Irgm1 Is a Negative Regulator of Interferon-Gamma Signaling in Hematopoietic Stem Cells.

Abstract Abstract 382 Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that give rise to all of the cellular elements of the blood and retain enormous proliferative potential in vivo. We have a growing understanding that the controls on HSC proliferation are tied in part to regulation by the immune system—specifically, that HSC proliferation and mobilization can be stimulated by the immune cytokines interferon-alpha and interferon-gamma (IFNg). Our previous work has demonstrated that HSC quiescence and function are aberrant in mice lacking the immunity-related GTPase Irgm1 (also Lrg47). Indeed, the bone marrow of Irgm1-deficient animals at baseline mimics the bone marrow of wild type animals that have been stimulated with IFNg. We hypothesized that the HSC defects in Irgm1-deficient animals are due to overabundant IFNg signaling, and that Irgm1 normally serves to dampen the stimulatory effects of IFNg on HSCs. To test this hypothesis, we used RNA expression profiling to compare gene expression in wild type versus Irgm1-deficient mice. We found that interferon-dependent signaling is globally upregulated in the HSCs of Irgm1-deficient mice. Next we generated Irgm1-/-IFNgR1-/- and Irgm1-/-Stat1-/- double knock out animals. In contrast to the phenotype of Irgm1 single knock out mutants, the hyperproliferation and self-renewal defects in HSCs were both rescued in the double knock out animals, indicating that IFNg signaling is required for manifestation of the Irgm1-deficient phenotype. Futhermore, we found that Irgm1 is expressed in HSCs in a Stat1- and IFNgR-dependent fashion, suggesting that it forms a negative feedback loop for IFNg signaling in the HSC population. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFNg-dependent stimulation in HSCs. These findings demonstrate that IFNg provides a significant stimulus for HSC proliferation even in the absence of infection, and that IFNg-dependent signaling must be tightly regulated to preserve HSC self-renewal capacity. This study provides evidence that the Irgm1 protein can serve as a link between immunity and regulation of hematopoiesis at the level of the stem cell. We speculate that utilization of Irgm1 for its immune functions may detract from its ability to regulate HSC self-renewal capacity, thus ultimately contributing to myelosuppression and increased risk of death from chronic infections such as tuberculosis. Disclosures: No relevant conflicts of interest to declare.

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Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

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Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2018-99-114144 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1269-1269 ◽

Cited By ~ 1

Author(s):

Lynn S. White ◽

Deepti Soodgupta ◽

Rachel L. Johnston ◽

Jeffrey A. Magee ◽

Jeffrey J. Bednarski

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Liver Cells ◽

Lower Percentage ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

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Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽

10.1182/blood.v104.11.1686.1686 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1686-1686

Author(s):

Hideyuki Oguro ◽

Atsushi Iwama ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Target Genes ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem ◽

Ex Vivo Culture ◽

Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

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Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽

10.1182/blood.v114.22.2525.2525 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2525-2525

Author(s):

Takuo Katsumoto ◽

Issay Kitabayashi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Liver Cells ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Interferon-Gamma Impairs the Self-Renewal of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2351.2351 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2351-2351

Author(s):

Alexander M. de Bruin ◽

Berend Hooibrink ◽

Martijn A. Nolte

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Interferon Gamma ◽

Chimeric Mice ◽

Self Renewal ◽

Hematopoietic Stem ◽

The Impact

Abstract Abstract 2351 Regulation of hematopoiesis during stress situations, such as bacterial or viral infections, is crucial for the maintenance of sufficient numbers of cells in the blood. It has become clear that activated immune cells provide such feedback signals to the bone marrow. An important mediator in this respect is the pro-inflammatory cytokine Interferon-gamma (IFNγ), which is produced in the bone marrow by activated T cells during the course of an infection. As such, we have previously shown that T cell-derived IFNγ can directly influence the output of myeloid and erythroid cells. To address whether IFNγ can also influence the function of hematopoietic stem cells (HSCs), we cultured highly purified HSCs from murine bone marrow with or without IFNγ and found that IFNγ strongly reduced the absolute number of HSCs in these cultures, both phenotypically and functionally. We confirmed that the functional impact of IFNγ was due to a direct effect on HSCs and not mediated by more differentiated progenitors. In addition, IFNγ does not directly influence the quiescent state of purified HSC, nor their cell cycle entry. By labeling HSCs with CFSE, we found that IFNγ reduces HSC expansion in vitro by decreasing their proliferative capacity, but not their ability to differentiate. To investigate the impact of IFNγ on HSCs in vivo, we infected WT and IFNγ−/− mice with lymphocytic choriomeningitis virus (LCMV) and found that IFNγ severely impaired HSC recovery upon infection. Finally, to exclude indirect effects of IFNγ on other cell types we generated chimeric mice with bone marrow from both WT and IFNγR−/− mice. Infection of these mixed-chimeric mice with LCMV resulted in decreased recovery of WT HSCs, but not of IFNγR−/− HSCs in the same mouse, which formally demonstrates that IFNγ directly impairs the proliferation of HSCs in vivo. Based on these experiments we conclude that IFNγ reduces HSC self renewal both in vitro and in vivo. Importantly, we thereby challenge the current concept in literature that IFNγ would induce the proliferation of HSCs (Baldridge et al, Nature 2010). Our findings thus provide challenging new insight regarding the impact of immune activation on hematopoiesis and will contribute significantly to the scientific discussion concerning this process. Moreover, our data also provide an explanation for the occurrence of anemia and bone marrow failure in several human diseases in which IFNγ is chronically produced. Disclosures: No relevant conflicts of interest to declare.

