Ing4 Suppresses Quiescence and Inflammation in Hematopoietic Stem Cells

Hematopoietic stem and progenitor cell (HSPC) development and maintenance is regulated through a complex regulatory network. In a screen of epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in HSPC specification. Ing4 acts to regulate transcription through interactions with transcription factors, including HIF, NF-kB, and p53. It is often mis-expressed in many human cancers and has been shown to promote stem cell-like characteristics in malignant cells, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. The transcription factor NF-kB is a regulator of inflammatory response and serves an important role in embryonic HSPC emergence, survival, differentiation and proliferation. The Ing4 protein binds to the p65/RelA subunit of NF-kB, inhibiting DNA binding and suppressing NF-kB cytokines and inflammatory pathways. In the absence of Ing4 there is an overexpression of NF-kB target genes that have inhibitory effects on hematopoietic programming. Given the regulatory role of Ing4 in both hematopoiesis and cancer, it is likely critical to the regulation of stem cell self-renewal, maintenance and specialization. To better define the role of Ing4 on hematopoiesis we use two Ing4 loss-of-function models: zebrafish and mouse. For the zebrafish model of Ing4 deficiency, Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the aorta, gonad, mesonephros (AGM) region of the developing zebrafish embryo, demonstrating a lack of HSPC specification. 36 hours post fertilization (hpf) Ing4 morphants display increased expression of NF-kB target genes when Ing4 is absent. Genetic epistasis experiments performed to block translation of RelA, IL-1b, and additional NF-kB target gene mRNA revealed recovered HSC marker expression in the aorta. To discover small molecule inhibitors that would mimic these effects, we conducted an in vivo chemical screen of NF-kB pathway inhibitors assessing their ability to rescue HSC specification in Ing4 morphant zebrafish. Ing4 morphants treated with NF-kB inhibitors had reduced NF-kB cytokine expression, as well as a dose-dependent rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the effects of Ing4 loss on hematopoiesis. To more thoroughly profile the effects of Ing4 loss on HSC specification and the bone marrow niche, an Ing4-/-mouse model was used. These mice are developmentally normal but are hypersensitive to stimulation with LPS due to increased inflammatory signaling. Peripheral blood analysis reveals an increase in Mac-1 cells in the Ing4-/- mouse. Ing4-/- bone marrow progenitors are skewed toward granulocyte-myocyte progenitor cells (GMPs) lending to the shift in cell populations present in the peripheral blood. Ing4 loss further disrupts the mouse hematopoietic program resulting in a dramatic increase in the number of short term-HSCs (ST-HSC) (WT: 11.4%, Null: 31.7%), a modest increase in long term-HSCs (LT-HSC) (WT: 2.4%, Null: 5.52%), and a dramatic decrease in multipotent progenitors (MPPs) (WT: 47.9%, Null: 19.3%). We also found significant alterations in stress hematopoiesis following competitive HSC transplant where sorted Ing4-/- LT-HSCs failed to engraft. Following myeloablative insult, we found no significant change in Ing4-/- LT-HSC (-1.18%) when compared with ST-HSC (-14.43%) indicating reduced sensitivity to 5-FU ablation in the Ing4-/- LT-HSC group. Cell cycle analysis identified 92.9% of Ing4-/- LT-HSCs are in G0 compared to 76.2% of wildtype LT-HSCs. ST-HSCs were also more quiescent with 27% of Ing4-/- ST-HSCs in G0 compared to 11.1% of wildtype ST-HSCs. Previously published work reports hyper proliferative HSCs that exhibit loss of quiescence as a result of proinflammatory NF-kB signaling. We believe that the interaction between Ing4 and the HIF-1a pathway may play a role in the observed phenotype of Ing4-/- LT-HSCs resulting in increased quiescence and disruption of the balance between self-renewal and differentiation critical to reconstitution of the hematopoietic compartment. Overall, our findings suggest that the regulatory effects of Ing4 play a crucial role in hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures Zon: CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Cellarity: Consultancy; Celularity: Consultancy.

