Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Exp Hematol. 2019 Oct 24;79:3–15.e4. doi: 10.1016/j.exphem.2019.10.003

KRasG12D expression in the bone marrow vascular niche affects hematopoiesis with inflammatory signals

Cindy L Hochstetler 1,2, Yuxin Feng 1, Mehmet Sacma 3, Ashley K Davis 1, Mahil Rao 4, Chia-Yi Kuan 5, Li-Ru You 6,7, Hartmut Geiger 1,3, Yi Zheng 1,2,*
PMCID: PMC6921092  NIHMSID: NIHMS1542136  PMID: 31669153

Abstract

The bone marrow (BM) niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained. Previous studies have shown that genetic mutations in various components of the niche can affect hematopoiesis and promote hematologic abnormalities, but the impact of abnormal BM endothelial cells (BMECs), a crucial niche component, on hematopoiesis remains incompletely understood. To dissect how genetic alterations in BMECs could affect hematopoiesis, we have employed a novel inducible Tie2-CreERT2 mouse model, with a tdTomato fluorescent reporter, to introduce an oncogenic KRasG12D mutation specifically in the adult endothelial cells. Tie2-CreERT2;KRasG12D mice had significantly more leukocytes and myeloid cells in the blood with mostly normal BM HSPC populations and developed splenomegaly. Genotyping PCR found KRasG12D activation in BMECs but not hematopoietic cells, confirming that the phenotype is due to the aberrant BMECs. Competitive transplant assays revealed that BM cells from the KRasG12D mice contained significantly lower functional hematopoietic stem cells (HSCs) and immunofluorescence imaging showed that HSCs in the mutant mice were localized farther away from BM vasculature and closer to the endosteal area. RNA-seq analyses found an inflammatory gene network, especially TNFα, as a possible contributor. Together, our results implicate an abnormal endothelial niche in compromising normal hematopoiesis.

Keywords: KRasG12D, Endothelial Cells, HSPCs

Germ line or acquired mutations in genes of the Rasopathy pathway are often associated with hematologic abnormalities and patients that harbor such mutations are at an increased risk of developing blood disorders1. In fact, in juvenile myelomonocytic leukemia (JMML), Rasopathy mutations have been implicated in ~85% clinical cases, with a Ras mutation being present in about 25% of all cases2–6. Mutations in Kras, Nras, Nf1, and Ptpn11 can promote cellular transformation and lead to the development of myeloproliferative disorders (MPD) and/or myelodysplastic syndromes (MDS)7, 8 that affect hematopoietic cell output. A study using the interferon-inducible Mx1-Cre mouse model to overexpress Ptpn11 in the hematopoietic stem and progenitor cells (HSPCs) led to the development of a fatal MPD and anemia9. Loss of heterozygosity in the Nf1 tumor suppressor gene is associated with JMML. The transplantation of fetal Nf1−/− HSPCs into recipient mice led to an MPD phenotype that mimicked human JMML, suggesting that such mutations can be causal for the clonal expansion of aberrant HSPCs in JMML10. Significantly, oncogenic KRasG12D expression resulted in a lethal myeloproliferative disorder with 100% penetrance11. These studies suggest that deregulated Ras signaling can promote hematologic abnormalities1.

HSPCs, which are responsible for hematopoietic cell production, are maintained in a specialized bone marrow microenvironment, the niche12, 13. Anatomically and functionally defined, the bone marrow niche constitutes a home where HSPCs reside and their balance between self-renewal and differentiation is governed by a host of cell intrinsic and extrinsic factors14–17. The existence of several niche components, including the osteoblastic18, 19 and vascular cells20, has been well documented. These niche cells help maintain and regulate the hematopoietic landscape within the bone marrow and in peripheral blood in a dynamic fashion. Different models have been proposed regarding how niche abnormalities may contribute to blood disease pathogenesis21. Several groups have shown that genetic mutations in the BM niche cells can lead to the development of myeloproliferative disorders. Mice without retinoic acid γ or with inactivated retinoblastoma protein in their hematopoietic system develop MPD22, 23, whereas the deletion of the endonuclease Dicer1 in mesenchymal osteoprogenitors initiated an MDS-like disease24. The activation of mutated Ptpn11 in Nestin+ cells resulted in increased myeloid progenitors in the bone marrow and increased myeloid cells in the blood25. These studies have thus shown that blood abnormalities can be derived from niche anomalies. However, while it is increasingly recognized that bone marrow endothelial cells (BMECs) act as a major niche component required for HSPC interaction26–29, the functional and mechanistic relationship between an abnormal endothelial niche and blood pathogenesis remains unclear.