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Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo

Blood ◽

10.1182/blood.v120.21.sci-41.sci-41 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-41-SCI-41

Author(s):

Andreas Trumpp ◽

Marieke Essers

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Strong Increase ◽

Interferon Α ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract SCI-41 Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs. Disclosures: No relevant conflicts of interest to declare.

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Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.796.796 ◽

2005 ◽

Vol 106 (11) ◽

pp. 796-796

Author(s):

Hui Yu ◽

Hongmei Shen ◽

Xianmin Song ◽

Paulina Huang ◽

Tao Cheng

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

The Impact

Abstract The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

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S6K1 Regulates Self-Renewal of Leukemia Initiating Cells and Normal Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v126.23.442.442 ◽

2015 ◽

Vol 126 (23) ◽

pp. 442-442

Author(s):

Joydeep Ghosh ◽

Anindya Chatterjee ◽

Baskar Ramdas ◽

Michihiro Kobayashi ◽

Peilin Ma ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Absolute Number ◽

The Self ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mtorc1 Pathway ◽

Deficient Mice

Abstract The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult hematopoietic stem cells (HSCs) and regulation of progenitor population. Translocations in MLL have been detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. Various genes, including AF4, AF5, AF9, ELL and ENL have been identified as partners for translocation in MLL-rearranged leukemia. In hematopoietic cells, expression of MLL fusion proteins result in a block of differentiation. AML patients who successfully undergo treatment but relapse suggest the importance of targeting leukemia initiating cells (LICs) within the MLL leukemia. LICs remain in a quiescent state and are capable of survival despite treatment with chemotherapeutic agents or targeted molecular inhibitors. In mouse models, LICs are defined by the capability of successfully propagating disease upon serial transplantation. In order to prevent disease relapse, identification of molecules, which regulate the self-renewal of LICs, is essential. The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway is a key regulator of self-renewal of both HSCs and LICs. Deletion of Raptor, a subunit of mTORC1, does not affect initiation and progression of acute myeloid leukemia (AML), but Raptor deficiency results in delayed propagation of AML. Upon its activation, mTORC1 phosphorylates and activates p70 ribosomal protein S6 kinase (S6K1) and inhibits the activity of eukaryote translation initiation factor 4E binding protein 1 (4E-BP1). S6K1 has been shown to be hyperactivated in hematopoietic cells expressing oncogenic MLL-AF9 fusion protein. In our study, we have assessed the role of S6K1 in the initiation, progression and propagation of AML using a genetic model of S6K1 knockout mice (S6K1-/-). We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and transplanted them into lethally irradiated recipients. Recipients of both WT and S6K1-/- HSC/Ps bearing MLL-AF9 displayed high white blood cell (WBC) count, splenomegaly and developed AML. There was no difference in survival between the WT and S6K1-/- recipients. In order to determine whether S6K1 regulates the self-renewal of LICs, we transplanted lethally irradiated mice with cells from WT and S6K1-/- primary recipients who developed AML. Recipients of S6K1 deficient AML cells survived significantly longer compared to controls (n=17/group, p<0.001). S6K1 deficient HSC/Ps expressing MLL-AF9 showed reduced activation of Akt as well as decreased mTORC1 activity, suggesting that deletion of S6K1 results in reduced activation of PI-3K-Akt-mTORC1 pathway both upstream and downstream of mTORC1 which indicates that S6K1 might be involve in a feedback loop within this pathway. To determine the role of S6K1 in normal HSC development and maintenance, we analyzed bone marrow derived HSCs in WT and S6K1-/- mice. S6K1 deficiency did not alter the frequency of long term HSCs (LT-HSCs) as defined by CD150+ CD48- Lin- Sca1+ c-Kit+ surface markers, but the absolute number of LT-HSCs were significantly reduced in S6K1 deficient mice (p<0.02). The absolute number of multipotent progenitors (MPPs) (p<0.001), common myeloid progenitors (CMPs) (p<0.01) and megakaryocyte-erythroid progenitors (MEPs) (p<0.01) were also significantly reduced in S6K1 deficient mice. Deficiency of S6K1 resulted in reduced quiescence of LT-HSCs (p<0.05). Expression level of p21, an inhibitor of cell cycle progression, was significantly decreased in LT-HSCs derived from S6K1-/- mice compared to LT-HSCs from control group. To study the role of S6K1 in HSCs' function, we performed competitive repopulation assay. Sorted LT-HSCs from bone marrow cells derived from either WT or S6K1-/- mice were transplanted with competitor cells into lethally irradiated recipients. S6K1 deficient LT-HSCs displayed reduced repopulating ability in secondary recipients (n=9-10/group, p<0.001). Expression level of p21 was downregulated in donor-derived HSCs isolated from secondary recipients of S6K1 deficient HSCs compared to control. Overall, our study establishes S6K1 as a critical regulator of self-renewal of both LICs and HSCs. Deficiency of S6K1 in AML cells results in delayed propagation of disease and deficiency of S6K1 in HSCs results in decreased self-renewal potential. Disclosures No relevant conflicts of interest to declare.

