Cited2 Regulates Hematopoietic Stem Cell Quiescence Through HIF-1α Dependent and Independent Pathways

Abstract Abstract 912 Hematopoietic stem cells (HSCs) are thought to be localized in hypoxic microenvironment of the bone marrow (BM) and can remain quiescent or differentiate into multiple blood cell lineages. A number of factors have been found to regulate HSC quiescence in either cell-intrinsic or cell-extrinsic manner. Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2), a member of a newly identified transcriptional modulator, is a cytokine inducible gene and plays various roles during mouse development. In particular, Cited2 is essential for fetal liver hematopoiesis. In this study, we used conditional knockout strategy to delete Cited2 in order to further investigate its function in adult hematopoiesis. Sequential injection of poly(I)-poly(C) (pI-pC) efficiently deleted the Cited2 gene in Cited2fl/fl;Mx1-Cre mice. In this mouse model (Cited2−/− mice), the white blood cell (WBC) count, BM cellularity and Lin−Sca-1+c-Kit+ (LSK) cell number were within the normal range. However, the long-term HSC (LT-HSC; defined as Flt3−CD34−LSK or CD48−CD150+LSK) frequency was significantly decreased. Cited2−/− mice also exhibited increased apoptosis in both LSK and CD34−LSK cells. In addition, Cited2 deficiency led to loss of HSC quiescence evidenced by cell cycle analysis and BrdU incorporation assay. HSC reconstitution capacity was significantly impaired assessed by 5-fluorouracil (5-FU) treatment and transplantation experiments. Transcriptional profiling revealed that multiple HSC quiescence and hypoxia related genes were affected, including p57, Hes1 and Egr1. Recent studies have shown that both HIF-1α-deficient and HIF-1α-stabilized HSCs result in impaired hematopoietic reconstitution, suggesting that precise regulation of HIF-1α level is essential for maintaining HSC quiescence and transplantation activity. Cited2 has been shown to be a negative regulator for HIF-1α through competitive binding to CBP/p300 with higher affinity. In addition, we previously showed that HIF-1α haploinsufficiency (HIF-1α+/−) partially rescues the heart defects in Cited2−/− embryos and HIF-1α deletion (HIF-1α−/−) rescues aberrant vasculature in Cited2−/− embryonic lens. These findings prompted us to explore whether defects of Cited2−/− HSC in adult mice are mediated by dysregulated HIF-1 activity by generating Cited2fl/flHIF-1αfl/fl;Mx1-Cre mice. Additional deletion of HIF-1α partially rescued impaired HSC quiescence and reconstitution capacity caused by Cited2 deficiency. Cited2−/− HIF-1α−/− HSCs displayed comparable apoptosis to Cited2−/− counterparts. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to a normal level. Taken together, these results suggest that Cited2 regulates HSC quiescence through HIF-1 dependent and independent pathways. Disclosures: No relevant conflicts of interest to declare.

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HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency

Blood ◽

10.1182/blood-2011-10-387902 ◽

2012 ◽

Vol 119 (12) ◽

pp. 2789-2798 ◽

Cited By ~ 39

Author(s):

Jinwei Du ◽

Yu Chen ◽

Qiang Li ◽

Xiangzi Han ◽

Cindy Cheng ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Knockout Mice ◽

Gene Dosage ◽

Fetal Liver ◽

Conditional Knockout ◽

Negative Regulator ◽

Transcriptional Level ◽

Double Knockout ◽

Hematopoietic Stem

Abstract Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1–dependent and HIF-1–independent pathways.

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NF-Ya Is Essential for Hematopoieic Stem Cell Proliferation.

Blood ◽

10.1182/blood.v114.22.1507.1507 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1507-1507 ◽

Cited By ~ 1

Author(s):

Gerd Bungartz ◽

Russell Garrett ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Regulatory Subunit ◽