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Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.797.797 ◽

2013 ◽

Vol 122 (21) ◽

pp. 797-797

Author(s):

Krasimira Rozenova ◽

Jing Jiang ◽

Chao Wu ◽

Junmin Wu ◽

Bernadette Aressy ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Failure ◽

Adaptor Protein ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

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Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽

10.1182/blood.v106.11.2190.2190 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2190-2190 ◽

Cited By ~ 1

Author(s):

Pieter K. Wierenga ◽

Ellen Weersing ◽

Bert Dontje ◽

Gerald de Haan ◽

Ronald P. van Os

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Late Antigen ◽

Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

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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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Modulation in the Expression of p70S6 Kinase Impairs the Engraftment and Self-Renewal of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v124.21.252.252 ◽

2014 ◽

Vol 124 (21) ◽

pp. 252-252

Author(s):

Joydeep Ghosh ◽

Baskar Ramdas ◽

Anindya Chatterjee ◽

Peilin Ma ◽

Michihiro Kobayashi ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Mtorc1 Pathway ◽

And Function

Abstract Regulation of hematopoietic stem cell (HSC) function(s) via the mammalian target of rapamycin complex1 (mTORC1) and its upstream regulators including PI3K and Akt has been described before. To this end, we and others have shown that hyperactivation and deficiency of the PI3K-mTORC1 pathway results in altered development, maintenance and function(s) of HSCs. However, the role of downstream effector of mTORC1, p70S6 kinase (S6K1), in HSC development and functions is unknown. Previous studies have implicated S6K1 as a regulator of ageing, by virtue of its ability to regulate cellular metabolic processes as well as protein translation. In certain cells, however S6K1 regulates cell survival and also acts as a negative regulator of PI3K-mTORC1 pathway, thus creating a negative feedback loop. Thus, how S6K1 impacts HSC ageing and stem cell functions remains an enigma. We have assessed the role of S6K1 in HSC development and function under steady-state as well as during recovery of hematopoietic system following myelosuppressive stress. We used a genetic model of S6K1 knockout mice (S6K1-/-). S6K1 deficiency in bone marrow hematopoietic cells resulted in decrease of absolute number of bone marrow hematopoietic progenitor cells as well as HSCs (Lin- Sca1+ c-Kit+; LSK) were significantly reduced relative to controls (n=14 in each group, p<0.04). Interestingly, in vitro, hematopoietic progenitor cells from S6K1-/- mice showed increased colony forming ability in response to cytokines which was associated with hyperactivation of Akt and ERK MAP kinase. To determine whether the reduced number of HSCs in S6K1-/- mice was due to deficiency of S6K1 in bone marrow microenvironment, we transplanted WT hematopoietic bone marrow cells into lethally irradiated WT or S6K1-/- mice. S6K1-/- mice transplanted with WT hematopoietic cells showed similar bone marrow cellularity and HSC numbers compared to controls suggesting that the bone marrow hypocellularity and reduced HSCs numbers in S6K1-/- mice were due to a cell intrinsic defect. To assess whether the reduced HSC number in S6K1-/- mice impacted the recovery of hematopoietic system following stress, WT and S6K1-/- mice were treated with a single dose of 5-fluorouracil (5-FU). In response to myelosuppressive stress, S6K1 deficiency resulted in increased frequency of HSCs in bone marrow despite a significant reduction in overall cellularity (n=12 in each group, p<0.02). Following administration of 5-FU, S6K1 deficiency resulted in increased cell cycle progression of HSCs in bone marrow and showed increased expression of CDK4 and CDK6 as compared to control suggesting that 5-FU administration results in upregulation of cell cycle regulatory genes in S6K1 deficient HSCs. Moreover, S6K1-/- mice showed more sensitivity to repeated injections of 5-FU (n=11 WT, 15 S6K1-/-, p<0.01). Given the differential role of S6K1 in HSCs and mature progenitors, we assessed the effect of S6K1 deficiency in HSC function. We performed competitive repopulation assay using S6K1 deficient HSCs. When transplanted into lethally irradiated primary and secondary recipients, S6K1 deficient HSCs show significantly reduced engraftment relative to controls (n=11-13 in each group; p<0.05). Interestingly, overexpression of S6K1 in wild type HSCs also resulted in reduced engraftment of HSCs in primary and secondary transplant recipients, suggesting that S6K1 overexpression in HSCs leads to decreased self-renewal. In summary, our study identifies S6K1 as a critical regulator of hematopoietic stem cell development and functions both under steady-state conditions as well as under conditions of genotoxic stress. Using both gain of function and loss of function approaches, we demonstrate that the level of expression and activation of S6K1in HSCs plays a critical role in the maintenance of HSC self-renewal and engraftment. Disclosures No relevant conflicts of interest to declare.

