Targeting of the bone marrow microenvironment improves outcome in a murine model of myelodysplastic syndrome

Abstract

In vitro evidence suggests that the bone marrow microenvironment (BMME) is altered in myelodysplastic syndromes (MDSs). Here, we study the BMME in MDS in vivo using a transgenic murine model of MDS with hematopoietic expression of the translocation product NUP98-HOXD13 (NHD13). This model exhibits a prolonged period of cytopenias prior to transformation to leukemia and is therefore ideal to interrogate the role of the BMME in MDS. In this model, hematopoietic stem and progenitor cells (HSPCs) were decreased in NHD13 mice by flow cytometric analysis. The reduction in the total phenotypic HSPC pool in NHD13 mice was confirmed functionally with transplantation assays. Marrow microenvironmental cellular components of the NHD13 BMME were found to be abnormal, including increases in endothelial cells and in dysfunctional mesenchymal and osteoblastic populations, whereas megakaryocytes were decreased. Both CC chemokine ligand 3 and vascular endothelial growth factor, previously shown to be increased in human MDS, were increased in NHD13 mice. To assess whether the BMME contributes to disease progression in NHD13 mice, we performed transplantation of NHD13 marrow into NHD13 mice or their wild-type (WT) littermates. WT recipients as compared with NHD13 recipients of NHD13 marrow had a lower rate of the combined outcome of progression to leukemia and death. Moreover, hematopoietic function was superior in a WT BMME as compared with an NHD13 BMME. Our data therefore demonstrate a contributory role of the BMME to disease progression in MDS and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function and overall survival.

Figure 1

NHD13 mice develop cytopenias, macrocytosis, and myeloid dysplasia, with increased rate of progression to leukemia or death. (A-D) Male and female NHD13 (blue diamond) and wild-type (WT; gray square) littermates were followed with serial complete blood count measurements (WT, n = 2-12; NHD13, n = 3-14). Data from leukemic animals censored at the first time point at which they were noted to be leukemic. Results analyzed for statistical significance using 2-way analysis of variance (ANOVA). P = .0025 for granulocytes; P < .0001 for all other parameters. (E) Normal neutrophil in the peripheral blood of a WT mouse. (×1000 oil immersion). (F) Dysplastic neutrophil in the peripheral blood of representative NHD13 mouse (×1000 oil immersion). (G) Flow cytometric analysis of mononuclear cells in peripheral blood (myeloid, CD11b⁺; lymphoid, CD3e⁺ or B220⁺). (H) Survival curves showing progression to leukemia or death in NHD13 or WT mice (log-rank [Mantel-Cox] test: P = .09). (I) Schematic of transplant setup. Spleen cells (1 x 10⁶) from NHD13 mouse (CD45.2) noted to have high peripheral WBC counts and massive splenomegaly were transplanted to WT CD45.1 recipients. (J) Leukemic donor cells demonstrated engraftment of >90% in recipients at 3 weeks posttransplant. (K) Representative spleens from nontransplanted WT (left) and recipients of NHD13 cells (right) demonstrating splenomegaly consistent with leukemic development at 3 weeks posttransplant. (L) Representative peripheral blood smears from recipients showing blasts consistent with leukemic development at 3 weeks posttransplant.

Figure 2

Heterogeneity of the phenotypic myeloid progenitor pool in NHD13 mice. Bone marrow (BM) was harvested from 25-week-old male and female NHD13 (n = 5) and WT (n = 4) littermates via the crushing technique. BM was analyzed for progenitor frequencies via flow cytometry. (A) Representative flow cytometric gating schema on the whole BM from a 25-week-old WT mouse in which common lymphoid progenitors (CLPs) (Lin⁻/Flt3⁺/IL7R⁺) and myeloid progenitors (MPs) (Lin⁻/c-Kit⁺/Sca1⁻) are a subset of the lineage-negative (live) cell population, the latter of which can be divided into common myeloid progenitors (CMPs) (FcγR⁻/CD34⁺), megakaryocyte-erythrocyte progenitors (MEPs) (FcγR⁻/CD34⁻), and granulocyte-macrophage progenitors (GMPs) (FcγR⁺/CD34⁺). (B-D) Quantification of progenitor populations in WT compared with NHD13 littermates. In all graphs, each dot represents an individual mouse, and color denotes the same individual mouse in each gate. *P < .05; ***P < .001.