In this study, we crossed previously described inducible Tie2Cre-ER mice30 with mice bearing the conditional lox-stop-lox KRasG12D mutation to specifically introduce a KRasG12D mutation into adult endothelial cells. We also introduced a fluorescent tdTomato reporter to track our genetic mutation. We describe how such a mutation in the vascular niche affects normal hematopoiesis. The mutant mice displayed splenomegaly and decreased HSC reconstitution potential. Molecularly, we observed a surprising inflammatory signature in the BMECs expressing KRasG12D. Our findings demonstrate that an altered BM vascular niche can change HSC function and implicate that oncogenic KRas may elicit an inflammatory response in the BMECs, contributing to hematologic abnormalities.

Methods

Animals

All animal work was performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Medical Center. To induce Cre expression, male and female mice at the adult stage (6–10 weeks of age) were injected with Tamoxifen (Cayman Chemical 13258) for 4 consecutive days. More details are available in the supplemental materials.

Cell Preparation

BM cells were harvested from femurs, tibiae, and ileac crests. The bones and spleens were flushed and/or crushed, filtered, and single cell suspensions were made. For cell sorting, the BMECs were flushed with HBSS, 2% fetal bovine serum, and 2mM EDTA.

Data Availability

RNA sequencing data from the sorted CD31+ tdT+ BMECs has been deposited to the National Center for Biotechnology Information’s Gene Expression Omnibus (GSE137649).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism software. Unless otherwise specified, unpaired Student’s t-test and a normal analysis of variance were used. Data were expressed as the mean ± the standard error of the mean. A p value of <0.05 was considered statistically significant.

Additional methods are described in the Supplemental Materials.

Results

KRasG12D expression in the murine bone marrow microenvironment causes an MPD-like phenotype and lethality

To determine the effect of an oncogenic gene mutation on the hematopoietic system, we used an Mx1-Cre; KRasG12D mouse model. The mutant mice showed a drastic increase in total white blood cell counts (Supplementary Figure 1A). They had increased neutrophil and monocyte counts (Supplementary Figures 1B and 1C), but no change in lymphocyte counts (Supplementary Figure 1D). Overall, they had more myeloid cells, especially in the neutrophil population (Supplementary Figures 1E and 1F), with a concurrent decrease in lymphocyte percentage (Supplementary Figure 1G). Blood smears confirmed the presence of more leukocytes and polychromasia cells (Supplementary Figures 1H and 1I). The mutant mice also had reduced hemoglobin (Supplementary Figure 1J), and platelets (Supplementary Figure 1K). These observations are consistent with previously published results of KRasG12D expression in the broad hematologic compartments leading to myeloproliferative disorders (MPD)31.

To examine the effect of KRasG12D expression in the BM microenvironment on hematopoiesis, we transplanted whole BM cells from syngeneic WT CD45.1 BoyJ cells into lethally irradiated adult CD45.2 Mx1-Cre;KRasG12D (WT:KRas) or Mx1-Cre;KRasWT (WT:WT) recipients (Fig. 1a). Genotyping PCR analysis of the peripheral blood cells after poly I:C injection of KRasG12D recipient mice showed the absence of activated KRasG12D, suggesting that the observed effect was due to KRasG12D expression in the non-hematologic lineage microenvironment (Fig. 1b). A Kaplan-Meier log-rank test showed that WT:KM mice all died within 100 days after transplantation whereas all the WT:WT recipients remained alive at the end of the period (Fig. 1c). Five weeks post transplantation, blood composition was found unchanged in the WT:WT recipients, but the KRas:WT mice rapidly developed a myeloproliferative phenotype, as evidenced by the increased percentage of neutrophils in the blood (Fig. 1d) and concurrent decrease in the percentage of lymphocytes (Fig. 1e). We also observed rapid anemia and thrombocytopenia onsets in the WT:KRas mice, as they had significantly lower counts of red blood cells (Fig. 1f), hemoglobin (Fig. 1g), and platelets (Fig. 1h). Thus, KRasG12D expression in the BM microenvironment is sufficient to cause an MPD-like phenotype and lethality.

Fig. 1. Inducible KRasG12D expression in the BM microenvironment causes an MPD-like phenotype.

Fig. 1

Open in a new tab

a Five million whole BM cells from WT mice were transplanted into lethally irradiated MxCre;KRasG12D or control mice. The recipients were injected with pIpC 1–2 weeks later to induce KRasG12D activation. b Genotyping data shows the presence of only WT allele in the mononuclear blood cells of WT:WT and WT:Kras transplanted mice five weeks after transplantation. KRasG12D lane: mononuclear cells from MxCre;KRasG12D mice prior to transplant; WT:KRas lane: WT donor transplanted to a MxCre;KRasG12D recipient; WT:WT lane: WT donor transplanted into a WT recipient. c Survival curve depicts faster death of WT:KM recipients. Complete blood counts by Hemavet (Drew Scientific) shows an increase in the percentage of d neutrophils with concurrent decrease in e lymphocytes, as well as f-g anemia development and h decreased platelet counts. n=6–12. *p<0.05, **p<0.01, and ***p<0.001.