Mouse Development ◽

Potent Inducer ◽

Hematopoietic Stem ◽

M Phase

Abstract Abstract 1507 Poster Board I-530 Proliferation, self-renewal and differentiation of hematopoietic stem cells (HSCs) must be tightly regulated in order to sustain hematopoiesis over a lifetime and to prevent uncontrolled expansion of cells. Several genes have been implicated in the regulation of HSC behavior, such as HoxB4, Notch1, Lef-1 and others. Previous studies from our and other laboratories have demonstrated that the heterotrimeric transcription factor NF-Y is a potent inducer of many of these regulatory genes by over-expressing NF-Ya, the regulatory subunit of NF-Y. Furthermore, Bhattarchaya et al. showed that the deletion of NF-Ya in mice leads to lethality before day E8.5, highlighting its importance in mouse development. While there is no doubt that NF-Y plays a role in the progression of the cell cycle in vitro, different groups – targeting different subunits for deletion or silencing – have obtained different results. In order to comprehensively investigate the in vivo function of NF-Y in the hematopoietic system, we utilized a conditional knockout mouse model. We found that the bone marrow (BM) cellularity decreases sharply starting as soon as one day after the ablation of NF-Ya. Our data indicate that the cell loss can be attributed to a combination of cell cycle arrest in G2/M-phase of the cell cycle and apoptosis at 24 hours after the gene deletion. Since NF-Y has been identified as a master regulator of genes involved in HSCs behavior, we focused on the effects of the NF-Ya deletion within the HSC compartment. We found a down regulation of HoxB4, Notch-1, Lef-1 and Bmi-1 following NF-Ya deletion. However, 24h after induction of NF-Ya deletion the HSC population appeared unaffected and their numbers remained stable, likely due to their predominantly quiescent status. To investigate the capability of stem cells to progress though the cell cycle, we activated HSCs using the interferon inducer poly IC and observed that, once activated, also HSCs accumulate in the G2/M-phase of the cell cycle. Finally, to test whether the deletion of NF-Ya impairs or absolutely abrogates HSC function, we performed long term experiments including competitive BM transplantation and colony formation assays that demonstrate that NF-Y activity is absolutely essential for HSC function. Altogether, our data identify NF-Y plays a pivotal role in the survival of hematopoietic cells and the progression of cells though the G2/M-phase of the cell cycle in vivo. Additionally, while we found that NF-Y ablation leads to reduced expression of many genes important for HSC behavior, this had no immediate effects on the maintenance of these cells due to their quiescent nature. Disclosures No relevant conflicts of interest to declare.

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Murine Fetal Bone Marrow HSPCs Undergo a Dramatic Shift in Frequency at Birth

Blood ◽

10.1182/blood-2019-129073 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2471-2471

Author(s):

Trent Hall ◽

Pramika Sriram ◽

Shannon McKinney-Freeman

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Absolute Number ◽

Global Changes ◽

Bone Marrow Niche ◽

Mouse Development ◽

Hematopoietic Stem ◽

Fetal Bone ◽

And Function

Hematopoietic stem cells (HSCs) give rise to all cells of the hematopoietic system and are classically defined by their ability to stably engraft and reconstitute the blood system of ablated recipients after transplantation. The first transplantable HSCs arise from hemogenic endothelium at embryonic day 10.5 (E10.5) of mouse development and migrate to the fetal liver (FL), where they undergo a robust expansion followed by a second migration to the fetal bone marrow (FBM) at E15.5. The dynamics of hematopoiesis within the FBM has been largely unexplored. To gain a better understanding of FBM hematopoiesis, we catalogued the frequency, absolute numbers, phenotype and function of HSPCs in murine FBM from E15.5 through post-natal day 28 (P28). To avoid assumptions regarding HSPC location during fetal and neonatal development, we pooled bone marrow from the entire fetal skeleton for these studies. HSCs were rare in the FBM, ranging from 70-150 total HSCs at E15.5-E17.5, followed by a burst at E18.5 to 2,200 total HSCs. The frequency and absolute number of HSCs in the bone marrow steadily increased from E18.5 to P6, followed by a continual increase in the absolute number of HSCs from P6 to adulthood. This may reflect the dynamics of HSC cycling, or an influx or expansion of more differentiated progenitors in the fetal and neonatal bone marrow. We also found that the most prevalent hematopoietic stem and progenitor cell (HSPC) population within Lineage-Sca-1+c-Kit+ (LSK) cells in E15.5-E18.5 FBM was Flt3-CD48+CD150+ cells (MPP2). MPP2 cells, which are a megakaryocyte-biased multipotent progenitor population, comprised up to 75% of the LSK compartment at these time points, compared to 3% in adult bone marrow. The percentage of MPP2 cells dropped abruptly and dramatically right before birth (e.g. to 17% at E19) and continued to drop until adulthood. Transplantation of limiting numbers of MPP2 cells revealed that E16.5, E18.5, and P0 MPP2s displayed no repopulating potential, while adult MPP2s showed transient reconstitution of irradiated recipients. Fetal bone marrow MPPs displayed no colony potential in single-cell methylcellulose colony assays until E18.5 and P0, with a bias for erythroid-megakaryocyte colonies. Therefore, fetal bone marrow MPP2s are functionally distinct from their adult counterparts. Whole fetal bone marrow transplants showed that the first transplantable FBM HSCs appeared at E16.5, with up to 75% reconstitution in the peripheral blood (PB) of irradiated recipients. E16.5 FBM HSCs also displayed secondary transplantation activity, while E15.5 FBM HSCs displayed limited ability to reconstitute the PB of primary recipients. In sum, our studies reveal that until birth the predominant HSPC population in the fetal bone marrow is an immunophenotypic MPP2 that is functionally distinct from adult MPP2s, and that HSCs do not begin to accumulate in significant numbers until right before birth. These studies suggest the presence of key mechanisms during birth that influence the HSPC landscape of the fetal and neonatal bone marrow, and work is currently underway to systematically characterize global changes in the bone marrow niche during parturition. Disclosures No relevant conflicts of interest to declare.