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Reversing Clonal Hematopoiesis and Associated Atherosclerotic Disease By Targeted Antibody-Drug-Conjugate (ADC) Conditioning and Transplant

Blood ◽

10.1182/blood-2020-143176 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-35

Author(s):

Karin Gustafsson ◽

Catherine S Rhee ◽

Elizabeth W Scadden ◽

Vanessa Frodermann ◽

Rahul Palchaudhuri ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Peripheral Blood ◽

Publicly Traded ◽

Antibody Drug Conjugate ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

The Past ◽

Antibody Drug

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recently, age-related clonal hematopoiesis (CH) has been recognized as a risk factor for CVD of comparable magnitude to smoking, hypertension and hypercholesteremia. While these other risk factors can be mitigated by pharmacological intervention or lifestyle changes, there are no such strategies in place for CH. As CH is initiated by mutations in hematopoietic stem cells (HSCs), a hematopoietic stem cell transplantat (HSCT) could serve as a curative therapy. However, stem cell transplantation is associated with significant toxicity due in part from current conditioning regimens. There is also no evidence that depletion of the disease-driving clones impacts established atherosclerosis. We developed an antibody drug conjugate (ADC) targeting murine CD45. In the context of stem cell transplantation, the CD45-ADC efficiently depletes endogenous HSCs as well as mature leukocytes while enabling rapid engraftment of an infused stem cell graft. In addition, the CD45-ADCs are not based on broad-acting genotoxic agents that lead to long-lasting health risks. We decided to test if CD45-ADC and HSCT could halt atherosclerosis progression through elimination Tet2 knockout HSCs and their disease propagating myeloid progeny. To model CH associated atherosclerosis, LDLR knockout mice were transplanted with 20% CFP labeled wild-type (WT) or Tet2 knockout bone marrow. A single dose of isotype- or CD45-ADC was delivered after 6 weeks of atherosclerosis development and was followed by an infusion of WT CD45.1 bone marrow. As has been reported before, we observed in the isotype-ADC treated animals that Tet2 deficiency leads to a competitive advantage over WT cells. Tet2 knockout cells contributed to peripheral blood chimerism at successively increasing levels and mice harboring the knockout graft showed a significant expansion of their HSC population. Despite their obvious advantage, Tet2 deficient HSC were as efficiently depleted as their WT counterparts upon CD45-ADC and HSCT. Peripheral blood and bone marrow chimerism were similar in WT and Tet2 knockout hosts and the expanded HSC pool was successfully curbed 6 weeks following the intervention. More importantly, CD45-ADC also depleted cells in the atherosclerotic plaques as efficiently as in blood in both WT and Tet2 mutant recipients. This resulted in a significant reduction of myeloid cell infiltration in CD45-ADC conditioned and transplanted knockout hosts and ultimately lead to drastically reduced plaque size in these animals. In conclusion, these data demonstrate that CD45-ADC and HSCT efficiently replaces the disease driving myeloid cells in the atherosclerosis plaques leading to an overall reduction in disease burden. CD45-ADC and transplantation may thus offer a novel therapy for CH and its associated morbidities. Disclosures Palchaudhuri: Magenta Therapeutics: Current Employment. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Proctor:Magenta Therapeutics: Current Employment. Gillard:Magenta Therapeutics: Current Employment. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