An inducible, endothelial cell specific KRasG12D expressing mouse model

The effects of genetic mutations in BM niche components have been intensively studied recently and it has been shown that these mutations can induce hematologic malignancies24, 25. Endothelial specific knockout of SCF or CXCR4 has also been shown to alter the BM microenvironment for HSC maintenance32, 33. To investigate how a genetic mutation, such as KRasG12D, when restricted in BMECs, may alter the BM microenvironment and possibly affect hematopoiesis, we utilized a recently generated inducible Tie2-CreERT2 mouse model allowing expression of genes selectively in adult mice ECs. This mouse model is highly efficient in response to Tamoxifen injection to express Cre activity only in the endothelial lineage, not blood lineages34. Six to ten weeks old adult Tie2-CreERT2;KRasG12D mice, thereby denoted as KRasG12D mice, and control Tie2-CreERT2;KRasWT, denoted as WT mice, were injected with TAM. This led to specific activation of oncogenic KRas in endothelial cells (Fig. 2a). We tracked tdTomato (tdT) fluorescent reporter to confirm the specific activation of KRasG12D in endothelial cells. Immunofluorescent staining showed that the fluorescent reporter was strictly confined in the vasculature which expressed endothelial marker CD31 (Fig. 2b–d), and co-localized with the endothelial marker Ve-Cadherin, VEGFR2, and DiI-Ac-LDL labeled BM sinusoids35 (data not shown). Immunofluorescent staining of flushed BM showed similar vascular expression of tdT marker in WT and mutant mice (Fig. 2e–f). FACS analysis also showed no difference in the CD31+ tdT+ cell frequencies in WT and mutant bone marrow or spleen (Fig. 2g–h). To exclude the possibility that the induced phenotype might be due to gene leakage in the hematopoietic system, we seeded BM cells into M3434 Methocult media and isolated the colonies 7–10 days later. Genotyping PCR found activated KRasG12D allele in the CD31+ tdT+ endothelial cells but not in the hematopoietic cells (Fig. 2i–k). We thus utilized this new mouse model to study the effect of the KRasG12D mutation in BMECs on hematopoiesis.

Fig. 2. Inducible expression of KRasG12D in the BM endothelial cells of the Tie2-CreERT2 mouse model.

Fig. 2

Open in a new tab

a Schematic representing the model used. b-d Immunofluorescent staining to determine the expression of CD31 in tdT+ BM after Tamoxifen induction. b CD31 staining, c tdT, and d overlay of CD31 and tdT are shown. e-f Confocal microscopy detected no obvious change in the BM vasculature. g-h FACS analysis measuring CD31+ tdT+ cell frequency in the g BM and h spleen (n=5) of the Tie2-CreERT2 mice. i Genotyping PCR found no KRasG12D activation in hematopoietic cells assayed from colonies of whole bone marrow cells. j-k KRasG12D activation is shown in j freshly sorted CD31+ tdT+ lung endothelial cells and k in sorted CD31+ tdT+ BM endothelial cells.

Endothelial cell specific KRasG12D expression leads to leukocytosis and myeloid bias

Following TAM injection, retro-orbital bleeds were performed monthly to assess blood composition. The KRasG12D mice develop leukocytosis, as the absolute counts of both myeloid and lymphoid cells increased (Fig. 3a–d). FACS analysis showed a myeloid vs. lymphoid cell bias as evidenced by an increase in the percentage of myeloid cells (Fig. 3e–g) and a decrease in the percentage of lymphocytes (Fig. 3h).

Fig. 3. Endothelial KRasG12D expression leads to leukocytosis and myeloid bias.

Fig. 3

Open in a new tab

Complete blood counts revealed that KRasG12D mice had higher counts of a leukocytes, b neutrophils, c monocytes, and d lymphocytes. FACS analysis of the blood also showed an increase in the percentage of e total myeloid, f neutrophils, and g monocytes, with concurrent decrease in h lymphocytes. n≥5. *p<0.05, and **p<0.01.

