Loss of De Novo DNA Methylation Causes Expansion of the Mouse Hematopoietic Stem Cell Pool

Abstract Abstract 835 DNA methylation is one of the major epigenetic modifications in the vertebrate genome and is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed a dynamic expression profile of Dnmt3a and Dnmt3b in the hematopoietic system with both enzymes expressed at exponentially higher levels in hematopoietic stem cells (HSCs) compared to progenitors and differentiated cells and hypothesized that some of the unique characteristics of HSCs were epigenetically regulated by Dnmt3a and Dnmt3b. To study this, we crossed Dnmt3a and -3b conditional knock-out (KO) mice to Mx1-cre mice to generate inducible single- and double-KO (dKO) mice. We performed competitive transplantation of HSCs (side-population+c-Kit+Lineage-Sca-1+ = SPKLS) from these mice along with wild-type whole bone marrow competitor and induced deletion of the Dnmt3's in the donor cells by sequential pIpC injections in the wild-type recipients. No dramatic differences were observed in primary recipients in the absence of other hematopoietic perturbation, however when we re-transplanted Dnmt3a- and Dnmt3b-KO HSCs into secondary recipients, they exhibited surprisingly high peripheral blood reconstitution compared to control HSCs (>4-fold increase in engraftment). This was reflected in the bone marrow of these mice with a corresponding >4-fold expansion of the HSC pool (phenotypically defined by any of SPKLS; CD34-Flk2-KLS; CD150+CD48-KLS) with virtually all of these cells being derived from the Dnmt3a- and Dnmt3b-KO donor HSCs. Consistent with a previous study, we observed a decline in functional output of Dnmt3a/3b-dKO HSCs in secondary transplants in terms of peripheral blood chimerism, but surprisingly these mice also exhibited a modest expansion of the HSC pool (∼2-fold), the majority of which were derived from donor Dnmt3a/3b-dKO HSCs. In subsequent tertiary and quaternary transplantation, Dnmt3 single-KO HSCs remained highly superior in peripheral blood engraftment capacity relative to control HSCs and Dnmt3a/3b-dKO HSCs (Figure 1), although the expansion of the HSC pool in all Dnmt3-KOs continued to varying degrees. This enhanced HSC activity appears to be a cell autonomous mechanism as purified Dnmt3-KO SPKLS cells from transplanted mice have much greater hematopoietic colony forming potential in vitro compared to control HSCs on a per cell basis. However the observed HSC expansion does not appear attributable to either enhanced proliferation of Dnmt3-KO HSCs or more resistance to apoptosis. The serially-transplanted Dnmt3-KO HSCs are not overtly transformed, in that the levels of differentiated blood cells are still normal and the mice appear to be healthy. This may be akin to a pre-malignant state seen in human myelodysplastic syndrome. We have performed microarray expression profiling of serially-transplanted Dnmt3-KO HSCs and identified several candidate genes which are currently being investigated as the mechanism for HSC expansion. Our data suggest ablation of de novo DNA methylation in HSCs uncouples normal self-renewal and differentiation. These studies present further evidence for the contribution of epigenetic regulation to stem cell activity and provide a tantalizing link between potential aberrant methylation in HSCs contributing to leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

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DNMT3b’s Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽

10.1182/blood.v112.11.1406.1406 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1406-1406

Author(s):

Matthew J Boyer ◽

Feng Xu ◽

Hui Yu ◽

Tao Cheng

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Peripheral Blood ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b’s function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

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Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice

Blood ◽

10.1182/blood.v118.21.4831.4831 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4831-4831

Author(s):

Stefanie Bugl ◽

Stefan Wirths ◽

R Müller Martin ◽

Märklin Melanie ◽

Tina Wiesner ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Nk Cell ◽

Conflicts Of Interest ◽

Control Group ◽

Early Event ◽

Wild Type ◽

Hematopoietic Stem ◽

Lymphoid Progenitors ◽

Fate Decision

Abstract Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

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PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v128.22.496.496 ◽

2016 ◽

Vol 128 (22) ◽

pp. 496-496

Author(s):

Stefan P. Tarnawsky ◽

Mervin C. Yoder ◽

Rebecca J. Chan

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Wild Type ◽

Hematopoietic Stem ◽

Donor Cells ◽

Stem And Progenitor Cells ◽

Leukemia Relapse ◽

Conditioning Regimens

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

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AMD3100 and G-CSF Synergize To Promote Repopulating Stem Cell Mobilization in Fanca −/− and Fancc −/− Mice.