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Ing4 Regulates Stress Hematopoiesis and Represses Self-Renewal in Multipotent Progenitor Cells

Blood ◽

10.1182/blood-2019-129054 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 724-724

Author(s):

Zanshe Thompson ◽

Melanie Rodriguez ◽

Seth Gabriel ◽

Georgina Anderson ◽

Vera Binder ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Cell Transplant ◽

Self Renewal ◽

Hematopoietic Stem ◽

Multipotent Progenitor Cells ◽

Equity Ownership

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells and is a frequent target of inactivation in various cancer types. The tumor suppressive activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In zebrafish, loss of Ing4 results in loss of HSC specification and a significant increase in NF-kB target gene expression. Knockdown of NF-kB expression in Ing4 deficient zebrafish recovered HSC marker expression in the aorta suggesting that NF-kB inhibition could remediate the loss of Ing4 expression. Small molecule NF-kB pathway inhibitors with varying mechanisms were also observed to rescue of HSC marker staining in the zebrafish aorta. Ing4 deficient embryos incubated with a lower dose of inhibitor had a 31% recovery of marker staining and 82% of embryos incubated in the highest dose recovered HSC marker staining emphasizing a dose dependent rescue of HSC specification through NF-kB suppression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- bone marrow has aberrant hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with a concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Analysis of differentiation in Ing4 null bone marrow also reveals skewed hematopoiesis. We see a 14% increase in granulocytes in the null mouse marrow and observe similar skewing in CFU assays. Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. In further characterization of these cells, we found an increase in MPPs that express lower levels of CD34 (55.5% vs 67.7%). CD34 expression is a marker of HSCs. This CD34+/mid population also express CD229 (85% positive), which is barely detectable in wildtype marrow (less that 0.01%). CD229 is also an HSC marker. Based on these exciting findings, we hypothesize that we have identified a subset of CD34+/midCD229+ MPPs in Ing4 deficient mice that retain self-renewal characteristics. Our data suggest that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures Zon: CAMP4: Equity Ownership; Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership.

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N-Cadherin Induces Hematopoietic Stem Cells in a Quiescent State in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v106.11.470.470 ◽

2005 ◽

Vol 106 (11) ◽

pp. 470-470 ◽

Cited By ~ 1

Author(s):

Kentaro Hosokawa ◽

Fumio Arai ◽

Toshio Suda

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Adhesion ◽

Stromal Cells ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Quiescent Hscs