To further ensure the Tie2-CreERT2;KRasG12D model limited KRasG12D expression in the endothelia, we transplanted WT CD45.1 whole BM cells into lethally irradiated CD45.2 KRasG12D (WT:KRas) or KRasWT (WT:WT) recipients, and injected the recipient mice with TAM after engraftment (Supplementary Figure 2A). Similar to the native Tie2-CreERT2;KRasG12D model, the KRasG12D recipients developed splenomegaly (Supplementary Figure 2B). At 16 weeks post-transplant, the WT:KRas mice had a significantly higher percentage of myeloid cells, especially neutrophils, and a lower percentage of lymphoid cells (Supplementary Figure 2C–F). Furthermore, the KRas:WT recipients showed signs of anemia, as evidenced by a lower hemoglobin count (Supplementary Figure 2G).

To determine the effects on blood composition in KRasG12D mice, we performed FACS analysis to assess the different progenitor compartments in the BM. Our results showed that most HSPCs appeared normal. There were no major changes within the different LK (Lin- Sca+ cKit-) and LSK (Lin- Sca+ cKit+) compartments (Fig. 4a–b). In addition, colony-forming assays showed that the BM of KRasG12D mice had a slightly increased colony forming activity (Fig. 4c). Pathology examination of the BM by H&E saw no major changes (Supplementary Figure 3A–B). These results indicate that endothelia expression of KRasG12D does not significantly affect HSPC populations and activities in the BM compartments.

Fig. 4. Endothelial KRasG12D expression does not significantly alter the HSPCs in the BM but promotes extramedullary hematopoiesis.

Fig. 4

Open in a new tab

FACS analysis of the BM progenitor cells showed a no major change in the LK or b LSK compartments. c Colonies were scored 7–10 days after total bone marrow cells were plated in Methocult. n≥10. d KRasG12D mice had enlarged spleens (n=3–4). FACS analysis of spleen progenitor cells showed e expansion of the GMP compartment and a small increase in the f ST-HSC compartments. g Colonies were scored 7–10 days after spleen cells were plated in Methocult. KRasG12D mice had an increased colony forming potential. n≥10. *p<0.05, and **p<0.01.

On the other hand, the KRasG12D mice showed clear splenomegaly (Fig. 4d). Endothelial KRasG12D expression altered the hematopoietic composition in the spleen, as the spleens of mutant mice had an increased percentage of myeloid cells (Supplementary Figure 3C), especially neutrophils (Supplementary Figure 3D), with a concurrent decrease in lymphoid cell percentage. However, there was no change in the lymphoid cell numbers (data not shown). FACS analysis further showed a significant expansion of the GMP population (Fig. 4e) and a small increase in the ST-HSC population (Fig. 4f). CFU assays found that the KRasG12D spleens had greater colony forming activity as evidenced by a significantly increased number of colony forming units (Fig. 4g). The CFU activity from the blood of KRasG12D mice was also significantly increased (Supplementary Figure 3E), but it is not clear whether GMP and ST-HSC populations are responsible for the increased colony numbers. Gross examination of the spleen by H&E revealed a slight disruption of the splenic architecture with a modest broadening of the white pulp regions (Supplementary Figure 3F–G). These results suggest that endothelial KRasG12D expression alters extramedullary hematopoiesis in the spleen, which may contribute to the observed myeloid bias and leukocytosis phenotypes.

Endothelial KRasG12D reduces HSC function and alters their BM vascular localization

To determine if there were functional consequences of HSCs residing in an abnormal BM vascular niche, we performed a competitive transplantation experiment followed by a secondary transplantation. At 13 weeks post TAM injection, we transplanted 2.5 million whole BM cells from pooled CD45.2 KRasG12D (KRas:WT) or KRasWT (WT:WT) mice, along with 2.5 million host supportive cells into lethally irradiated CD45.1 syngeneic BoyJ primary recipients (Fig. 5a). These mice were then bled monthly to assess donor chimerism. FACS analysis revealed that the KRas:WT mice had significantly less overall CD45.2% donor chimerism (Fig. 5b) and in all blood lineages analyzed (Fig. 5c–d). To analyze the functionality of the HSCs, 5 months after the primary transplantation, the BM cells from the recipients were transplanted into lethally irradiated CD45.1 secondary recipients. FACS analysis showed a similar effect as in the primary transplant recipients: 5 months post transplant, the KRas:WT recipients had significantly reduced overall donor chimerism (Fig. 5e), and in all blood lineages analyzed (Fig. 5f–g).

Fig. 5. Endothelial KRasG12D expression reduces HSC function.

Fig. 5

Open in a new tab

a Schematic for competitive transplant model. 2.5 million whole BM cells were transplanted from pooled KRasG12D or WT CD45.2 donors injected with TAM into lethally irradiated syngeneic BoyJ recipients. 2.5 million CD45.1 cells were also used as competitor cells. Five months later, 3 million cells from pooled donors were transplanted to lethally irradiated syngeneic BoyJ recipients in a non-competitive setting. Compared to WT:WT, primary KM:WT recipients had b significantly overall lower CD45.2% donor chimerism and in all blood lineages analyzed, namely c Mac1+Gr1+, and d B220. Compared to WT:WT, secondary KM:WT recipients had e significantly overall lower CD45.2% donor chimerism and in all blood lineages analyzed, namely f Mac1+Gr1+, and g B220. n=5–7. *p<0.05, **p<0.01, and ***p<0.001.

