Architectural and Functional Heterogeneity of Hematopoietic Stem/Progenitor Cells in Non-Del(5q) Myelodysplastic Syndromes

Introduction

Myelodysplastic syndromes (MDS) are clinically diverse malignant disorders of aging with a propensity to evolve to acute myeloid leukemia (AML) or bone marrow failure. In early MDS, whole genome sequencing identified mutations distributed in few clones. Evidence have been provided for the existence of a MDS-initiating cell in cases harboring a 5q deletion. However, the clonal heterogeneity and its consequences on the phenotypic diversity of non-del(5q) MDS is little documented. Here we focused on studying the hierarchical organization and the functionality of clones defined by molecular profiling. The clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment was investigated and dominant clones were examined as MDS-initiating cells.

Material and methods

Bone marrow (BM) samples were obtained from 20 patients with non-del(5q) MDS enrolled in the national Programme Hospitalier de Recherche Clinique MDS-04 after informed consent in accordance with ethics committee guidelines. BM samples,

cytaphereses from age-matched healthy individuals and cord bloods were used as controls. Targeted NGS of a selected panel of 39 genes was used to define the mutational landscape on BM mononuclear cells (MNC). To study the clonal architecture at the HSPC level, single CD34+CD38- cells were seeded in 96-well plates coated with MS-5 stromal cells and cultured in H5100 MyeloCult medium (StemCell Technologies, Vancouver, Canada) with cytokines for six weeks. For long-term culture-initiating cell (LTC-IC) assays, CD34+ progenitors were cultured for six weeks on MS-5-coated plates without cytokines and then tested for colony-forming cells. For clonogenic assays, CD34+CD38- cells were seeded in methylcellulose for two weeks. All animal experimentations were performed in NSG mice.

Results

In the 20 cases of non-del(5q) MDS, genomic lesions were traced down to single CD34+CD38- HSPC-derived colonies. Clonal organization was mostly linear in 13/17 patients and branched in 4 cases with retention of a dominant subclone. The clone detected in LTC-IC compartment and that reconstituted short-term human hematopoiesis in xenotransplantation models was usually the dominant clone, which gave rise to the myeloid and to a lesser extent to the lymphoid lineage. Other mutations not detected in LTC-IC can appear in CD34+CD38- compartment or at the level of lineage-committed progenitors. The pattern of mutations may differ between common myeloid (CMP), granulo-monocytic (GMP) and megakaryocytic-erythroid (MEP) progenitors. For instance, a major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knockdown by shRNA in normal CD34+ progenitors impaired their granulocytic and erythroid differentiation. By contrast, a STAG2 gene mutation, not detected in CMP or MEP, amplified in a GMP, which drove the transformation to AML.

Conclusion

In the present study, we characterized the first genetic hits that initiate disease in a dominant clone of the CD34+CD38- HSPC compartment, which exhibits LTC-IC activity and reconstitutes human short-term hematopoiesis in NSG mice. The genetic heterogeneity in non-del(5q) MDS arises within the HSPC compartment and in lineage-committed progenitors which ultimately support the transformation into AML. The clonal architecture of HSPC compartment and mutations selection along differentiation contribute to the phenotype of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation, and may guide the search for novel therapeutic strategies.