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p38α Is Required for Proper Proliferation of Hematopoietic Stem Cells during Stress

Blood ◽

10.1182/blood.v124.21.4317.4317 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4317-4317

Author(s):

Daiki Karigane ◽

Keiyo Takubo ◽

Shinichiro Okamoto ◽

Toshio Suda

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Cellular Senescence ◽

Expression Level ◽

Wild Type ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Hematopoietic stem cells (HSCs) are capable of self-renewal and multilineage-differentiation during lifespan. HSCs are maintained in a quiescent state to avoid cellular senescence. Previous reports utilizing pharmacological inhibitors or shRNAs against p38MAPK suggest a pivotal role of p38MAPK-pRb-Ink4a signaling in induction of HSC senescence by hematological stress or chronological aging. However, no genetic evidence exists for p38MAPK-mediated cellular senescence in vivo. Here we report unexpected roles of the dominant isoform of p38MAPK family, p38α, in adult hematological system. p38MAPK has four isozymes, α, β, γ and δ. Among them, p38α isozyme was highly expressed in various bone marrow hematopoietic cells, and the expression level of p38α in HSCs was higher than differentiated cells (p<0.01). Phosphorylation of p38MAPK was mainly observed in multipotent progenitors but not in HSCs in steady-state hematopoiesis, in addition, physiological aging (1 year old mouse bone marrow) did not affect phosphorylation status of p38MAPK in steady state. In contrast, p38MAPK was phosphorylated in HSCs after transplantation or 5-FU treatment. Mean fluorescence index (MFI) of phosphorylation of p38MAPK in HSCs is significantly higher at day 3 post 5-FU treatment (250 mg/kg) than steady-state. MFI of phosphorylation of p38MAPK in HSCs was higher at day 1 post transplantation than steady state, and returned to normal at day 7 post transplantation. These results showed phosphorylation of p38α was immediately induced after hematopoietic demand. p38α-deficient embryos die due to defective erythropoiesis in a non-cell-autonomous manner. Thus, we used a conditional knockout model; CAG-CreERT2:p38αfl/fl mouse to analyze the effects of p38α on adult hematopoiesis and HSCs. Expression level of p16Ink4a, one of the cellular senescence markers, was not significantly different between p38α-deficient mice and wild-type mice. Treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type, N=6, p<0.05). Loss of p38α in HSCs showed a defective transplantation capacity. Inducible loss of p38α in bone marrow chimera resulted in a gradual loss of peripheral blood chimerism of p38α-deficient cells. In addition, short-term BrdU incorporation assay showed that the cell cycle progression of p38α-deficient HSCs was suppressed (BrdU positive rate; 3.5±2.2%, N=9, p38α-deficient cells vs 6.5±2.6%, N=5, wild-type, p<0.05). Therefore, hematopoietic function was obviously lowered in p38α-deficient HSCs during hematopoietic stresses. These observations collectively support the requirement of p38α for proper proliferation of HSCs during stress hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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