To determine if the observed phenotypic and functional abnormality of the HSCs is reflected in their BM niche localization, we examined the endogenous HSCs by immunofluorescence in the native bones harvested 13 weeks post TAM injections. The HSCs in mutant mice were seen localized more distal to the blood vessels (Fig. 6a–d). Quantification revealed that the HSCs of mutant mice were located closer to the endosteal area (Fig. 6e) but were significantly further away from the vasculature (Fig. 6f).

Fig. 6. Endothelial KRasG12D alters HSC localization in the BM.

Fig. 6

Open in a new tab

a-d Immunostaining showed that the mice expressing endothelial KRasG12D had less long-term stem cells closer to blood vessels. Quantification showed that in mice expressing endothelial KRasG12D, the HSCs were located slightly closer to the e endosteal area, but significantly further away from f the vasculature. **p<0.01.

KRasG12D expressing BM endothelial cells gain an inflammatory signature

Since a change in the BM endothelial niche may alter the microenvironment in which HSCs reside, we asked how the BMECs have modified the immediate niche component that may affect the hematopoietic landscape. Thirteen weeks post TAM injection, WT and mutant mice were euthanized and their BM cells harvested. RNA was obtained from CD31+ tdT+ sorted cells and RNA-seq was performed. The genes were grouped and differentially expressed pathways were obtained using EGSEA36 (Fig. 7a). Gene set enrichment analysis (GSEA) showed a list of the differentially expressed genes (Fig. 7b). The enrichment plots showed that the cytokine-cytokine receptor interaction pathway and the TNFα signaling pathways were among the most significantly enriched (Fig. 7c–e). To further validate these results, CD31+ tdT+ cells in the BM were analyzed by FACs and Western blotting for the TNFα level. The KRasG12D mice contained significantly higher TNFα protein in their BMECs (Fig. 7f–g). Interestingly, p-ERK and p-Akt levels in the isolated KRasG12D-expressing BMECs did not show significant differences from those of the WT samples (Fig. 7h), suggesting the growth promoting MAPK pathway was not altered in the BMECs. Consistently, the mutant BM vasculatures appeared normal compared with that of WT mice (Fig. 2e–f), indicating that even under the influence of a strong genetic mutation such as KRasG12D, the BM vasculatures strive to retain normal angiogenic activity.

Fig. 7. KRasG12D BM endothelial cells contain increased inflammatory signatures.

Fig. 7

Open in a new tab

BMECs were sorted on the basis of CD31+ tdT+ signals. a After sequencing, differentially expressed pathways were determined with EGSEA. n=4. b Using GSEA software, the most differentially expressed genes were made into a heat map. c-d Enrichment plots were then generated. e Heat map showing the differentially expressed genes within the TNFα pathway. BM cells were harvested and using FACS, CD31+ tdT+ cells were assessed for TNFα levels by f flow cytometry (n=5) and g Western blot. The TNFα levels were normalized to WT. h Phospho-ERK and phospho-AKT levels were also assessed. *p<0.05.

Discussion

HSCs are maintained in a tightly regulated BM niche that contains balanced secreted signals such as cytokines and chemokines. The secretory signals can be modulated by a change in the niche components. Multiple studies using the interferon-inducible Mx1-Cre model have shown that an oncogenic KRas mutation in the hematopoietic system could lead to leukocytosis and a phenotype that is reminiscent of MPD/MDS11, 37. To interrogate the contributions of the BM niche components, mutations were introduced in cells of the osteoblastic lineage18, 24, and mesenchymal stroma cells25, 38. These studies found that oncogenic mutations such as Dicer and Shp2 in these niche cell types are sufficient to cause hematologic malignancies. In our study, WT mouse BM transplantation into Mx1-Cre;KRasG12D mice caused increased myeloid cell bias with decrease in lymphoid cells. Consistent with what has previously been shown by Staffas et al31, our Mx1-Cre;KRasG12D recipient mice with WT blood cells had significantly less hemoglobin and platelets, and developed MPD/MDS-like syndromes that led to lethality. As germ-line and acquired mutations of the Rasopathy pathway have previously been linked with diverse blood disorders39, 40, we seek to understand how an initiating oncogenic KRasG12D mutation in the BMECs alone may affect the normal hematopoietic process. An aberrant BM vascular niche expressing genetic mutations such as KRasG12D led to increased leukocytosis with a myeloid vs. lymphoid differentiation bias, which is reminiscent of MPD/MPN. At later time points of our model, this phenotype is even more pronounced, and the mice also develop anemia (data not shown). However, we did not observe a transformation to leukemia, suggesting that a hit such as KRasG12D in the bone marrow endothelial microenvironment is not enough for full transformation.

