scholarly journals Loss of IKKβ Increases the Number and Self-Renewal Capacity of Hematopoietic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4345-4345
Author(s):  
Jing Zhang ◽  
Li Li ◽  
Albert S. Baldwin ◽  
Alan D. Friedman ◽  
Ido Paz-Priel
Keyword(s):  
Bone Marrow ◽  
Knockout Mice ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Pharmacologic Inhibition ◽  
Wild Type Control ◽  
Marrow Cells

Abstract NF-kB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IkB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the roles of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and decreased monocytes and. We now show that bone marrow cells from IKKβ deleted mice display accumulation of granulocyte-macrophage progenitors (GMPs, Lin-Sca-1-c-kit+CD34+CD16/32+), with 1.8-fold increase compared to wild-type control. Accordingly, methylcellulose-based colony-forming assay demonstrated that IKKβ-deficient marrow produced increased proportion of CFU-G and decreased CFU-M compared with wild type control. Importantly, pharmacologic inhibition of IKKβ activity in wild type murine bone marrow cells results in significant increase in the total number of colonies and the number and proportion of CFU-G colonies. In addition, loss of IKKβ is associated with a modest decrease of baseline hematocrit levels (52.5 ± 0.64 vs. 43.9 ± 4.3, p<0.0001) and blunted recovery after challenge with 5-FU. Accordingly, IKKβ deleted mice have 3-fold decreased megakaryocyte-erythrocyte progenitors population (MEPs, Lin-Sca-1-c-kit+CD34-CD16/32-). Using FACS analysis erythroid progenitor subsets in the marrow were characterized based on Ter119, CD71, and forward scatter. We found a significantly reduced frequency of proerythroblasts, basophilic and polychromatic erythroblasts. When cultured in methylcellulose in the presence of hEPO IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. Accordingly, pharmacologic inhibition of IKKβ in wild type marrow cells resulted in diminished BFU-E colonies formation. We next studied the role of IKKβ in early hematopoietic progenitors. Bone marrow from IKKβ knockout mice displays the accumulation of phenotypic hematopoietic stem cells (HSCs), including LT-HSCs (Lin-Sca-1+c-kit+CD135-CD34-) and ST-HSCs (Lin-Sca-1+c-kit+CD135-CD34+), with 1.5-fold increase as compared to the wild-type control. Functionally, Lin- bone marrow cells from IKKβ deleted mice show increased serial replating in colony-forming assays, indicating increased cell autonomous long-term self-renewal capacity. Accordingly, competitive transplantation studies demonstrated that deletion of IKKβ greatly increases the repopulation ability of HSCs resulting in a stable advantage of bone marrow derived from IKKβ knockout mice. Quantitative real-time PCR assay demonstrated that compared to Lin- bone marrow from wild-type control mice, Lin- bone marrow cells from IKKβ deficient mice have up-regulation of genes related to HSC self-renewal in early stage of hematopoiesis and granulocytic lineage commitment such as GFI1, HOXA9, PU.1, C/EBPα, CEBPε; but down regulation of genes involved in megakaryocytic-erythroid lineage determination such as GATA1, GATA2, Tal-1, and Klf1. In summary, our data indicate that loss of IKKβ results in a cell autonomous alteration of expression of key regulators of hematopoiesis, leads to increased self-renewal of HSC, and drives hematopoietic development towards GMP lineage, favoring granulopoiesis over monopoiesis. Disclosures No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Hui Z. Zhang ◽  
Svetlana Rogulina ◽  
Wendy Chen ◽  
Barbara A. Degar ◽  
Bernard G. Forget
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 372-372
Author(s):  
Michael J. Nemeth ◽  
Stacie M. Anderson ◽  
Lisa J. Garrett-Beal ◽  
David M. Bodine
Keyword(s):  
Bone Marrow ◽  
Hind Limb ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Bone Marrow Cellularity ◽  
Marrow Cellularity ◽  
Marrow Cells

Abstract Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p < 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p < 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited < 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

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

Blood ◽  
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&lt;0.05) which persisted at 3 months (22.8 vs 1.5, p&lt;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.

Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2629-2629
Author(s):  
Ying Zhao ◽  
Flora Ling ◽  
Hong-Cheng Wang ◽  
Xiao-Hong Sun
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Chronic Inflammation ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Inflammatory Conditions ◽  
The Impact ◽  
Lps Treatment ◽  
Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

The F-Box Protein NIPA Regulates the Hematopoietic Stem Cell Pool

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2330-2330
Author(s):  
Stefanie Kreutmair ◽  
Anna Lena Illert ◽  
Rouzanna Istvanffy ◽  
Melanie Sickinger ◽  
Christina Eckl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
F Box Protein ◽  
Stem Cell Pool

Abstract Abstract 2330 Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation and the expression of numerous cell cycle regulators must be tightly controlled. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. To examine the function of NIPA on vivo, we generated NIPA deficient animals, which are viable but sterile due to a defect in recombination and testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in NIPA deficient animals. Peripheral blood counts taken at different ages revealed no apparent difference between NIPA knockout and wild type mice in numbers and differentiation. In contrast, looking at the hematopoietic stem cell pool, FACS analyses of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA deficient animals, where LSKs were reduced to 40% of wild type littermates (p=0,0171). This effect was only apparent in older animals, where physiologically higher LSK numbers have to compensate for the exhaustion of the stem cell pool. Additionally, older NIPA deficient mice have only half the amount of multi myeloid progenitors (MMPs) in contrast to wild type animals. To examine efficient activation of stem cells to self-renew in response to myeloid depression, we treated young and old mice with the cytotoxic drug (5-FU) four days before bone marrow harvest. As expected, 5-FU activated hematopoietic progenitors in wild type animals, whereas NIPA deficient progenitors failed to compensate to 5-FU depression, e.g. LSKs of NIPA knockout mice were reduced to 50% of wild type levels (p<0.001), CD150+CD34+ Nipa deficient cells to 20% of wild type levels (p<0.0001). Interestingly, these effects were seen in all NIPA deficient animals independent of age, allowing us to trigger the self-renewal phenotype by activating the hematopoietic stem cell pool. Using competitive bone marrow transplantation assays, CD45.2 positive NIPA deficient or NIPA wild type bone marrow cells were mixed with CD45.1 positive wild type bone marrow cells and transplanted into lethally irradiated CD45.2 positive recipient mice. Thirty days after transplantation, FACS analysis of peripheral blood and bone marrow showed reduced numbers of NIPA knockout cells in comparison to NIPA wild type bone marrow recipient mice. This result was even more severe with aging of transplanted mice, where NIPA deficient cells were reduced to less than 10% of the level of wild type cells in bone marrow of sacrificed mice 6 months after transplantation, pointing to a profound defect in repopulation capacity of NIPA deficient HSCs. Taken together our results demonstrate a unique and critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2430-2430
Author(s):  
Zhong-Fa Yang ◽  
Wang Junling ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Bone Marrow Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Hematopoietic Stem ◽  
Threonine Kinase ◽  
Marrow Cells