Blood ◽

10.1182/blood.v108.11.3760.3760 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3760-3760

Author(s):

Anna C. Pulliam ◽

M. Joe Hobson ◽

Fengchun Yang ◽

Hal E. Broxmeyer ◽

D. Wade Clapp

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Chemokine Receptors ◽

Cell Activity ◽

Fold Increase ◽

Phenotypic Analysis ◽

Hematopoietic Stem ◽

Test Cells ◽

Myeloid Progenitors

Abstract Fanconi Anemia (FA) is an autosomal recessive DNA repair disorder clinically characterized by congenital abnormalities, progressive bone marrow (BM) failure and a propensity for malignancies. It is advantageous to bank FA patients’ cells for autologous treatment of pancytopenia, transplantation following chemotherapy or future gene therapy trials. Because the amount of cells able to be collected by BM harvesting is limited and the process is often problematic in children, mobilization of stem cells into the circulation would be an ideal means of collecting repopulating hematopoietic stem and progenitor cells (HSPC). Traditional mobilization protocols utilizing granulocyte colony-stimulating factor (G-CSF) as a mobilizing agent result in poor yields of phenotypic HSPC in FA patients. We have shown that analogous to the human patients with FA, administration of recombinant G-CSF is not sufficient to effectively mobilize myeloid progenitors from the BM of Fancc −/− mice (Broxmeyer et al, JEM, 2005). Antagonists to chemokine receptors, together with G-CSF, can enhance mobilization of murine wild type (WT) HSPC. AMD3100 is a selective antagonist of CXCR4, which is thought to function in the retention of HSPC in the BM. We hypothesized that the combination of AMD3100 and G-CSF would enhance mobilization of HSPC in Fanca −/− and Fancc −/− mice. Peripheral blood was collected to assess the number of myeloid progenitors and HSPC repopulating ability. Myeloid progenitor numbers were assessed using progenitor assays. Either G-CSF or AMD3100 alone resulted in poor mobilization of hematopoietic progenitors. In contrast, administration of AMD3100 and G-CSF together increased the number of colonies of myeloid progenitors up to 18 fold above that of either G-CSF or AMD3100 alone in both Fanca −/− and Fancc −/− mice. The stem cell activity of mobilized peripheral blood cells was evaluated using competitive repopulation assays, in which a common pool of isogenic ‘competitor cells’ and ‘test cells’ from mice of different genotypes or treatment conditions are co-transplanted. Fanca −/− and Fancc −/− cells mobilized with both G-CSF and AMD3100 yielded a 3–4 fold increase in peripheral blood chimerism as compared to the cells mobilized with G-CSF or AMD3100 alone. The latter mice also had a decreased survival. Phenotypic analysis of the peripheral blood of primary recipients confirmed the engraftment of multi-lineage test cells. Transplantation of chimeric bone marrow into secondary recipients maintained the test cell chimerism, confirming that long-term repopulating cells were mobilized. Collectively, these data from two FA murine models demonstrate that the AMD3100 and G-CSF protocol is an effective strategy to mobilize FA deficient repopulating HSPC. This has potential implications for more efficient banking of FA patients’ HSPC for future use or for use in gene transfer protocols.

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Hepatic Leukemia Factor Is An Important Regulator Of Hematopoietic Stem Cell Activity and Identity

Blood ◽

10.1182/blood.v122.21.2410.2410 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2410-2410

Author(s):

Karolina Komorowska ◽

Hanna K.A Mikkola ◽

Jonas Larsson ◽

Mattias Magnusson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Cell Activity ◽