Abstract Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout the lifetime of individuals. Interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining the stem cell properties, including self-renewal capacity and the ability of differentiation into single or multiple lineages. The niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules, and regulate stem cell fates. Recently, it was clarified that long-term bone marrow (BM) repopulating (LTR) HSCs exist frequently in BM trabecular bone surface, and that N-cadherin + spindle-shaped osteoblasts (OBs) are identified as a major niche component. We found that side-population (SP) in c-Kit +Sca-1 +Lin −(KSL) fraction, which is the quiescent HSC in the OB niche, expressed N-cadherin. Expression of N-cadherin in both of the quiescent HSCs and OBs thought to be essential for an adherens junction between HSCs and OBs in the niche. However, the role of N-cadherin in hematopoiesis is still unclear. In this study, we focused on the function of N-cadherin in the maintenance of the stem cell specific property, such as cell adhesion, quiescence, and LTR-activity. To clarify the function of N-cadherin in hematopoiesis, we prepared the retroviruses expressing wild-type N-cadherin, transfected retroviruses into OP9 stromal cell line and KSL cells, and performed the coculture. After coculture of KSL cells with OP9 cells, long-term culture-initiating cells (LTC-ICs) were maintained on OP9 cells overexpressing WT-N-cadherin (OP9/WT-NCAD). In addition, overexpression of WT-N-cadherin in both of the KSL cells and stromal cells enhanced cobblestone formation. N-cadherin overexpressing KSL cell showed slow-cell division from the single cell, when they cultured on OP9/WT-N-cedherin or N-cadherin-Fc protein coated plates, suggesting that N-cadherin-mediated cell-cell adhesion between HSCs and stromal cells enhances the quiescence of HSCs and keeps HSCs in immature state in in vitro. To clarify the role of N-cadherin in the BM reconstitution ability of HSC, we transfected control-IRES-GFP, WT-N-cadherin-IRES-GFP and N-cedherin/390Δ-IRES-GFP retrovirus into the Ly5.1 BM mononuclear cells and transplanted into lethally irradiated Ly5.2 mice. N-cedherin/390Δ, which is a mutant N-cadherin with a deletion at the extracellular domain, exhibits a dominant negative effect on the activity of endogenous cadherins. Control and WT-N-cadherin expressing cell reconstitute the recipient mice BM, while N-cadherin/390Δ expressing cells did not. It suggests that the adhesion between HSCs and BM niche cell is indispensable for the LTR-activity. In addition, we found that WT-N-cadherin overexpressing HSCs were enriched in the SP fraction after 4 months of BM transplantation, indicating that N-cadherin-mediated cell adhesion induced HSCs in the quiescent and kept quiescent HSCs in the niche. Altogether, these observations suggest that N-cadherin is a critical niche factor for the maintenance of the quiescence and self-renewal activity of HSCs. N-cadherin promotes tight adhesion of HSCs to the niche and keeps HSCs in the quiescent state

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Ing4 Regulates Hematopoiesis through Suppression of NF-Kb

Blood ◽

10.1182/blood-2018-99-120033 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 642-642

Author(s):

Zanshe Thompson ◽

Vera Binder ◽

Michelle Ammerman ◽

Ellen Durand ◽

Leonard I. Zon ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Target Gene ◽

Target Genes ◽

Zebrafish Embryos ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In the absence of Ing4, there is a significant increase in NF-kB target gene expression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis, where Ing4 deficiency impairs hematopoietic stem cell (HSC) function, but enhances multipotent progenitor cell (MPP) regenerative capacity. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. To define the role of Ing4 in zebrafish HSPCs, we designed an anti-sense morpholino oligo against Ing4 and injected into zebrafish embryos at the single cell stage. Embryos were screened using in situ hybridizations for c-myb and runx1 expression, which are highly expressed in the aorta, gonad, mesonephros (AGM) region in the developing zebrafish embryo. We found that Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the AGM, demonstrating a lack of HSPC specification. Analysis of ephrinB2 expression showed normal specification of the aorta in Ing4 morphant embryos, signifying that the step of HSPC specification is affected in the absence of Ing4. Overexpression of human Ing4 in zebrafish embryos resulted in increased HSPC marker staining suggesting that normal expression levels of Ing4 are required for HSC specification. As Ing4 is an epigenetic regulator that binds specific gene loci, we examined the chromatin occupancy of Ing4 in human peripheral blood CD34+ progenitor cells. Using ChIP-seq for Ing4 in CD34+ cells, we show that Ing4 binds to many regulators of blood development including MYB, LMO2, RUNX1, and IKAROS, and several NF-kB target genes. In other tissues, Ing4 negatively regulates NF-kB, so accordingly, loss of Ing4 results in an overabundance of NF-kB signaling. To address NF-kB target gene expression in Ing4-deficient zebrafish embryos, we performed qPCR analysis at 36hpf. These assays showed an increase in the expression of a subset of NF-kB target genes (IKBKE, IL-19, IL-1b, IL-20R). Simultaneous knockdown of both Ing4 and RelA, through combined morpholino injections against both factors, resulted in the rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the loss of Ing4. A mouse model for Ing4 deficiency was generated to further evaluate the role of Ing4 in differentiated immune cells. These mice are developmentally normal but are hypersensitive to stimulation with LPS. Interestingly, we found that Ing4-/- mice showed skewed hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. Based on these exciting findings, we hypothesize that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures No relevant conflicts of interest to declare.

