Mdm2 levels are essential for the survival of MSCs
We first sought to generate a BM-specific MSC reporter mouse to mark MSCs and perivascular cells in vivo. Because of its previously characterized expression in other settings, we used the Osx-Cre allele 17, 18, 19 combined with mTmG allele 20 to generate double-transgenic Osx-Cre;mTmG reporter mice. The mTmG allele expresses membrane-localized red fluorescence globally in the absence of Cre recombinase and green fluorescence specifically in Cre recombinase-expressing cells. Consistent with previous reports 17, 18, GFP-expressing cells were exclusively present in MSCs that give rise to BM stromal cells and eventually differentiate to osteoblasts and adipocytes (Supplementary Figure S1A). In adult BM, GFP marked the osteoblast lineages including trabecular, endosteal, and periosteal cells. The Lepr+ MSCs and perivascular cells within the BM were green fluorescent protein positive (GFP+) (Supplementary Figure S1B). Flow cytometric analysis of markers for MSCs in GFP+ cells derived from BM showed that the GFP+ cells were partially positive for CD73, CD44, and CD90, suggesting that the Osx-Cre;mTmG reporter accurately marked the population of MSCs (Supplementary Figure S1C). Thus, the population of GFP+ cells marked by Osx-Cre represent the major characteristics of MSCs in postnatal mice.
Next, we evaluated the role of Mdm2 in MSCs by conditional deletion of Mdm2 using Osx-Cre as the driver. We interbred progeny of Osx-Cre;mTmG mice with the Mdm2fl allele 21 so that we were able to delete Mdm2 specifically in MSCs and trace them with GFP (Figure 1A). Then, we evaluated the bone and hematopoietic phenotype of littermate embryos derived from crossing of the Mdm2fl/fl mice with the Osx-Mdm2fl/+ mice. Mice with homozygous deletion of Mdm2, Osx-Cre;Mdm2fl/fl, died shortly after birth due to skeletal malformation and compromised breathing (Figure 1D). Developing bones in the Osx-Mdm2fl/fl mice displayed enlarged chondrocytes without ossification and trabecular bone formation (Figure 1B). However, the Osx-Mdm2fl/+ mice did not show any abnormalities in trabecular bone formation and the structure of the growth plate (Figure 1C). Importantly, hematopoiesis was eliminated in the metaphysis of developing bones in Osx-Mdm2fl/fl mice, suggesting that Mdm2 is essential for hematopoietic support by MSCs (Figure 1E). The hematopoietic cells were distributed throughout the metaphysis of developing bones in Osx-Mdm2fl/+ mice (Figure 1F).
Since mice with homozygous deletion of Mdm2 in MSCs were not viable, we focused on Osx-Mdm2fl/+ mice and further characterized their bone development. We used terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining to estimate DNA strand breaks as a marker of cell apoptosis in bone cells. Many MSCs, but not mature osteocytes, in Osx-Mdm2fl/+ femurs were TUNEL positive, whereas few TUNEL positive cells were seen in the femurs of Osx-Mdm2+/+ mice (Figures 1G and 1H). To evaluate the osteoblast differentiation potential of MSCs in Osx-Mdm2fl/+ mice, we performed bone densitometry. The trabecular bone volume in Osx-Mdm2fl/+ mice was significantly decreased significantly more than that of similarly-aged Osx-Mdm2wt mice (Figure 1I and 1J), suggesting that osteoblast differentiation of MSCs was also attenuated in Osx-Mdm2fl/+ mice. Thus, genetic depletion of Mdm2 in MSCs was associated with increased MSCS apoptosis levels and osteopenia in adult mice.