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Prdm16 Is a Critical Regulator of Adult Long-Term Hematopoietic Stem Cell Quiescence

Blood ◽

10.1182/blood-2020-139489 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Kristbjorn Orri Gudmundsson ◽

Yang Du ◽

Nhu Nguyen ◽

Kevin Oakley ◽

Yufen Han ◽

...

Keyword(s):

Conflicts Of Interest ◽

Fetal Liver ◽

Annexin V ◽

Conditional Knockout ◽

Cell Cycle Regulators ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Knockout Mouse Model ◽

Underlying Mechanisms

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). PRDM16 encodes a zinc-finger transcription factor homologous to MECOM and was first cloned from chromosome translocations in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Disruption of Prdm16 during mouse embryonic development has been shown to cause a profound loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. Using a novel Prdm16 conditional knockout mouse model, we show that Prdm16 deletion in adult mouse hematopoietic system have a less severe effect on HSCs, causing a gradual decline in adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-FU injections. Annexin V staining showed no difference in apoptosis between wild type and knockout adult long-term HSCs (LT-HSCs). In contrast, BrdU analysis revealed that loss of Prdm16 significantly increases cycling of LT-HSCs with majority of the cells found in the S to G2/M phase. Consistently, RNA-seq analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs. Two such genes were further identified as direct targets of Prdm16. Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence. Disclosures No relevant conflicts of interest to declare.

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Tyrosine Phosphorylation of Runx1 In Megakaryocytes by Src Family Kinases

Blood ◽

10.1182/blood.v116.21.742.742 ◽

2010 ◽

Vol 116 (21) ◽

pp. 742-742 ◽

Cited By ~ 2

Author(s):

Hui Huang ◽

Zachary Waldon ◽

Gordon Chan ◽

Helen Zhu ◽

Hanno Steen ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Tyrosine Phosphatase ◽

Conditional Knockout ◽

Regulatory Function ◽

Wild Type ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Proteomic Approach

Abstract Abstract 742 Runx1 and its cofactor, CBF-beta, are the most frequent targets of chromosomal translocations in human leukemias. Point mutations in Runx-1 also occur in some cases of myelodysplastic syndrome and undifferentiated leukemia. During normal hematopoiesis, Runx1 is required for the ontogeny of all definitive hematopoietic stem cells and for the proper maturation of megakaryocytes (Mks) and lymphocytes. Despite these critical roles, the regulation of Runx1 activity via cell signaling pathways remains incompletely understood. Here, we report that Runx-1 is tyrosine phosphorylated in Mks. This occurs on multiple residues and is mediated by src-family tyrosine kinases (SFKs). Loss of Runx1 tyrosine phosphorylation correlates with phorbol ester induced differentiation of L8057 megakaryoblastic cells, suggesting a negative regulatory function. Consistent with this model, retroviral expression of a tyrosine non-phosphorylatable mutant Runx1 molecule increases primary murine fetal liver Mk maturation and Runx1 target gene expression to a greater extent than wild type Runx1. Moreover, treatment of wild type primary Mks with SFK inhibitors markedly enhances Mk maturation, as previously reported (Lannutti BJ, et al 2005 Blood;105:3875-3878). Treatment of L8057 cells with the pan-tyrosine phosphatase inhibitor Na3VO4, significantly increases Runx1 tyrosine phosphorylation levels, suggesting that tyrosine phosphorylation of Runx1 is dynamically regulated under steady-state conditions. Using a proteomic approach, we found that Runx1 physically interacts with the non-receptor tyrosine phosphatase SHP-2 (Ptpn11). We validated this interaction and showed that it occurs via direct interactions involving the Runx1 runt domain. ShRNA mediated knock down of SHP-2 in L8057 cells increases Runx1 tyrosine phosphorylation levels. Conditional knockout of SHP-2 in Mks using SHP-2fl/fl, PF4-Cre mice leads to reduced peripheral blood platelet counts and delayed platelet recovery following transient anti-GPIb antibody induced immune thrombocytopenia. Lastly, we show that treatment of TPA-induced L8057 cells with Na3VO4 markedly diminishes binding between Runx1 and the key Mk transcription factor GATA-1. Taken together, our data suggest that tyrosine phosphorylation of Runx1 via SFKs inhibits Runx1 function. Dephosphorylation, at least in part via SHP-2, relieves this inhibition and promotes Mk maturation. These effects are likely mediated through altered protein-protein interactions. Disclosures: No relevant conflicts of interest to declare.