Hematopoietic Stem ◽

Fold Reduction ◽

Stem Cell Activity

Abstract The transcription factor Hepatic Leukemia Factor (HLF) was originally identified in a chromosomal translocation with the gene E2A causing a subset of childhood B-lineage acute lymphoid leukemia. Moreover, HLF has been described as a regulator of circadian rhythm and recent findings have implicated HLF as a candidate “stemness” gene in both normal and malignant stem cells. Accordingly, overexpression of HLF in human hematopoietic stem cells (HSC) results in an enhanced reconstitution capability in NOD-SCID mice. However, little is known about HLF’s physiological role in hematopoiesis and HSC regulation. Using quantitative PCR, we found that HLF is highly expressed in mouse (C57Bl/6) HSC and is downregulated upon differentiation (HSC 3.2 (±0.95) fold (p<0.001), LSK 1.9 (±0.47) fold (p<0.05), CMP, GMP MEP all less then 0.1 fold, all values are compared to HPRT). This encouraged us to further investigate HSC function in the absence of HLF. The conventional HLF knockout (KO) mice (C57bl/6 background) were viable, born at normal Mendelian ratios and showed normal hematopoietic parameters (bone marrow cellularity: WT 2.7x107 (±5.4 x106), KO 3.3x107 (±6.4 x106), p>0.2 n=9). In addition, the HLF KO mice demonstrated normal lineage distribution of both mature cells in the peripheral blood and bone marrow as well as the frequency of immunophenotypic HSC (Lin-Sca1+ckit+CD34-Flt3-: WT 0.0005 (±0.5x10-4)%, KO 0.0005 (±0.1x10-3)%; n>10). However, in a serial competitive transplantation assay using whole bone marrow (200 000 cells 1:1 ratio), HLF KO cells demonstrated a significant reduction in reconstitution capacity in primary recipients (WT 56 (±15)%, KO 40.2 (±16)%, p=0.028, n>10), which was further increased in the secondary recipients (WT 87.2 (±26)%, KO 8.7 (±5.8)%, p<0.001, n>10). Almost no engraftment was detected from the HLF KO cells in tertiary recipients. To further evaluate stem cell activity in the absence of HLF, we next enumerated the number of competitive repopulating units (CRU) by limiting dilution assay, which revealed a 2.6 fold reduction, of CRU in the HLF KO mice compared to WT controls (WT 1.6 (±0.4)/105 bone marrow cells, KO 0.6 (±0.2)/105 bone marrow cells). Similarly, transplantation of sorted HSC (Lin-Sca1+ckit+CD34-Flt3-) also showed a 2.4 fold (WT 47.3 (±24)%, KO 19.4 (±25)%, p=0.16, n=9) reduced engraftment of total cells but with enhanced T cell frequency in peripheral blood (WT 19.5 (±6.2)%, KO 40.8 (±7.4)%, p=0.01, n=9). Since we also found that HLF was highly expressed in fetal liver derived HSC, we transplanted fetal liver HLF KO cells from E14.5 in a competitive repopulation setting. In line with the phenotype seen in the adult HLF KO mice, the fetal liver HLF KO cells demonstrated impaired reconstitution ability (WT 52.8 (±16)%, KO 0.9 (±1.4)%, n>10). Intriguingly, the phenotype was stronger than in the adult HLF KO HSC, indicating that HLF is particularly important during the expansion phase of HSC in embryonic development. The underlying mechanism of the reduced HSC activity is still unclear, but preliminary findings show that HLF KO HSC have enhanced ROS levels (WT 337 (±33), KO 510 (±55), p<0.05, n=3) and increased cycling HSC (G0: WT 66.5 (±6.4)%, KO 58.5 (±4.7)%; G1/S/G2/M: WT 33.6 (±6.6)%, KO 41.7 (±4.9)%, n=3). We are currently performing global gene expression analysis to further understand the mechanism of HLF in HSC regulation. Interestingly, we also found that HLF appears to regulate the identity of HSC by modulating the expression of the SLAM code on the cell surface of the HLF KO HSC. In contrast to the normal frequency of LSK Flt3-CD34- cells, the HLF KO mice displayed a 3.5 fold reduction in the frequency of LSK CD150+CD48- cells (WT 1.94x10-4 (±4.4x10-5)%, KO 0.56x10-4 (±1.5x10-5)%, p<0.001 n>10). Strikingly, transplantation of as many as 150 LSK CD150+CD48-HLF KO cells showed a complete lack of repopulating capacity in vivo. This did not correlate to the number of functional HSC seen when transplanting whole bone marrow and indicates that HLF affects the identity of HSC by modulating the expression of the SLAM markers. Taken together, we show here for the first time that HLF has a fundamental role in HSC biology during both fetal and adult hematopoiesis by regulating HSC activity and identity. Disclosures: No relevant conflicts of interest to declare.

