Understanding the Impact of Inflammation on Hematopoietic Stem 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.

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SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Blood ◽

10.1182/blood.v120.21.2315.2315 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2315-2315 ◽

Cited By ~ 1

Author(s):

Pauline Rimmele ◽

Carolina L. Bigarella ◽

Valentina d'Escamard ◽

Brigitte Izac ◽

David Sinclair ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Abstract 2315 SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures: No relevant conflicts of interest to declare.

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Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽

10.1182/blood.v70.6.1758.1758 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1758-1763 ◽

Cited By ~ 12

Author(s):

T Nakano ◽

N Waki ◽

H Asai ◽

Y Kitamura

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Electrophoretic Pattern ◽

Phosphoglycerate Kinase ◽

X Chromosomes ◽

Hematopoietic Stem ◽

Colony Forming Units ◽

Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

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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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Loss of P-Selectin Promotes the Function of Aged Hematopoietic and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1577.1577 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1577-1577

Author(s):

Yaoyu Chen ◽

Sullivan Con ◽

Yiguo Hu ◽

Linghong Kong ◽

Cong Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Wild Type ◽

Post Transplant ◽

Marrow Cells

Abstract Abstract 1577 Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between the blood cells and their microenvironment. Adhesion molecules like P-selectin are essential to hematopoiesis, and their dysregulation has been implicated in leukemogenesis. We have previously shown a role for P-selectin in chronic myeloid leukemia and demonstrated that in its absence the disease process accelerates. Recently, there has also been speculation that P-selectin may play a role in the aging hematopoietic stem cells (HSCs), as its expression in upregulated as a mouse ages. In this study, we show that the loss of P-selectin function dysregulates the balance of stem cells and progenitors and that these differences become more pronounced with age. We compared the percentages of HSCs, long-term (LT)-HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), CMPs, GMPs and MEPs in bone marrow by flow cytometry between wild type (WT) and Selp-/- mice. An age-dependent LT-HSC expansion was observed in WT mice. However, this expansion was prevented by the loss of Selp as observed in Selp-/-mice. Further, we demonstrate that with age LT-HSCs in particular express more elevated levels of P-selectin. LT-HSCs and ST-HSC/MPPs were isolated from the bone marrow of young (2 months old) and old (15 months old) WT mice and examined P-selectin expression by FACS. A significant increase in P-selectin expression was observed in LT-HSCs of old mice, and this increase was not observed in the ST-HSC+MPP subpopulations. We also show that the loss of P-selectin gene has profound effects of stem cell function, altering the capacity of these cells to home. Despite impaired homing capacity, stem cells lacking P-selectin possess a competitive advantage over their wild type counterparts. Using a stem cell competition assay, HSCs derived from Selp-/- mice (CD45.2+) and WT control mice (CD45.2+GFP+) were mixed in 1:1 ratio and transplanted into irradiated WT recipients (CD45.1). The initial findings were potentially indicative of the ability of cells derived from GFP mice to more efficiently home and engraft. Despite this initial advantage, cells derived from Selp-/- eventually exhibited a competitive and statistically significant advantage over the cells derived from GFP mice. At 30 days post-transplant, 49.9±1.4% of the CD45.2 subpopulation was GFP+. At 86 days post-transplant, 25.7±3.3 % of the CD45.2 cells derived from the peripheral blood were GFP+. Similarly, 23.0±3.7% of the CD45.2 cells derived from the bone marrow of these mice were GFP+. Indeed, we demonstrate that recipients of P-selectin deficient bone marrow cells more efficiently repopulate the bone marrow than controls and that this advantage extends and expands in the long-term. Finally, we demonstrate that recipients of leukemic cells lacking P-selectin develop a more accelerated form of leukemia accompanied by significant increases in stem and progenitor cells. Bone marrow cells from donor WT and Selp-/- mice were infected with retrovirus expressing BCR-ABL-GFP, and irradiated WT recipients were transplanted with 2×105 of these transduced donor cells. At 14 days post-transplant, recipient mice from each of the groups were sacrificed, and bone marrow cells were harvested and analyzed by flow cytometry. Recipients of leukemic Selp-/- cells possessed 3.5-fold more LSCs than recipients of wild-type cells. There were 3.1-fold more LT-LSCs and 3.8-fold more ST-LSCs and MPPs in recipients of Selp-/- cells than WT cells. In addition, recipients of leukemic Selp-/- cells possessed significantly more CMP (16.9-fold) and MEP (4.5-fold) cells. Because P-selectin expression increases with age on LT-HSCs, we sought to determine the role that age plays in CML development and progression. Bone marrow cells derived from 15-month-old donor Selp-/- and WT mice were transduced with BCR-ABL, respectively, followed by transplantation of the transduced cells into recipient mice. All recipients of BCR-ABL transduced Selp-/- cells died by 23 days after induction of CML and had a median survival of 19 days, whereas recipients of the transduced WT cells survived significantly longer. This pro-leukemic role for cells lacking P-selectin expression is leukemic stem cell-specific rather than stromal cell-specific and supports an essential role for P-selectin on leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.2330.2330 ◽

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.

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

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v122.21.2430.2430 ◽

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.