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The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.32.32 ◽

2004 ◽

Vol 104 (11) ◽

pp. 32-32

Author(s):

Lei Wang ◽

Linda Yang ◽

Marie–Dominique Filippi ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Fetal Liver ◽

Brdu Incorporation ◽

Low Density ◽

Actin Assembly ◽

Wild Type ◽

Hematopoietic Stem ◽

Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

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Placenta Is a Niche for Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.2671.2671 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2671-2671

Author(s):

Hanna K.A. Mikkola ◽

Christos Gekas ◽

Francoise Dieterlen-Lievre ◽

Stuart H. Orkin

Keyword(s):

De Novo ◽

Fetal Liver ◽

Yolk Sac ◽

Mouse Development ◽

Adult Type ◽

Cell Potential ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Hematopoietic System ◽

Hematopoietic Organs

Abstract The hematopoietic system in the embryo develops in anatomically distinct sites, facilitating rapid generation of erythroid cells and formation of a pool of pluripotent HSCs. The origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct the genesis, maturation, expansion and differentiation of HSCs. In avians, de novo hematopoiesis occurs not only in the yolk sac and the AGM but also in another mesodermal appendage, the allantois. In mammals, the allantois forms the umbilical cord and fetal placenta upon fusion with the chorion. The placenta has not been recognized as a hematopoietic organ, although Melchers reported fetal B-cell potential in murine placenta 25 years ago (Nature 1979, 277:219). Recently, Alvarez-Silva et al. showed that the placenta is a rich source for multipotential hematopoietic progenitors prior to the fetal liver (Development2003, 130:5437). We have performed spatial and temporal analysis of HSCs during mouse development and for the first time assessed HSC activity in the placenta. Hematopoietic organs from E10.5-18.5 embryos (CD45.1/CD45.2) were treated with collagenase and transplanted in limiting dilutions (3–1/1000 embryo equivalents, ee) into irradiated CD45.2+ adult hosts with CD45.1+ support BM cells. Reconstitution was analyzed by FACS and HSCs were quantified as repopulating units (RUs/ee = ([reconstituted recipients] /[total recipients]) /[transplanted dose]). Our data show that the placenta functions as a hematopoietic organ that during midgestation harbors a large pool of pluripotent HSCs. The onset of HSC activity in the placenta parallels that of the AGM starting at E10.5–11.0. However, the placenta HSC pool expands until E12.5–13.5 (>50 RUs) contrasting lack of HSC expansion in the AGM. The expansion of CD34+c-kit+ HSCs in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver and is not accompanied with myeloerythroid differentiation. A far greater expansion of placenta HSCs compared to that of clonogenic progenitors (17-fold vs. 2-fold at E11.5–12.5) suggests that the placenta provides a favorable niche for HSCs. Indeed, placenta HSCs possess functional properties of authentic adult-type HSCs by providing high level multilineage reconstitution for >5 months and exhibiting self-renewal capacity upon serial transplantation. Importantly, placenta HSCs are distinct from circulating HSCs that appear in low numbers after E11.5. HSC activity in the placenta declines towards the end of gestation while HSCs in the fetal liver and blood continue to increase, possibly reflecting mobilization of placenta HSCs to the fetal liver and other developing hematopoietic organs. The early onset of HSC activity in the placenta suggests that the allantois and its derivatives may participate in de novo genesis and maturation of HSCs together with the AGM and possibly the yolk sac. As the main blood volume from the dorsal aorta reaches the fetal liver via umbilical vessels and the placenta, placenta may also provide a niche where nascent HSCs, or pre-HSCs, from the AGM colonize for maturation and expansion prior to seeding fetal liver. While further studies are needed to define the precise origin of placenta HSCs and the function of placenta microenvironment as an HSC supportive niche, the unique kinetics and magnitude of HSC activity suggest an important, previously unappreciated role for the placenta in establishing the definitive hematopoietic system.