Figure 3

Phenotypic and functional loss of HSPC pool in NHD13 mice. (A) Representative flow plots gating for LSKs in the marrow of 5 female 20-week-old WT mice (concatenated data; left) and 5 female 20-week-old NHD13 mice (concatenated data; right). (B) Schematic of competitive repopulation assay. Whole BM from 4 22-week-old NHD13 or WT littermate donors was transplanted in a 1:1 ratio with normal WT whole BM into each of 2 lethally irradiated WT recipients. After harvesting primary recipient BM at 16 weeks posttransplant, 500 000 whole BM cells were transplanted into lethally irradiated secondary WT CD45.1 recipients. (C) Serial blood flow cytometric analysis was performed at 4-week intervals. Mice transplanted with NHD13 marrow (n = 8) had decreased donor-derived HSPC function compared with those transplanted with WT marrow (n = 7) as measured by percent of donor cells in the peripheral blood. Results analyzed for statistical significance using 2-way ANOVA with point matching; P < .01 for all parameters. Bonferroni posttests significant for all except CD11b curves. (D) Serial blood flow cytometric analysis in secondary transplant recipients. Primary donor NHD13 cells contributed proportionately less to circulating B and T cells in secondary recipients (P < .0001 by 2-way ANOVA with point matching) and more to circulating myeloid cells (P < .0001). (E) At 16 weeks post–primary transplant, percent lineage contribution to CD45.2 blood cells is shown from NHD13 (n = 7) and WT (n = 7). (F) Sixteen weeks post–secondary transplant, percent lineage contribution to CD45.2 blood cells is shown from NHD13 (n = 8) and WT (n = 7). (G) Donor contribution to LSK pool in the BM of primary recipients harvested 16 weeks posttransplant. Results analyzed for significance via Student t test. Each dot represents an individual mouse. ***P < .001.

Figure 4

Increased endothelial cell populations and decreased megakaryocytes in NHD13 mice. (A) Flow cytometry gating strategy used to identify BM Lin⁻/CD45⁻ nonhematopoietic cells, Lin⁻/CD45⁻/CD31+/Sca1+ cells enriched for arteriolar endothelial cells (AECs), and Lin⁻/CD45⁻/CD31+/Sca1⁻ cells enriched for sinusoidal endothelial cells (SECs). The first 3 plots represent WT mice, and the last plots show overlay of populations from concatenated NHD13 (red) and WT (black) mice data. (B) Expression of NUP98/HOXD13 transgene in hematopoietic, endothelial, and stromal cells. (C-D) Frequency of AECs within total BM pool (C) and Lin⁻/CD45⁻ nonhematopoietic pool (D) of NHD13 and WT mice. (E-F) Frequency SECs within total BM pool (E) and Lin⁻/CD45⁻ nonhematopoietic pool (F) of NHD13 and WT mice. (G) Endomucin immunohistochemistry for metaphyseal region of representative WT and NHD13 mice. Bars represent 20 µm. (H) Peripheral blood serum VEGF level measured by Luminex xMAP assay. (I) GP1bβ immunohistochemistry for metaphyseal region of representative WT and NHD13 mouse. Bars represent 20 µm. (J) Quantification of GP1bβ⁺ megakaryocyte numbers per area of interest (AOI) in femora of WT and NHD13 mice. For all graphs, *P < .05; **P < .01. Each dot represents an individual mouse; mean and standard error of the mean are shown.