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Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽

10.1182/blood.v124.21.1591.1591 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1591-1591

Author(s):

Juliana M. Xavier ◽

Lauremilia Ricon ◽

Karla Priscila Vieira ◽

Longhini Ana Leda ◽

Carolina Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Progenitor Cells ◽

Bone Marrow Niche ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Short Term ◽

Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (meanÂ± SD) (2.07 Â± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 Â± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 Â±1.39; Arhgap21+/-: 2.17 Â± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 Â± 3.85; Arhgap21+/-: 14.07 Â±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 Â±0.03; Arhgap21+/-0.12Â±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 Â±1.1; Arhgap21+/-94.3Â±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

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TNF-α-Induced Bone Marrow Stromal Progenitor Alterations Enhance Leukemic Stem Cell Growth and Treatment Resistance Via Increased CXCL1-CXCR2 Signaling

Blood ◽

10.1182/blood-2018-99-117801 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 875-875

Author(s):

Puneet Agarwal ◽

Hui Li ◽

Kwangmin Choi ◽

Robert S. Welner ◽

Jianbo He ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Critical Role ◽

Mesenchymal Cell ◽

Osteogenic Potential ◽

Pcr Analysis ◽

Hematopoietic Stem ◽

Cxcl1 Expression

Abstract Mesenchymal cells within the bone marrow (BM) microenvironment play an important role in regulation of hematopoietic stem cell (HSC) fate. However, the effects of leukemia development on distribution and function of mesenchymal cell subpopulations are not well understood. Here we used the SCL-tTA-BCR-ABL transgenic CML mouse model to examine how CML development affected murine BM mesenchymal subpopulations within a recently delineated skeletal stem cell (SSC) hierarchy, and evaluate how mesenchymal subpopulations affected HSC and leukemia stem cell (LSC) growth. We observed a significant increase in bone-forming "Thy" and stroma-forming "6C3" progenitor subsets in CML compared to normal BM. CML Thy and 6C3 progenitors demonstrated increased proliferation and CFU-F potential. In addition, CML Thy cells exhibiting increased osteogenic potential whereas CML 6C3 cells showed increased adipogenic potential compared to their normal counterparts. CML LSC and normal HSC were cocultured with Thy and 6C3 cells, purified from CML and normal mice for 3 days and transplanted into irradiated normal recipients to evaluate long-term engraftment. Normal HSC engraftment was enhanced by coculture with normal Thy and 6C3 cells but not by their CML counterparts. On the other hand, LSC engraftment was enhanced by CML 6C3 cells compared to normal 6C3 cells, but not by CML Thy cells compared to normal Thy cells. These results indicate that CML stromal progenitors demonstrate enhanced support of LSC and reduced support of normal HSC. Q-PCR analysis showed that expression of major hematopoietic regulatory molecules, including CXCL12, G-CSF, SCF, IL-1, IL-6, and IGF-1, was significantly reduced in CML 6C3 and Thy progenitors.RNA-Seq analysis demonstrated that expression of TNFaand NF-kbrelated gene sets was significantly increased in CML compared with normal 6C3 cells. Ligand-receptor interactome analysis, based on differential gene expression in LSC and normal HSC, and CML and normal 6C3 cells, revealed upregulation of the chemokines CXCL1 and CXCL5 in CML 6C3 cells, and of their cognate receptor CXCR2 in LSC. We have previously shown that TNFalevels are increased in CML compared to normal BM (Zhang et al., 2012). Here we found that treatment of WT mice with TNFα led to expansion of 6C3 cells and increased CXCL1 expression on 6C3 cells. In contrast, treatment of CML mice with anti-TNFα antibodies led to reduction in 6C3 cell numbers and reduced CXCL1 expression in 6C3 cells. These results support a critical role for TNFα signaling in expansion and increased CXCL1 expression by stromal progenitors in CML BM. We evaluated the role of paracrine CXCL1-CXCR2 signaling in growth and TKI resistance of LSC. Treatment with CXCL1 and CXCL5 resulted in expansion of LSC and leukemic progenitors respectively, and this effect was blocked by the CXCR2 inhibitor SB225002. Treatment with SB225002 significantly reduced proliferation of LSC cocultured with CML 6C3 cells. SB225002 administration to mice engrafted with CML cells resulted in significant reduction in peripheral blood WBC counts, neutrophil percentage, and leukemic short-term HSC (STHSC) and granulocyte-macrophage progenitors (GMP) in the BM. The combination of SB225002 and the TKI Nilotinib (50mg/kg) resulted in significantly greater reduction in peripheral blood WBC and neutrophils, and in BM LSC compared to Nilotinib or SB225002 alone.SB225002 treatment also significantly reduced proliferation and enhanced apoptosis of human CML CD34+CD38- cells cocultured with human CML BM mesenchymal stromal cells, and that the combination of SB225002 and Nilotinib significantly enhanced apoptosis and inhibited proliferation of CML CD34+CD38- cells compared to Nilotinib alone. We conclude that increased TNFα signaling results in expansion of stromal progenitors in CML BM, which differentially support LSC compared to normal HSC. TNFα-signaling leads to overexpression of CXCL1 by stromal progenitors, which interacts with CXCR2 overexpressed on LSC to enhance their growth. Inhibition of CXCR2 signaling reduces LSC proliferation and survival, and enhances LSC elimination in combination with TKI. These observations support further exploration of targeting of CXCL1-CXCR2 interactions as a novel and effective strategy to target BM microenvironment-protected TKI-resistant LSC. Disclosures No relevant conflicts of interest to declare.