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Essential Role for Rbm15 (Ott1) in the Maintenance of Hematopoietic Stem Cell Function and Cell Cycle Control.

Blood ◽

10.1182/blood.v110.11.1223.1223 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1223-1223

Author(s):

Chao Niu ◽

Jiwang Zhang ◽

Stephan W. Morris

Keyword(s):

Cell Cycle ◽

Cell Function ◽

Critical Role ◽

White Blood Cells ◽

Conditional Knockout ◽

Brdu Incorporation ◽

Fusion Partner ◽

Ki 67 ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract RBM15 is the 5′ fusion partner in RBM15-MKL1 (aka OTT1-MAL), a putative oncoprotein in non-Down syndrome infants and children with acute megakaryoblastic leukemia (FAB-M7) containing t(1;22). RBM15 belongs to the “spen” family, which is characterized by the presence of three RNA recognition motifs and a spen paralog and ortholog C-terminal (SPOC) domain. RBM15-homologous Drosophila proteins are involved in regulation of a variety of signaling cascades including MAPK, Wnt, Notch, cyclin E and Hox pathways, but the normal functions of mammalian RBM15 remain largely uncharacterized. We determined that Rbm15 is highly expressed in hematopoietic stem cells (HSCs) as well as T-lineage cells. To study Rbm15 specifically in the regulation of HSC function, we generated Rbm15 conditional knockout mice using the Cre-LoxP system to overcome embryonic death observed with a conventional knockout. Using an inducible Mx1-Cre transgenic line, we conditionally deleted Rbm15 (deletion efficiency ∼96–100%) in HSCs. Both the percentage and absolute number of long-term HSCs (Lin-Sca1+ckit+/Flk2-) were increased in Rbm15-deleted (Rbm15lx/lx;Mx1-Cre+) mice (0.23 ± 0.02% of total nucleated marrow cells [TNMC], 2.3-fold higher) compared to matched littermate controls (Rbm15lx/lx;Mx1-Cre-) (0.10 ± 0.01% TNMC) (P<0.0001, n=18 mice per group). By contrast, total white blood cells (WBCs) were significantly decreased in the peripheral blood (PB) of Rbm15-deleted animals compared to controls (Rbm15-deleted: 5.33 ± 0.40 × 103/uL, Rbm15-intact: 10.26 ± 0.49 × 103/uL; P <0.0001, n=31 per group). Functional analysis of Rbm15-deleted HSCs by competitive repopulation showed these cells to be markedly impaired in their reconstitution of hematopoiesis in lethally-irradiated recipient mice, with only 9.73 ± 2.32% donor-derived cells in the PB of transplanted animals compared to a 47.52 ± 7.26% contribution by donor cells from littermate controls (P=0.00015; n=10 mice per group; 1:1 ratio of donor:wild-type competitor marrow). The serial transplantation ability of Rbm15-deleted HSCs was also severely decreased, with a decline in their engraftment and contribution to the blood of recipient mice noted beginning with the 3rd round of transplantation, culminating in essentially complete failure to engraft in the 4th round (wild-type donor-derived PB cells in 4th round ∼60.95% vs. ∼2.71% Rbm15-deleted donor-derived PB cells; P=0.00003). These defects in HSC function may be due in part to altered HSC cell cycle status in the absence of Rbm15. For example, in representative experiments using Ki-67/Hoechst 33342 and BrdU/7AAD staining, 24.1% and 19.3% of Rbm15-deleted HSCs were found to be in G1 and G2/M phases, respectively - a marked increase compared to Rbm15-intact controls (8.3% and 4.3%, respectively); furthermore, Rbm15-deleted HSCs exhibited significantly less BrdU incorporation (6.8%) compared to control HSCs (27.1%) in in vivo labeling studies. Consistent with these altered cell cycle parameters, hematopoiesis in Rbm15-deleted mice recovered significantly more slowly than controls following 5-FU exposure, with only half the number of total nucleated marrow cells at day 9 and half the number of mature PB WBCs at day 12 following 5-FU as compared to Rbm15-intact littermates (p=0.003, n=3 per group). Collectively, these data demonstrate a critical role for Rbm15 in maintaining HSC integrity and suggest a yet-to-be fully elucidated function for Rbm15 in modulating HSC cell cycle kinetics.

