar to osteocalcin (OCN)-labeled mouse mature osteoblasts. In animal models, genetic perturbations specifically in OSX but not OCN labeled cells resulted in hematological malignancy. Based on that observation, we hypothesized that population 2B is the cell subset most likely to harbor molecular perturbations in the BM of MDS patients. We analyzed populations 2A, 2B, 1(1A+1B) and another population 3 which is devoid of colony forming cells and encompasses all CD271-CD146- non hematopoietic stromal cells in 16 human MDS patients. BM from 11 patients undergoing hip replacement surgeries was used as a source of age- and gender-matched normal controls.
Despite the heterogeneity of the disease, principal component analysis revealed that the transcriptional profile of normal samples clearly separated from MDS patients mostly in populations 2B and 1 (Figure 1B). Functional annotation of the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) demonstrated minimal overlap of enriched terms between the four populations. Thus our data demonstrate transcriptionally distinct subsets of BM mesenchymal cells and identify signatures of transcriptional deregulation which differentiate these cell subsets in MDS patients from normal individuals.
Osteopontin, an extracellular matrix protein expressed by osteoblasts among other cells, was the most significantly differentially expressed gene in population 2B of MDS patients.To study the effect of the gene expression perturbations on MDS evolution in vivo, we created a chimeric model where BM cells expressing the Nup98/HoxD13 (NHD13) translocation were transplanted in competition with wild type cells in wild type (WT) or osteopontin knock out (Opn-KO) recipients. Animals were conditioned using immunotoxin to avoid the damaging effect of irradiation on the microenvironment. Following chimerism of donor cells in peripheral blood over 24 weeks demonstrated a competitive advantage of NHD13 cells in the Opn-KO recipients leading to a significant difference in survival when compared to NHD13 cells transplanted in wild type mice (Figure 1C, D).
Altogether, our data provide evidence that specific mesenchymal progenitor subsets in the BM of MDS patients harbor molecular perturbations that contribute to the competitive advantage of MDS over normal hematopoietic cells.

Disclosures
Sykes: Clear Creek Bio: Equity Ownership, Other: Co-founder.