Mdm2 haploinsufficiency in MSCs promotes thrombocytopenia after IR-induced cellular stress
We examined whether the Mdm2 level in MSCs was important in hematopoiesis. The population of hematopoietic stem cells defined as CD150+Lin–/c-Kit+/Sca-1+ was not significantly altered in the BM of Osx-Mdm2fl/+ mice compared with control Osx-Mdm2+/+ mice, suggesting that the hematopoietic support of MSCs was not functionally compromised in Osx-Mdm2fl/+ mice (Supplementary Figure S2A). To determine whether the Mdm2 level in MSCs was important in hematopoietic recovery after cellular damage by irradiation, the Osx-Mdm2fl/+ and control mice were irradiated (6 Gy), and peripheral blood was analyzed by cell counter. We included a cohort of hematopoietic-specific Mdm2 haploinsufficient (Vav-Cre;Mdm2fl/+) mice, known to be IR-sensitive, as positive controls (Figure 2A). Analysis of the peripheral blood one week after IR showed a significant decrease in peripheral platelet counts after IR in all mice (Figure 2B). There were no significant differences between Osx-Mdm2fl/+ (Mdm2 haploinsufficient MSCs) and Vav-Cre;Mdm2fl/+ (Mdm2 haploinsufficient hematopoietic stem cells) in response to IR, as both groups developed severe thrombocytopenia. However, platelet counts were significantly lower in Osx-Mdm2fl/+ mice than in the Mdm2wt controls (p < 0.001), suggesting that Mdm2 haploinsufficiency in MSCs contributed to platelet production by megakaryocytes during the recovery phase. Analysis of BM sections derived from irradiated mice revealed a striking pancytopenia in Osx-Mdm2fl/+ as well as Vav-Cre;Mdm2fl/+ mice compared with controls (Figure 2C). The megakaryocytes were absent in Vav-Cre;Mdm2fl/+ mice (Figure 2D). However, the number of megakaryocytes were comparable between Osx-Mdm2fl/+ and the controls, suggesting that the thrombocytopenia after irradiation was due to the functional impairment of megakaryocytes in platelet production rather than their depletion (Figures 2D and 2E). In contrast to Osx-Mdm2fl/+mice, which survived after irradiation, all Vav-Cre;Mdm2fl/+ mice died around 2 weeks after IR, likely due to eradication of the hematopoietic stem cells (Figure 2F). TUNEL staining of BM samples derived from Osx-Mdm2fl/+ mice after IR showed apoptosis of hematopoietic cells, whereas the MSCs were completely devoid of TUNEL positivity, indicating that Mdm2 haploinsufficient MSCs survived the DNA damage following irradiation, and that the thrombocytopenia observed in these mice was not due to loss of MSCs (Figure 2G). In addition, immunostaining showed strong p53 accumulation in MSCs of Osx-Mdm2fl/+ mice (Figure 2H), suggesting that Mdm2 haploinsufficiency results in accumulation of p53 after IR stress. Thus, inhibition of Mdm2 in MSCs contributes to thrombocytopenia after IR.
Deletion of Trp53 in MSCs prevents the myelosuppressive side effects of MDM2i
We next examined whether the myelosuppressive effects of MDM2i are due to p53 levels in MSCs. Mice with specific deletion of Trp53 (Osx-Trp53fl/fl) or Mdm2 (Osx-Mdm2fl/+) in MSCs and control mice with hematopoietic-specific deletion of Trp53 (Vav-Cre;Trp53fl/fl) were treated with DS5272, an orally active murine Mdm2i 22, and peripheral blood and BM samples were analyzed after the last dose of treatment (Figure 3A). Mdm2i treatment resulted in leukopenia in peripheral blood of mice in a p53-dependent manner (Figure 3B). Heterozygous deletion of Mdm2 in MSCs exacerbated the myelosuppressive effect of MDM2i, however, deletion of Trp53 in MSCs (Osx-Cre;Trp53fl/fl) completely reversed the cytopenia associated with Mdm2i treatment (Figure 3B), In fact, p53 deletion in MSCs was equally efficient as deletion of Trp53 in hematopoietic cells (Vav-cre-Trp53fl/fl), suggesting that increased p53 levels in MSCs contributed to hematopoietic toxicities of MDM2i therapy. Analysis of BM samples revealed moderate cytopenia in Mdm2wt mice and striking cytopenia in Osx-Mdm2fl/+ mice after MDM2i treatment (Figure 3C). Importantly, the BM of mice with deletion of Trp53 in MSCs (Osx-Cre;Trp53fl/fl) displayed areas of active hematopoiesis and high cellularity (Figure 3C). The megakaryocytes were completely depleted in Osx-Mdm2fl/+ mice, whereas Osx-Cre;Trp53fl/fl mice displayed a significantly higher number of megakaryocytes in the BM (p < 0.01, n = 3). Collectively, these data demonstrate that p53 levels in MSCs are functionally important in hematopoietic failure after MDM2i therapy.