Recent studies have demonstrated that HSCs and progenitors mostly reside close to the BM vasculature, specifically in the perisinusoidal niche41, which are the exclusive sites for HSPC migration from the BM42. Our HSC/BMEC co-imaging data showed that HSCs of KRasG12D mice were located further from the vasculature but closer to the endosteum. Such a location may explain the observed reduction in HSC function of the mutant mice. The observed splenomegaly phenotype in the mutant mice could be a result of more egress of immature HSPCs into the circulation, and it is likely that the altered spleen contributed to the increased leukocytosis.

Interestingly, under homeostatic conditions, while the KRasG12D-expressing BM vasculature appeared normal in morphology and the BMECs did not bear detectable change in canonical MAPK signaling, our RNA sequencing analysis revealed that multiple gene expression changes in the KRasG12D-expressing BMECs occur, with a significant increase in the inflammatory signatures, especially in the TNFα pathway. The cytokine-cytokine receptor interaction network was also significantly enriched. We speculate that the increased TNFα secretion from the mutant BMECs and the resulting inflammatory microenvironment are contributing factors to the hematopoiesis change observed in the mutant mice. Walkley et al.22 formerly showed that myeloid proliferation derived from a microenvironment-induced defect was partially caused by a significant increase in TNFα expression. However, similar to their case, we did not observe any significant increase in other cytokines typically linked with inflammation (data not shown). A more recent study by Zhou et al.43 showed that, in a Fanconi anemia model, WT bone marrow transplantation into Fancc−/−;Fancg−/− double knockout mice resulted in significantly decreased colony forming potential of BM cells but a marked increase in the myeloid cells and their associated progenitors in a co-culture model. This phenotype was accompanied by a significant increase in TNFα concentration levels measured from the supernatant of their mesenchymal stem progenitor cells and adipocytes. This suggests that TNFα can contribute to the expansion of myeloid cells. While the increase in TNFα may partially underlie the BM transplant results which showed a reduced long-term reconstitution capability of the HSCs, it is likely that additional factors in the altered BMEC microenvironment also play important roles as the inflammatory gene changes were more profound. To this end, our results are also consistent with other reports showing that TNFα had inhibitory effects on hematopoiesis and HSPC exposure to extraneous TNFα pre-transplant impaired HSPC reconstitution capability44, 45.

Previous clinical studies have revealed that abnormal niche components can promote blood disorders. A study by Verstovsek et al. found that the bone marrows of patients with primary myelofibrosis (MF) have a large number of abnormal neoplastic fibrocytes and transplantation of these BM cells into immunodeficient mice causes the recipient mice to develop a phenotype that resembles MF46. Furthermore, a study looking at splenic vascular endothelial cells (SVECs) in MF patients showed that those SVECs may contribute to the expansion of MF CD34+ cells47. Additionally, when BM samples were collected from patients with aplastic anemia (AA), researchers found that the BM mesenchymal cells from the AA patients had an abnormal morphology and appeared “ragged”48. These studies thus strongly support the concept that an abnormality in the niche can trigger changes in hematopoiesis.

Taken together, our data provide direct evidence that an alteration in BMECs can abrogate normal hematopoiesis, affecting HSC function, and myeloid vs. lymphoid lineage balance. As some Ras pathway inhibitors have shown promising results in animal models, our studies suggest BMECs affected by genetic mutations can be therapeutic targets. Significantly, oncogenic mutations in BMECs may promote an inflammatory surrounding that contributes to blood pathogenesis.

Supplementary Material

1

Highlights.

  1. Endothelial KRasG12D expression affects normal hematopoiesis with a myeloid bias.

  2. Endothelial KRasG12D causes reduced HSC function in a competitive transplant setting.

  3. KRasG12D in endothelial cells elicits an inflammatory signal in the bone marrow niche.

Acknowledgments

We thank the Flow Cytometry Core, the Comprehensive Mouse and Cancer Core, and the Pathology Research Core at Cincinnati Children’s Hospital Medical Center for their technical assistance. We also thank James Johnson for technical assistance and Feng Zhang for assistance to obtain expression counts. This work was supported in part by a Cancer Free Kids grant, a National Institute of Environmental Health Sciences T32ES007250 training grant and a National Heart, Lung, and Blood Institute F31HL136229 training grant to C.L.H, National Institutes of Health grants RO1 DK104814, R01 CA204895 and R01 HL134617 to Y.Z.