Figure 5

Expansion of multipotent stromal and OBCs in NHD13 mice that are nonfunctional. (A) Flow cytometry gating strategy used to identify BM Lin⁻/CD45⁻ nonhematopoietic cells, Lin⁻/CD45⁻/CD31⁻/CD51⁺/Sca1⁻ OBCs, and Lin⁻/CD45⁻/CD31⁻/CD51⁺/Sca1⁺ MSCs. The first 3 plots represent WT mice, and the last 2 plots show overlay of populations from concatenated NHD13 (red) and WT mice (black) data. (B-C) Frequency of MSCs within total BM pool (B) and Lin⁻/CD45⁻ nonhematopoietic pool (C) of NHD13 and WT mice. (D-E) Number of total CFU fibroblasts (identified by staining with crystal violet) (D) and alkaline phosphatase–positive CFU fibroblasts (E) from 1 × 10⁶ BM cells after 10 to 14 days in culture. Data from 2 separate experiments and NHD13 data points represent mean of triplicate cultures normalized to mean of WT group. (F-G) Frequency of OBCs within total BM pool (F) and Lin⁻/CD45⁻ nonhematopoietic pool (G) of NHD13 and WT mice. (H) Peripheral blood serum osteocalcin levels measured by enzyme-linked immunosorbent assay. (I-J) Femoral trabecular number (I) and thickness (J) as measured by micro–computed tomography. (K-L) Number of alkaline phosphatase–positive CFU-OBs (K) and Von Kossa–positive bone nodules (L) formed by 4 × 10⁶ BM cells after 17 days of culture in mineralization media. Each data point represents mean number of CFU-OBs from triplicate cultures. (M) Peripheral blood serum CCL3 levels measured by Luminex xMAP assay. (N) Number of histologically identified osteoclasts as TRAP⁺ endosteal-associated cells. (O) Peripheral blood serum C-telopeptides (CTX) levels measured by enzyme-linked immunosorbent assay. All mice used in analyses are 20 ± 3 weeks of age. For all graphs, *P < .05; **P < .01; mean and standard error of the mean are shown.

Figure 6

Impact of the BMME on survival and leukemia. (A) Schematic of transplants into WT recipients. (B) In WT recipients, survival curves indicate mitigation of progression to leukemia or death. Data collected from 8 separate experiments. Log-rank (Mantel-Cox) test: P = .98. The BM age demarcated on the x-axis is indicative of primary donor BM age based on date of birth of the primary donor. No leukemia was diagnosed in these groups. (C) Schematic of competitive repopulation assay in which marrow from an NHD13 mouse was competitively transplanted into either irradiated NHD13 or WT recipients. (D) Survival curves showing progression to leukemia or death in primary WT recipients of competitively transplanted NHD13 marrow and in primary NHD13 recipients of competitively transplanted NHD13 marrow. Data collected from 6 separate experiments. Leukemia was formally diagnosed in 1 WT recipient and 4 NHD13 recipients. The same donor/competitor ratios were present in both experimental groups. Log-rank (Mantel-Cox) test: P = .01. In all transplants, competitor marrow was 6- to 8-week-old WT marrow. The BM age demarcated on the x-axis is indicative of primary donor BM age based on date of birth of the primary donor.

Figure 7

Hematopoiesis is improved when MDS marrow is exposed to a WT BMME. (A) Schematic of competitive repopulation assays in which NHD13 donor BM was transplanted with WT competitor BM into NHD13 recipients (blue diamonds) and WT recipients (gray squares). (B-F) Donor BM was from a 20-week-old NHD13 mouse transplanted into either lethally irradiated 23-week-old NHD13 mice (n = 3) or lethally irradiated WT littermates (n = 4). Comparison of engraftment of NHD13 marrow (B) in WT recipients vs NHD13 recipients (2-way ANOVA: P < .0001). At 16 weeks following transplant, whole BM of recipients was analyzed to determine contribution of NHD13 donor marrow to the total LSK pool (C) in WT and NHD13 recipients. Serial hematologic parameters hemoglobin (Hgb) (D), WBC count (E), and platelet count (Plt) (F) were measured in recipient mice every 4 weeks from time of transplant. (G-K) Donor BM was from a 15-week-old NHD13 mouse transplanted into either lethally irradiated 15-week-old NHD13 mice (n = 3) or WT littermates (n = 5). Comparison of engraftment of NHD13 marrow (G) in WT recipients vs NHD13 recipients (2-way ANOVA: P > .05). At 16 weeks following transplant, whole BM of recipients was analyzed to determine contribution of NHD13 donor marrow to the total LSK pool (H) in WT and NHD13 recipients. Serial hematologic parameters Hgb (I), WBC count (J), and Plt (K) were measured in recipient mice every 4 weeks from time of transplant. (L) Frequency of myeloid vs lymphoid cells within CD45.1⁺ WT competitor-derived cells in peripheral blood of all WT and NHD13 recipients at 16 weeks posttransplant. For hematologic data, upper and lower borders of gray boxes represent the interquartile range of Hgb, WBC, or Plt values for nontransplanted WT mice (from Figure 1).