p53 in MSCs contributes to response to murine MDM2i
Previously, we reported that MSCs derived from AML patients display significantly higher p53 protein levels 16. To determine whether p53 levels in MSCs contribute to the response to MDM2i, we established a traceable syngeneic leukemia model in Osx-Cre;mTmG andOsx-Cre;mTmG;Trp53fl/fl mice and analyzed their survival. Mice were transplanted with leukemia cells originally derived from p53-null mice and transformed by lentivirus-mediated delivery of an oncogenic AML-ETO fusion gene 23 as well as a fluorescent transgene to express turquoise fluorescence (Figure 4A). We validated the Cre activity and engraftment of leukemia cells by fluorescent microscopic analysis of BM isolated from syngeneic AML mice 10 days after transplant (Figure 4B). Mice were treated with vehicle or DS-5272 starting on day 3 after transplant for 10 days. Deletion of p53 in MSCs significantly prolonged mice’s survival beyond discontinuation of therapy (p < 0.003), suggesting that survival of AML cells in response to DS-5272 might depend on p53 levels in MSCs (Figure 4C). Of note, Mdm2 inhibition with nutlin-3a has been shown to disrupt p73-MDM2 interaction in p53-null cells 24. To assess the early molecular pathways in p53-null MSCs after Mdm2 inhibition, we isolated GFP+ cells from Osx-Cre;Trp53fl/fl mice (p53 null) and determined the gene expression profiles of vehicle- or nutlin-treated cells after 24 hours (Figure 4D). RNA-Seq and subsequent Ingenuity Pathway Analysis of p53null MSCs revealed upregulation of genes involved in glycolysis as well as Hif-1α signaling (Figures 4E-4G). The top seven pathways identified by the upregulated differentially expressed genes are illustrated in Figure 4G. Mdm2 inhibition induced Slc2a1 (Glut1), Pdk1, and Fgfr3 genes, known to promote osteoblast differentiation 25, 26, 27. Scd2 (stearoyl-CoA desaturase 2) plays a role in the regulation of energy metabolism and lipid synthesis and was significantly upregulated by Mdm2 inhibition. The top upstream activated regulator was CD38, a key cellular metabolic driver of aging 28 (Figure 4H). In addition to CD38, Hif-1, Egln, IL5, and IL15 were enriched as upstream regulators of genes induced in p53-null MSCs treated with Mdm2i. These expression changes suggest that Mdm2 inhibition might promote osteoblast differentiation of p53-null MSCs partly through metabolic pathways.
Heterozygous deletion of Mdm2 in Osx-Trp53fl/fl mice results in osteosclerosis and myelofibrosis
To determine the direct effect of Mdm2 inhibition in Osx-Trp53fl/fl mice, we interbred progeny from Osx-Mdm2+/fl and Trp53fl mice with mTmG reporter mice to generate Osx-Cre;Mdm2+/fl;Trp53fl/fl;mTmG mice (referred to hereafter as Osx-Mdm2+/fl;Trp53fl/fl). This enabled us to image the population of MSCs (GFP+) irrespective of their cell surface markers that change upon their differentiation. As expected, homozygous deletion of Trp53 reversed the prenatal lethal phenotype of Osx-Mdm2fl/fl mice, and pups were born at Mendelian ratios without an obvious bone phenotype (data not shown). Unlike Osx-Mdm2+/fl mice, in which we had observed less trabecular bone volume, Osx-Mdm2+/fl;Trp53fl/fl mice displayed trabecular bone formation mainly due to trabecular ossifications, resulting in a sponge-like network of trabecular bone (Figures 5A-5C). Bone densitometric analysis confirmed a sclerotic trabecular bone phenotype in the Osx-Mdm2+/fl;Trp53fl/fl mice, accompanied by significant increase in trabecular bone volume (Figures 5C-5D). Bone histomorphometric further confirmed the ossification of trabecular bones in Osx-Cre;Mdm2+/fl ;Trp53fl/fl mice (Figure 5E). Osx-Mdm2+/fl;Trp53fl/fl mice became moribund as they aged and died of BM failure. Histopathologic analysis of the BM in Osx-Mdm2+/fl;Trp53fl/fl mice revealed massive endochondral bone formation that reduced the effective marrow space by approximately 80%. The architecture of the growth plate displayed typical chondrocytes with regular proliferating and hypertrophic zones. However, osseous trabeculae with new bone formation were present throughout the epiphysis, metaphysis, and diaphysis of the long bones as well as vertebrae (Figure 5F). The diaphyseal cortical bone diameter was increased and coalesced with the subjacent trabecular bone. Histologic examination by reticulin staining showed widespread reticulin positivity in the BM reminiscent of myelofibrosis (Figure 5G). Of note, the trabecular bone volume in mice with deletion of p53 in MSCs, Osx-cre;Trp53fl/fl, was comparable with that of p53 wild-type mice, suggesting that the observed phenotype in Osx-Mdm2+/fl;Trp53fl/fl mice was due to decreased levels of Mdm2 (Supplementary Figures S2B and S2C).
Next, we sought to determine whether the sclerotic BM was derived from MSCs. Since the sclerotic bones were densely mineralized, we decided to perform immunohistochemistry for GFP in the BM sections isolated from Osx-Cre;mTmG;Mdm2+/fl;Trp53fl/fl mice. As shown in Supplementary Figure S2D, the sclerotic BM was GFP positive, suggesting that the sclerotic trabecular bones were derived from MSCs. Collectively, these data demonstrate that genetic depletion of Mdm2 in MSCs lacking p53 promotes osteoblast differentiation leading to lethal osteosclerosis.