Footnotes

Conflict of Interest Disclosure

The authors have no conflict of interest to disclose.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Lauchle JO, Braun BS, Loh ML, Shannon K. Inherited predispositions and hyperactive Ras in myeloid leukemogenesis. Pediatr Blood Cancer 2006; 46: 579–85. [DOI] [PubMed] [Google Scholar]
  • 2.Side L, Taylor B, Cayouette M, Conner E, Thompson P, Luce M, et al. Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders. N Engl J Med 1997; 336: 1713–20. [DOI] [PubMed] [Google Scholar]
  • 3.Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003; 34: 148–50. [DOI] [PubMed] [Google Scholar]
  • 4.Flotho C, Valcamonica S, Mach-Pascual S, Schmahl G, Corral L, Ritterbach J, et al. RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML). Leukemia 1999; 13: 32–7. [DOI] [PubMed] [Google Scholar]
  • 5.Kalra R, Paderanga DC, Olson K, Shannon KM. Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras. Blood 1994; 84: 3435–9. [PubMed] [Google Scholar]
  • 6.Loh ML, Reynolds MG, Vattikuti S, Gerbing RB, Alonzo TA, Carlson E, et al. PTPN11 mutations in pediatric patients with acute myeloid leukemia: results from the Children’s Cancer Group. Leukemia 2004; 18: 1831–4. [DOI] [PubMed] [Google Scholar]
  • 7.Janssen JW, Steenvoorden AC, Lyons J, Anger B, Bohlke JU, Bos JL, et al. RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes. Proc Natl Acad Sci U S A 1987; 84: 9228–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.MacKenzie KL, Dolnikov A, Millington M, Shounan Y, Symonds G. Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice. Blood 1999; 93: 2043–56. [PubMed] [Google Scholar]
  • 9.Chan G, Kalaitzidis D, Usenko T, Kutok JL, Yang W, Mohi MG, et al. Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis. Blood 2009; 113: 4414–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ingram DA, Wenning MJ, Shannon K, Clapp DW. Leukemic potential of doubly mutant Nf1 and Wv hematopoietic cells. Blood 2003; 101: 1984–6. [DOI] [PubMed] [Google Scholar]
  • 11.Braun BS, Tuveson DA, Kong N, Le DT, Kogan SC, Rozmus J, et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci U S A 2004; 101: 597–602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978; 4: 7–25. [PubMed] [Google Scholar]
  • 13.Trentin JJ. Determination of bone marrow stem cell differentiation by stromal hemopoietic inductive microenvironments (HIM). Am J Pathol 1971; 65: 621–8. [PMC free article] [PubMed] [Google Scholar]
  • 14.Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004; 118: 149–61. [DOI] [PubMed] [Google Scholar]
  • 15.Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007; 131: 324–36. [DOI] [PubMed] [Google Scholar]
  • 16.Himburg HA, Harris JR, Ito T, Daher P, Russell JL, Quarmyne M, et al. Pleiotrophin regulates the retention and self-renewal of hematopoietic stem cells in the bone marrow vascular niche. Cell Rep 2012; 2: 964–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 2006; 441: 518–22. [DOI] [PubMed] [Google Scholar]
  • 18.Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003; 425: 841–6. [DOI] [PubMed] [Google Scholar]
  • 19.Xie Y, Yin T, Wiegraebe W, He XC, Miller D, Stark D, et al. Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 2009; 457: 97–101. [DOI] [PubMed] [Google Scholar]
  • 20.Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005; 121: 1109–21. [DOI] [PubMed] [Google Scholar]
  • 21.Raaijmakers MH. Niche contributions to oncogenesis: emerging concepts and implications for the hematopoietic system. Haematologica 2011; 96: 1041–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Walkley CR, Olsen GH, Dworkin S, Fabb SA, Swann J, McArthur GA, et al. A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency. Cell 2007; 129: 1097–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Walkley CR, Shea JM, Sims NA, Purton LE, Orkin SH. Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 2007; 129: 1081–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA, et al. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature 2010; 464: 852–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Dong L, Yu WM, Zheng H, Loh ML, Bunting ST, Pauly M, et al. Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment. Nature 2016; 539: 304–08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hooper AT, Butler JM, Nolan DJ, Kranz A, Iida K, Kobayashi M, et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 2009; 4: 263–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Doan PL, Russell JL, Himburg HA, Helms K, Harris JR, Lucas J, et al. Tie2(+) bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury. Stem Cells 2013; 31: 327–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Butler JM, Nolan DJ, Vertes EL, Varnum-Finney B, Kobayashi H, Hooper AT, et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 2010; 6: 251–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Gori JL, Butler JM, Chan YY, Chandrasekaran D, Poulos MG, Ginsberg M, et al. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. J Clin Invest 2015; 125: 1243–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Feng YLM, Guo F, Sampson L, You LR, Kuan CY, Zheng Y. Novel method to study mouse bone marrow endothelial cells in vivo and in vitro. . Blood 2012; 120:617. [Google Scholar]