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Leukemia-Associated Mutations Of DNMT3A Inhibit Differentiation Of Hematopoietic Stem Cells and Promote Leukemic Transformation Through Abberant Recruitment Of Bmi1

Blood ◽

10.1182/blood.v122.21.469.469 ◽

2013 ◽

Vol 122 (21) ◽

pp. 469-469

Author(s):

Junji Koya ◽

Keisuke Kataoka ◽

Takako Tsuruta-Kishino ◽

Hiroshi Kobayashi ◽

Kensuke Narukawa ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Research Funding ◽

Bone Marrow Cells ◽

Cpg Island ◽

Hematopoietic Stem ◽

Cell Accumulation ◽

Exogenous Expression ◽

Marrow Cells

Whole genome sequencing has revealed DNMT3A mutation is present in over 20% of cytogenetically normal acute myeloid leukemia (CN-AML) and R882 is the most frequent and recurrent mutated site. Cumulating clinical data have emphasized the importance of the mutation as a poor prognostic factor of AML. Since the functional role of DNMT3A mutation in leukemogenesis remains largely unknown, we aimed to elucidate the impact of DNMT3A mutation on the development and maintenance of AML. To investigate the effect of exogenous expression of DNMT3A R882 mutant (Mut) in hematopoiesis, we transplanted 5-FU primed mouse bone marrow cells transduced with empty vector (EV), DNMT3A wild type (WT), or DNMT3A Mut to lethally irradiated mice. Recipients transplanted with DNMT3A Mut-transduced cells exhibited hematopoietic stem cell (CD150+CD48-Lin-Sca1+c-Kit+) accumulation and enhanced repopulating capacity compared with EV and DNMT3A WT recipients. To identify the downstream target genes of DNMT3A Mut that evoked hematopoietic stem cell accumulation, we sorted vector-transduced LSK cells from transplanted mice and conducted quantitative PCR (Q-PCR) of various hematopoiesis-related genes. Q-PCR revealed that Hoxb cluster expression was up-regulated and differentiation-associated genes, such as PU.1 and C/ebpa, were down-regulated in DNMT3A Mut-transduced LSK cells. Targeted bisulfite sequencing showed hypomethylation of the Hoxb2 promoter-associated CpG island in DNMT3A Mut-transduced cells compared with EV-transduced cells, which suggests dominant-negative effect of DNMT3A R882 mutation. DNMT3A Mut caused no change in methylation status of PU.1 promoter-associated CpG island, indicating that DNA methylation-independent mechanism underlies PU.1 downregulation. Given that DNMT3A interacts with several histone modifiers to regulate target gene transcription, we performed co-immunoprecipitation to investigate whether these interactions are altered by DNMT3A mutation. We found that DNMT3A Mut has the emhanced capacity to interact with polycomb repressive complex 1 (PRC1), which is thought to be a potential mechanism of the DNMT3A Mut-induced differentiation defect. Co-immunoprecipitation experiments showed that DNMT3A R882H and R882C mutant exhibited augmented interaction with BMI1 and MEL18, respectively. In addition, RING1B, an essential component of PRC1, co-localized with DNMT3A Mut more frequently than WT, irrespective of the type of amino acid substitution. Furthermore, heterozygosity of Bmi1 restored the PU.1 mRNA to the normal level and canceled the effect of stem cell accumulation in mice transplanted with DNMT3A Mut bone marrow cells. Chromatin immunoprecipitation in AML cell lines showed that BMI1 and RING1B were more efficiently recruited to the upstream regulatory element of PU.1 upon expression of DNMT3A Mut than WT, while the amount of DNMT3A recruited were comparable between DNMT3A WT and Mut. In the murine transplantation model, we found that exogenous PU.1 expression impaired repopulating capacity in both EV and R882H-transduced cells to the similar level. Exogenous expression of DNMT3A WT inhibited proliferation and induced terminal myeloid differentiation, whereas DNMT3A Mut-transduced cells remained immature in AML cell lines. DNMT3A Mut-transduced cells were resistant to ATRA-induced differentiation compared to EV-transduced cells. Furthermore, R882 mutation promoted blastic transformation of murine c-Kit+ bone marrow cells in vitro in combination with HOXA9 which is highly expressed in clinical cases harboring DNMT3A mutation. Morphological and surface marker analysis revealed these cells were F4/80+ monocytic blasts, consistent with clinical observation that DNMT3A mutation is found frequently in FAB M4/M5 leukemia. These results indicate a distinct role for DNMT3A Mut as well as a potential collaboration between DNMT3A Mut and HOXA9 in malignant transformation of hematopoietic cells. Interestingly, Bmi1 heterozygosity impaired this monoblastic transformation of R882H and HOXA9 co-transduced progenitors. Taken together, our results highlight the functional role of DNMT3A mutation in differentiation block of hematopoietic stem cells and in promoting leukemic transformation via aberrant recruitment of Bmi1 and other PRC1 components. Disclosures: Kurokawa: Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

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Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽

10.1182/blood-2021-153296 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 20-20

Author(s):

James Bartram ◽

Baobao (Annie) Song ◽

Juying Xu ◽

Nathan Salomonis ◽

H. Leighton Grimes ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Functional Decline ◽

Functional Heterogeneity ◽

Hematopoietic Stem ◽

Donor Cells ◽

Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2018-99-110836 ◽

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.

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