Disruption Of The Hematopoietic Stem and Progenitor Cell Pool and Bone Marrow Microenvironment In a Murine Model Of Myelodysplastic Syndrome

Myelodysplastic Syndromes (MDS) are a group of clonal disorders characterized by ineffective hematopoiesis. Recently, data have emerged supporting a role of the bone marrow microenvironment (BMME ) in the initiation of MDS. We and others have previously shown that cells within the BMME play a central role in normal regulation of hematopoietic stem and progenitor cells (HSPCs). To determine if the HSPC compartment in MDS is defective and also if HSPC function in MDS is regulated by the BMME, we studied a transgenic murine model that expresses the Nup98/HOXD13 (NHD13) translocation product. As was previously reported, these mice develop ineffective hematopoiesis resulting in progressive cytopenias with dysmorphic cells, a phenotype similar to that of human MDS. We investigated the composition of the HSPC pool in these transgenic (TG) mice at 20 to 22 weeks from birth, a time when an MDS phenotype was evident but acute leukemia had not yet developed. Immunophenotypic analysis by flow cytometry on marrow cells from TG and wild type (WT) age-matched littermates demonstrated a severe defect in the TG HSPC pool, with a severe decline in Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (WT vs. TG: 3.5 ± 1.2 x 10⁴ vs 4.4 ± 3.4 x10², p =0.0025) and in Lin-Sca1+cKit+Flt3+Thy1.1- multipotent progenitors (WT vs. TG: 5.6 ± 1 x 10⁵ vs 2.1 ± 0.4 x 10⁴, p<0.0001), as well as in total Lin-Sca1+cKit+ cells and short-term HSCs. To determine if the numerical changes in phenotypic HSPCs corresponded with decreased HSPC function, we performed a competitive repopulation assay using whole bone marrow, and found relative loss of function of HSPCs by 9 weeks after transplantation of marrow from 22-week old TG vs littermate WT donor mice into lethally irradiated WT recipients as measured by percent of donor cells in the blood (WT vs. TG: 37.7 ± 3.4 vs 14.7 ± 1.7, p<0.0001). Serial blood cell flow cytometric analysis demonstrated myeloid skewing (marked by percent of CD11b positive cells) of HSPCs transplanted from TG mice at the expense of lymphocytes by 5 weeks (WT vs. TG: 44.0 ± 4.3 vs 64.9 ± 4.5, p=0.0047), which persisted at 9 weeks (WT vs. TG: 43.6 ± 3.6 vs 69.1 ± 5.9, p=0.0023) and 13 weeks post transplant, a feature which has been previously associated with HSPC aging. Curiously, despite robust engraftment of normal competitor marrow, serial blood counts of recipients after competitive transplant showed that mice receiving 22-week old TG marrow developed leukopenia (9 weeks, WT vs. TG: 7.3 ± 0.47 vs 4.6 ± 0.41, p=0.0008) and lymphopenia (9 weeks, WT vs. TG: 6.0 ± 0.42 vs 3.4 ± 0.37, p=0.0003), suggesting a bystander effect initiated by the TG marrow resulting in ineffective hematopoiesis in the recipients. To determine if the MDS microenvironment contributes to ineffective hematopoiesis, we transplanted NHD13 TG and normal competitor marrow into lethally irradiated TG or WT recipient mice. NHD13 TG marrow engrafted significantly better in WT compared to TG recipients as seen by 4 weeks post transplant (Percent of total cells, WT vs. TG recipient: 14.2 ± 2.3 vs 1.1 ± 0.1, p = 0.0049; Percent of CD11b positive cells, WT vs. TG recipient: 17.1 ± 4.2 vs 1.7 ± 0.1, p = 0.0208; Percent of B220 positive cells, WT vs. TG recipient: 2.7 ± 0.3 vs 0.1 ± 0.0, p = 0.0008). These aggregate results indicate (1) severe disruption of the immunophenotypic HSPC pool in this murine TG model of MDS, (2) a functional defect of HSPCs in this MDS model as evidenced by decreased engraftment and myeloid skewing, (3) contribution of the MDS BMME to ineffective hematopoiesis downstream of immature MDS cells and (4) MDS-dependent signals initiating such microenvironmental effects. Our data strongly suggest that the malignant clone in MDS initiates signals that disrupt the normal marrow microenvironment. Furthermore, these data provide support for a strategy where rejuvenation of the marrow microenvironment and/or interference with MDS-initiated signals may result in mitigation of ineffective hematopoiesis. Further understanding of the HSPC defect in this murine model of MDS and of the role of the BMME in MDS could therefore inform new therapeutic targets for this disease.