Novel In Vivo Models Uncover Non-Cell-Autonomous Effects of Myelodysplastic Syndrome (MDS) on Marrow Osteolineage Cells and Hematopoietic Dysfunction

Myelodysplastic syndromes (MDS) remain a therapeutic challenge, leading to significant morbidity and mortality due to blood cytopenias and leukemic transformation. We and others have demonstrated that abnormalities of the bone marrow microenvironment (BMME) contribute to disease pathogenesis and progression. However, current models fail to assess in vivo the impact of the malignant cells on the BMME and on residual non-clonal hematopoiesis. We previously identified BMME abnormalities in Vav1-NUP98/HOXD13 (NHD13) mice, a well-established transgenic murine model that recapitulates pathologic hallmarks of human MDS including blood cytopenias, dysmyelopoiesis, and transformation. NHD13 mice had increased osteoblastic-lineage cells (OBC, lineage-/CD45-/CD31-/CD51+/Sca1-), as well as hematopoietic dysfunction and hematopoietic stem cell (HSC) loss.

To determine if MDS can initiate BMME alterations, we transplanted MDS marrow of NHD13 mice or wild-type (WT) littermate marrow (CD45.2+/CD45.1+) into irradiated WT recipients (CD45.2+/CD45.1-). Using flow cytometry to distinguish donor- vs. host-derived hematopoietic cells, we found that NHD13 and WT donor-derived cells contributed to approximately 60% of recipient marrow, generating an in vivo model of MDS cells among genetically-WT hematopoietic cells. This transplant model recapitulates human MDS, where MDS clones emerge and expand among normal hematopoietic cells in a BMME that is initially genetically normal. At 6 weeks post-transplant, OBCs were increased in recipients of MDS marrow (p<0.05), indicating that interaction with MDS cells induced osteolineage cells to take on features of an MDS-BMME. Concurrent with BMME remodeling,HSCs were decreased in recipients of MDS marrowcompared to recipients of WT marrow (p<0.05). Notably, HSCs in recipients of MDS marrow were also decreased compared to surviving irradiated but non-transplanted mice (p<0.05), consistent with disruption of normal HSC function by MDS cells. In line with acquired hematopoietic dysfunction, recipients of MDS marrow exhibited leukopenia and anemia compared to recipients of WT marrow. To assess the hematopoietic function of genetically-WT recipient cells in the presence of MDS cells, we quantified donor- vs. recipient-derived peripheral white blood cells (WBC). The number of recipient-derived WBCs in mice transplanted with MDS marrow was lower compared to irradiated, non-transplanted mice. This suggests that cytopenias in recipients of MDS marrow were not only due to defective cell-autonomous hematopoiesis by MDS cells, but also due to the impaired hematopoietic function of residual WT recipient cells. Together, these data indicate that MDS cells may decrease hematopoietic support, leading to dysfunctional hematopoiesis.

To assess hematopoietic function of non-irradiated WT hematopoietic cells in the presence and absence of MDS, we transplanted NHD13/GFP- marrow mixed 1:1 with WT/GFP+ marrow (NHD13/GFP-:WT/GFP+) or WT/GFP-:WT/GFP+ marrow into irradiated WT recipients. Although NHD13 donor-derived cells contributed to only 4% of reconstituted recipient marrow in this model, HSC depletion was unexpectedly observed in recipients of NHD13/GFP-:WT/GFP+ marrow compared to recipients of WT/GFP-:WT/GFP+ marrow, showing that, even when recipient marrow is comprised of over 90% normal hematopoietic cells, the HSC pool is reduced in the presence of MDS cells. These data demonstrate a strong non-cell-autonomous effect of MDS on hematopoietic function.

Because human MDS is associated with diverse genetic abnormalities, we sought to determine if MDS-dependent BMME remodeling transcends the genetic makeup of MDS by assessing the BMME in mice with inducible EVI1 overexpression (EVI1++), an oncogenic transcription factor important in myeloid progenitor function and commonly implicated in human MDS. Marrow from EVI++ mice (mixed 1:1 with WT marrow) was transplanted into WT mice. By 14 weeks post-transplant, recipients of EVI1++ cells exhibited BMME alterations, demonstrated by increased OBCs (p<0.05) compared to recipients of WT cells. Thus, exposure to both NHD13 and EVI1++ marrow induces OBC remodeling in our transplant models.

Together, these data uncover non-cell-autonomous effects of MDS causing hematopoietic dysfunction in vivo, and provide novel tools to discern the MDS signals and BMME changes impairing normal hematopoiesis.