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ROLE OF MACROPHAGES IN REGULATION OF HEMATOPOIETIC STEM CELL MIGRATION IN BONE MARROW PERIPHERAL BLOOD SYSTEM

Medical Immunology (Russia) ◽

10.15789/1563-0625-2010-1-2-7-12 ◽

2014 ◽

Vol 12 (1-2) ◽

pp. 7

Author(s):

B. G. Yushkov ◽

I. G. Danilova ◽

I. A. Pashnina ◽

I. A. Brykina ◽

M. T. Abidov

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Migration ◽

Hematopoietic Stem Cell ◽

Peripheral Blood ◽

Blood System ◽

Hematopoietic Stem ◽

Stem Cell Migration

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Homing and Engraftment of Mobilized Hematopoietic Stem Cells: Involvement of VLA-5.

Blood ◽

10.1182/blood.v104.11.4943.4943 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4943-4943

Author(s):

Pieter K. Wierenga ◽

Gerald de Haan ◽

Bert Dontje ◽

Ellen Weersing ◽

Ronald van Os

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Post Transplant ◽

Hematopoietic Reconstitution

Abstract VLA-5 has been implicated in the adhesive interactions of stem and progenitor cells with the bone marrow extracellular matrix and stromal cells and is therefore considered to play an important role in the hematopoietic reconstitution after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3% of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-GSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 38±3%. Despite this low frequency of VLA-5+ cells, however, even when equal numbers of progenitor cells are transplanted MPB cells provide a much faster hematopoietic recovery compared to BM cells. To shed more light on the role of VLA-5 in the process of homing and engraftment, we investigated whether differences in homing potential of the stem cell subsets might be responsible for this enhanced reconstitution. At 3 hours post-transplant, however, no differences in homing efficiency of progenitor and stem cells from MPB and BM grafts in both bone marrow and spleen could be detected. It should be realized that MPB and BM grafts demonstrate different ratios of stem/progenitor cells which might be another explanation for the observed differences in repopulation potential. Furthermore, MPB cells migrating in vitro towards SDF-1α showed potent reconstitution while VLA-5 expression was reduced on these cells. In fact, in vitro treatment with SDF-1α showed further decrease in VLA-5 expressing cells (from 38% to 4%) in the lin- fraction. When equal numbers of MPB were transplanted with and without SDF-1α pretreatment, no difference in hematopoietic reconstitution was observed suggesting a minor role of VLA-5 in homing and engraftment. On the other hand, after VLA-5 blocking an inhibition of 59±7% in the homing of MPB progenitor cells in the bone marrow could be found, whereas homing in the spleen of the the recipients is only inhibited by 11±4%. To elucidate whether the observed enhanced reconstitution could be explained by a selective homing of VLA-5+ cells or a rapid upregulation of VLA-5 expression, cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. It could be demonstrated that at 3 hours post-transplant cells from MPB grafts showed a rapid increase from 38±3% up to 66±9% of VLA-5+ cells in the bone marrow of the recipient. In the spleen no significant increase in VLA-5+ cells was observed. When MPB cells were transplanted after pretreatment with SDF-1α an increase from 2±1% up to 33±5% of VLA-5+ cells in the bone marrow was detected. When calculating the number of cells recovered from bone marrow, a selective homing of VLA-5+ cells cannot be excluded. Therefore, we also assessed the number of VLA-5+ cells in the PKH+ fraction in peripheral blood from the recipient immediately (½-1 hour) after transplantation but found no increase during that time period. So far it can be concluded that MPB cells show low number of VLA-5+ cells but these cells possess an enhanced hematopoietic reconstitution potential. Homing of progenitor cells to the spleen seems to be less dependent on VLA-5 expression than homing to the bone marrow. A rapid upregulation of VLA-5 expression on engrafting MPB cells early after transplantation does not occur and hence our data are suggestive for the preferential homing of VLA-5+ cells in the bone marrow after transplantation.

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