Microenvironmental Contribution to Dysfunctional Hematopoiesis in a Murine Model of Myelodysplastic Syndrome
Authors: Balderman Sophia R. , Frisch Benjamin J , LaMere Mark W , Goodman Alexandra N , Becker Michael W. , Calvi Laura M. 
Published: October 2019

Abstract
In vitro data provide evidence of an altered bone marrow microenvironment (BMME) in the myelodysplastic syndromes (MDS). To assess the role of the BMME in MDS in vivo, we used a well-established transgenic murine model with expression of the translocation product Nup98-HOXD13 (NHD13) in hematopoietic cells that leads to development of an MDS phenotype, fully penetrant by 5 months of age. In order to assess whether the BMME contributes to diminished hematopoiesis as a feature of MDS, we transplanted marrow from 5-month-old NHD13 mice and normal competitor marrow into irradiated NHD13 mice and their wild type (WT) littermates. Serial analysis of peripheral blood (PB) indicated engraftment of NHD13 marrow was improved in WT recipients relative to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p<0.0001). Flow cytometric analysis of marrow harvested at 16 weeks post-transplant revealed increased NHD13 donor contribution to the hematopoietic stem and progenitor cell (HSPC) pool in WT relative to NHD13 recipients (28.2 ± 4.3 vs. 2.4 ± 0.5 % of total Lineage-, cKit+, Sca1+ (LSK) cells, p<0.01). Surprisingly, leukopoiesis was improved after transplantation of NHD13 marrow into WT as compared to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p<0.01). These data establish that the MDS BMME interferes with the ability of MDS HSPCs to function similarly to normal HSPCs. After the identification of a microenvironmental defect in adult NHD13 mice, we further investigated the NHD13 BMME support for hematopoietic progenitors. By flow cytometric analysis, there were no differences in marrow multipotent progenitors (MPPs) and long term hematopoietic stem cells (LT-HSCs) from NHD13 mice vs. WT littermates at 3 weeks of age. However, in adults there was a progressively severe decline in the NHD13 HSPC pool. HSPCs were not diminished in the spleens of NHD13 mice, suggesting a specific BMME defect. The decrease in phenotypic HSPCs in NHD13 mice was confirmed functionally by competitive repopulation assays using NHD13 or WT donor marrow transplanted into irradiated WT recipients. NHD13-derived PB cells demonstrated marked myeloid skewing relative to WT-derived cells, indicative of a differentiation defect in NHD13-associated hematopoiesis. At 16 weeks post-transplant, recipient marrow was assayed for relative NHD13 and WT donor contributions to the HSPC pool. Consistent with the decreased NHD13 donor contribution to PB counts, NHD13 donor contribution to the HSPC pool in the marrow was diminished (59.4 ± 8.7 vs. 15.5 ± 5.6, % WT donor vs. NHD13 donor contribution to total LSK cells, p<0.001). Despite robust engraftment of WT competitor marrow, cytopenias and macrocytosis were observed in the recipients of NHD13 marrow, suggesting a bystander effect by the NHD13 clone on the function of the normal competitor marrow. To determine NHD13 long-term engraftment function, secondary transplantation of marrow harvested from the primary recipients of NHD13 and WT donors was performed using WT recipients. Serial PB flow cytometric data demonstrated improved overall engraftment of the NHD13 relative to WT donor marrow with persistent and even more marked myeloid skewing of NHD13 donor derived blood cells than was seen in the primary transplant. Consistent with PB data, at 16 weeks post-transplant, the contribution of NHD13 and WT donors to the HSPC pool was similar. Improved NHD13 HSPC number and function in the secondary recipients may be related to BMME rejuvenation through serial passage into a WT BMME. Our data indicate that in this model (1) MDS hematopoietic function is improved in a normal compared to MDS microenvironment (2) the HSPC pool is defective and (3) there is suppression of non-clonal hematopoiesis via a bystander effect, possibly mediated by the MDS BMME. In aggregate our data demonstrate a contributory role of the BMME to ineffective hematopoiesis in MDS, and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function.

Disclosures
Calvi: Fate Therapeutics: Patents & Royalties.