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Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cell Function and Promotes G-CSF Mediated HSC Mobilization

Blood ◽

10.1182/blood.v124.21.4334.4334 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4334-4334

Author(s):

Angela Herman ◽

Molly Romine ◽

Darlene Monlish ◽

Laura G. Schuettpelz

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Immune Cell ◽

Conflicts Of Interest ◽

Wild Type ◽

Hematopoietic Stem ◽

Tlr2 Signaling

Abstract Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function. To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2-/- + Tlr2+/+ marrow transplanted into Tlr2+/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r-/-) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs. Disclosures No relevant conflicts of interest to declare.

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Expression of HMGA2 Cooperates with Jak2V617F in the Development of Myelofibrosis

Blood ◽

10.1182/blood.v128.22.797.797 ◽

2016 ◽

Vol 128 (22) ◽

pp. 797-797

Author(s):

Avik Dutta ◽

Robert E Hutchison ◽

Golam Mohi

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Jak2v617f Mutation ◽

Sequencing Analysis ◽

Wild Type ◽

Control Vector ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

Abstract High Mobility Group AT Hook 2 (HMGA2) is a non-histone chromatin protein that regulates gene transcription and controls cell proliferation, survival and self-renewal of stem cells. HMGA2 is expressed at a low level in normal adult hematopoietic progenitors but is highly expressed in hematopoietic progenitors of patients with Myelofibrosis (MF). However, the contribution of HMGA2 to the pathogenesis of MF remains unknown. MF is the deadliest form of myeloprolifearative neoplasm (MPN) characterized by deposition of fibrous tissues in the bone marrow, increased megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. Median survival of patients with MF is less than 6 years. The JAK2V617F mutation has been found in 50-60% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, up-regulation of HMGA2 expression has been found in association with the JAK2V617F mutation in a significant percentage of patients with MF. To understand the role of JAK2V617F mutation in the pathogenesis of MPN, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mouse hematopoietic compartments is sufficient to induce a polycythemia vera (PV)-like MPN. Recently, we have shown that deletion of EZH2 promotes the development of MF in Jak2V617F knock-in mice and EZH2 deletion increases the expression of HMGA2 in hematopoietic progenitors of EZH2-deleted Jak2V617F mice. To directly assess the effects of concomitant expression of HMGA2 and heterozygous Jak2V617F in mice hematopoietic compartments, we expressed control vector or HMGA2 in wild type and heterozygous Jak2V617F knock-in mice BM by lentiviral transduction and performed bone marrow transplantation into lethally irradiated C57BL/6 recipient mice. Whereas recipients of vector-transduced Jak2V617F knock-in BM cells exhibited a PV-like MPN characterized by increased red blood cells (RBC), hemoglobin, hematocrit and platelets in their peripheral blood, recipients of HMGA2-transduced Jak2V617F knock-in BM showed reduced hemoglobin and hematocrit parameters compared with recipients of vector-expressing Jak2V617F BM cells. Interestingly, peripheral blood neutrophil and platelet counts were further increased in transplanted animals receiving HMGA2-transduced Jak2V617F BM cells. Expression of HMGA2 also resulted in significantly larger spleen size in the transplanted animals receiving HMGA2-expressing Jak2V617F BM cells. Flow cytometric analysis showed significant increase in megakaryocytic