Abstract Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 477-477
Author(s):  
Christopher B Cole ◽  
Angela M. Verdoni ◽  
David H Spencer ◽  
Timothy J. Ley
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 941-941
Author(s):  
Brian Wadugu ◽  
Amanda Heard ◽  
Joseph Bradley ◽  
Matthew Ndonwi ◽  
Jin J Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Splicing ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Wnt5a Inhibits Wnt3a-Mediated HSC Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2271-2271
Author(s):  
Michael Nemeth ◽  
David Bodine
Keyword(s):  
Bone Marrow ◽  
Formation Control ◽  
Bone Marrow Cells ◽  
Wnt Signaling Pathway ◽  
Retroviral Vectors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Canonical Wnt ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Activation of the canonical Wnt signaling pathway by Wnt3a has been implicated in hematopoietic stem cell (HSC) self-renewal (Reya et al., Nature, 2003). Wnt5a has been observed to inhibit Wnt3a signaling (Topol et al., J Cell Biol, 2004). We hypothesized that Wnt3a and 5a act as antagonists on HSC function. 1 x 106 lineage negative cells (lin−) were cultured for 4 days in the presence of 50 ng/ml SCF and Flt3L (control) plus 100 ng/ml rmWnt3a and/or 500 ng/ml rmWnt5a (all factors added on day 0 and day 2). Control lin− cell numbers expanded more than lin− cells cultured with Wnt3a, 5a, or both (control 8.3 ± 0.3-fold; Wnt3a 6.9 ± 0.2-fold (p &lt; .01); Wnt5a 4.8 ± 0.2-fold (p &lt; .001); Wnt3a and 5a 2.6 ± 0.6-fold (p &lt; .001); n = 3). After 4 days, cells were analyzed for myeloid colony formation. Control cells and cells cultured in Wnt3a had similar numbers of CFU-GM/5000 lin− cells (control 13.1 ± 11.1; Wnt3a 21.8 ± 15.3; p = .21; n = 8), while cells cultured in Wnt5a and Wnt3a and 5a had 2-fold and 5.9-fold more CFU-GM/5000 lin− cells than control (Wnt 5a 26.8 ± 13.3 (p = .04); Wnt3a and 5a 77.9 ± 48.3 (p &lt; .01); n = 8). To analyze repopulating ability, 4 x 105 lin− Ly5.1 cells, cultured under the same conditions, were transplanted with 2 x 106 Ly5.2 bone marrow cells into lethally-irradiated Ly5.2 recipients. 16 weeks after transplant, repopulation by control lin− cells increased 2-fold compared to lin− cells cultured in Wnt3a or Wnt5a (control 7.3 ± 3.8%; Wnt3a 3.37 ± 1.2% (p &lt; .01); Wnt5a 3.6 ± 1.1% (p &lt; .01); n = 9-10). However, lin− cells cultured in Wnt3a and 5a showed normal repopulating activity (n = 10; 8.7 ± 5.3%; p = .52). 1 x 104 HSCs (lin−, c-kitHI, Sca-1HI, IL-7Rα −) were cultured for 6 days with SCF, Flt3L, Wnt3a and 5a (factors added on day 0 and day 3) as described above. Control HSC numbers expanded more than HSCs cultured with Wnt3a, Wnt 5a, or both (control 20.7 ± 10.4-fold; Wnt 3a 7.0 ± 4.1-fold (p = .05); Wnt5a 1.7 ± 1.7-fold (p = .01); Wnt3a and 5a 1.2 ± 1.0-fold (p &lt; .01); n = 4). Similar numbers of control HSCs and HSCs cultured with Wnt3a or 5a were lin+ (control 21.7 ± 0.2%; Wnt 3a 15.4 ± 5.3% (p = .10); Wnt5a 14.4 ± 5.2% (p = .07); n = 3). However, culturing HSCs with Wnt3a and 5a resulted in a 50% decrease in the number of lin+ cells compared to control (12.3 ± 2.0% (p = .001)). Cultured Ly5.1 HSCs were transplanted with Ly5.2 bone marrow cells at a 1:100 ratio. There was no difference in repopulation between control HSCs and HSCs cultured with Wnt3a (control 5.8 ± 6.1%; Wnt3a 3.6 ± 0.4%; p = .43; n = 5). To examine the effects of enforced expression of Wnt ligands in HSCs, 5-FU treated bone marrow was transduced with Wnt3a-IRES-GFP, Wnt5a-IRES-dsRED, or IRES-GFP retroviral vectors. Sorted IRES-GFP+, Wnt3a-GFP+ and Wnt5a-dsRed+ cells (Ly5.1) were transplanted with equal numbers of mock-transduced cells and 3 x 105 Sca-1− bone marrow cells (Ly5.2) into lethally-irradiated Ly5.2 mice. 16 weeks later, recipients of IRES-GFP+ and Wnt5a-dsRed+ cells contained a similar number of engrafted cells expressing the vector (3.4 ± 1.8% GFP+ Ly5.1 and 3.5 ± 0.4% dsRed+ Ly5.1 respectively; n = 8). In contrast, no GFP+ Ly5.1 cells were detected in Wnt3a-GFP+ recipients (n = 8). 33.4 ± 3.7% of bone marrow cells were Ly5.1+ indicating successful engraftment and retroviral DNA was detected by PCR, suggesting that transduction had occurred but that only cells in which the vector was silenced survived. We conclude that activation of the canonical Wnt pathway in HSCs promotes differentiation of primitive hematopoietic cells and that other signals, such as Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.

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