  • 31.Staffas A, Karlsson C, Persson M, Palmqvist L, Bergo MO. Wild-type KRAS inhibits oncogenic KRAS-induced T-ALL in mice. Leukemia 2015; 29: 1032–40. [DOI] [PubMed] [Google Scholar]
  • 32.Ding L, Saunders TL, Enikolopov G, Morrison SJ. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 2012; 481: 457–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Comazzetto S, Murphy MM, Berto S, Jeffery E, Zhao Z, Morrison SJ. Restricted Hematopoietic Progenitors and Erythropoiesis Require SCF from Leptin Receptor+ Niche Cells in the Bone Marrow. Cell Stem Cell 2019; 24: 477–86 e6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Forde A, Constien R, Grone HJ, Hammerling G, Arnold B. Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements. Genesis 2002; 33: 191–7. [DOI] [PubMed] [Google Scholar]
  • 35.Li XM, Hu Z, Jorgenson ML, Slayton WB. High levels of acetylated low-density lipoprotein uptake and low tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) promoter activity distinguish sinusoids from other vessel types in murine bone marrow. Circulation 2009; 120: 1910–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Alhamdoosh M, Law CW, Tian L, Sheridan JM, Ng M, Ritchie ME. Easy and efficient ensemble gene set testing with EGSEA. F1000Res 2017; 6: 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lyubynska N, Gorman MF, Lauchle JO, Hong WX, Akutagawa JK, Shannon K, et al. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice. Sci Transl Med 2011; 3: 76ra27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Arranz L, Sanchez-Aguilera A, Martin-Perez D, Isern J, Langa X, Tzankov A, et al. Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms. Nature 2014; 512: 78–81. [DOI] [PubMed] [Google Scholar]
  • 39.Xu D, Liu X, Yu WM, Meyerson HJ, Guo C, Gerson SL, et al. Non-lineage/stage-restricted effects of a gain-of-function mutation in tyrosine phosphatase Ptpn11 (Shp2) on malignant transformation of hematopoietic cells. J Exp Med 2011; 208: 1977–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Le DT, Kong N, Zhu Y, Lauchle JO, Aiyigari A, Braun BS, et al. Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder. Blood 2004; 103: 4243–50. [DOI] [PubMed] [Google Scholar]
  • 41.Acar M, Kocherlakota KS, Murphy MM, Peyer JG, Oguro H, Inra CN, et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 2015; 526: 126–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Itkin T, Gur-Cohen S, Spencer JA, Schajnovitz A, Ramasamy SK, Kusumbe AP, et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 2016; 532: 323–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhou Y, He Y, Xing W, Zhang P, Shi H, Chen S, et al. An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia. Haematologica 2017; 102: 1017–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Drutskaya MS, Ortiz M, Liepinsh DJ, Kuprash DV, Nedospasov SA, Keller JR. Inhibitory effects of tumor necrosis factor on hematopoiesis seen in vitro are translated to increased numbers of both committed and multipotent progenitors in TNF-deficient mice. Exp Hematol 2005; 33: 1348–56. [DOI] [PubMed] [Google Scholar]
  • 45.Ishida T, Suzuki S, Lai CY, Yamazaki S, Kakuta S, Iwakura Y, et al. Pre-Transplantation Blockade of TNF-alpha-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells. Stem Cells 2017; 35: 989–1002. [DOI] [PubMed] [Google Scholar]
  • 46.Verstovsek S, Manshouri T, Pilling D, Bueso-Ramos CE, Newberry KJ, Prijic S, et al. Role of neoplastic monocyte-derived fibrocytes in primary myelofibrosis. J Exp Med 2016; 213: 1723–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Qiu J, Salama ME, Hu CS, Li Y, Wang X, Hoffman R. The characteristics of vessel lining cells in normal spleens and their role in the pathobiology of myelofibrosis. Blood Adv 2018; 2: 1130–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Li J, Yang S, Lu S, Zhao H, Feng J, Li W, et al. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia. PLoS One 2012; 7: e47764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 2012; 7: 562–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005; 102: 15545–50. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1542136-supplement-1.docx (16.3MB, docx)

Data Availability Statement

RNA sequencing data from the sorted CD31+ tdT+ BMECs has been deposited to the National Center for Biotechnology Information’s Gene Expression Omnibus (GSE137649).

RESOURCES