precursors (CD41+) but decrease in erythroid precursors (CD71+/Ter119+) in the BM and spleens of transplanted animals receiving HMGA2-expressing Jak2V617F BM compared with control vector-expressing Jak2V617F BM. Furthermore, the frequency of hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+) was significantly increased in recipients of HMGA2-transduced Jak2V617F knock-in BM compared with control vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM. Histopathologic analysis revealed extensive fibrosis in the BM and spleens from recipients of HMGA2-expressing Jak2V617F mice at 32 weeks after transplantation while BM and spleens from recipients of vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM showed very little or no fibrosis at this age. Together, these data suggest that expression of HMGA2 promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F. To gain insights into the mechanisms by which expression of HMGA2 accelerates the development of MF in Jak2V617F mice, we performed RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells. Gene set enrichment and pathway analyses revealed that the genes related to chemokine, TGF-β, MAP Kinase, PI3 kinase-Akt, mTOR and WNT signaling pathways were up-regulated in HMGA2-expressing Jak2V617F mice LSK compared with vector-expressing Jak2V617F LSK cells. We also found that HMGA2 directly binds to the promoter regions of some of these target genes and regulate their expression. Further studies will validate the targets of HMGA2 and determine their contribution in MF mediated by Jak2V617F. In conclusion, our studies show that expression of HMGA2 cooperates with Jak2V617F in the development of MF. Disclosures No relevant conflicts of interest to declare.

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BMP4 regulates the hematopoietic stem cell niche

Blood ◽

10.1182/blood-2009-02-206433 ◽

2009 ◽

Vol 114 (20) ◽

pp. 4393-4401 ◽

Cited By ~ 63

Author(s):

Devorah C. Goldman ◽

Alexis S. Bailey ◽

Dana L. Pfaffle ◽

Azzah Al Masri ◽

Jan L. Christian ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Activity ◽

Wild Type ◽

Hematopoietic Stem ◽

Hematopoietic Microenvironment ◽

Hematopoietic Stem Cell Niche ◽

Cell Niche ◽

And Function

Abstract Bone morphogenetic protein 4 (BMP4) is required for mesoderm commitment to the hematopoietic lineage during early embryogenesis. However, deletion of BMP4 is early embryonically lethal and its functional role in definitive hematopoiesis is unknown. Consequently, we used a BMP4 hypomorph to investigate the role of BMP4 in regulating hematopoietic stem cell (HSC) function and maintaining steady-state hematopoiesis in the adult. Reporter gene expression shows that Bmp4 is expressed in cells associated with the hematopoietic microenvironment including osteoblasts, endothelial cells, and megakaryocytes. Although resting hematopoiesis is normal in a BMP4-deficient background, the number of c-Kit+, Sca-1+, Lineage− cells is significantly reduced. Serial transplantation studies reveal that BMP4-deficient recipients have a microenvironmental defect that reduces the repopulating activity of wild-type HSCs. This defect is even more pronounced in a parabiosis model that demonstrates a profound reduction in wild-type hematopoietic cells within the bone marrow of BMP4-deficient recipients. Furthermore, wild-type HSCs that successfully engraft into the BMP4-deficient bone marrow show a marked decrease in functional stem cell activity when tested in a competitive repopulation assay. Taken together, these findings indicate BMP4 is a critical component of the hematopoietic microenvironment that regulates both HSC number and function.

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