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Lnk Constrains Oncogenic JAK2-Induced Myeloproliferative Disease in Mice.

Blood ◽

10.1182/blood.v114.22.1437.1437 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1437-1437

Author(s):

Alexey Bersenev ◽

Chao Wu ◽

Joanna Balcerek ◽

Wei Tong

Keyword(s):

Signaling Pathways ◽

Conflicts Of Interest ◽

Adaptor Protein ◽

Janus Kinase ◽

Negative Regulator ◽

Myelogenous Leukemia ◽

Cell Surface Receptor ◽

Cytokine Signaling ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 1437 Poster Board I-460 Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation. Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2. Disclosures No relevant conflicts of interest to declare.

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A Novel MBT-Containing Protein, Hemp, Plays Essential Roles In Skeletal Formation and Hematopoietic Stem Cell Function.

Blood ◽

10.1182/blood.v116.21.1597.1597 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1597-1597

Author(s):

Phyo Wai Htun ◽

Keiyo Takubo ◽

Hideaki Oda ◽

Feng Ma ◽

Kenjiro Kosaki ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Thoracic Vertebrae ◽

Wild Type ◽

Hematopoietic Stem ◽

Epigenetic Regulator ◽

Skeletal Formation

Abstract Abstract 1597 Hemp (hematopoietic expressed mammalian polycomb, also denoted as mbt-containing 1) gene was originally identified in the hematopoietic stem cell (HSC)-enriched fraction of the mouse fetal liver (FL). It encodes a protein containing a putative Cys2-Cys2 zinc-finger region, followed by four tandem malignant brain tumor (MBT) repeats, which is frequently observed in polycomb gene (PcG) proteins. The structural characteristics strongly suggest that Hemp functions as an epigenetic regulator, but its biological role remains unknown. To address this issue, we generated hemp-deficient (hemp–/–) mice. Hemp–/– mice died soon after birth. Although no abnormalities were detected in internal organs, skeletal analysis exhibited a variety of malformations. Severe deformities were observed in the thoracic cavity, strongly suggesting that hemp–/– mice died of respiratory failure. Interestingly, they showed malformations of cervical and thoracic vertebrae, which were different from typical homeotic transformations observed in PcG-deficient mice. These results suggest that Hemp governs downstream target genes in distinct manners from conventional PcG proteins. The hematopoietic analysis of hemp in the FL showed that hemp is preferentially expressed in CD150+LSK and CD150–LSK HSC fractions in the hematopoietic hierarchy. Hemp–/– FL contained a significantly reduced number of hematopoietic cells and produced fewer number of hematopoietic colonies as compared to hemp+/+ FL. The decreases correlated with reduced number of CD150+LSK HSCs in hemp–/– FL, which generated much fewer hematopoietic colonies in the HPP-CFC assay. In addition, the competitive repopulation assay exhibited that the hematopoietic reconstitution ability of hemp–/– FL CD150+LSK HSCs was significantly impaired. Moreover, microarray analysis revealed that expression levels of several genes, such as Prdm16, Sox4, and Erdr1 were altered in hemp–/– FL HSCs. Since hemp–/– mice died at neonate, the role of Hemp in adult hematopoiesis remains to be elucidated. To address this issue, we generated hemp conditional knockout (cKO) mice. Acquired deletion of Hemp in the hematopoietic tissues was successfully achieved by crossing hempflox/flox mice with MxCre mice and stimulating the compound mice with pIpC. Analysis of the hematopoietic tissues revealed that the cell numbers of Mac+Gr1– and Mac+Gr1+ fractions in the hemp cKO bone marrow (BM) were significantly increased and decreased, respectively, as compared to those of the wild-type BM. However, no apparent differences have so far been observed between hemp cKO and wild-type littermates in functional analyses, such as colony forming activity and competitive repopulation ability of the BM cells. Here, we report that a novel MBT-containing protein, Hemp, plays essential roles in skeletal formation and HSC function during embryogenesis and also contributes to myeloid differentiation in adult hematopoiesis. Since Hemp likely functions as an epigenetic regulator, further studies will be required to clarify whether and what methylated lysine residues Hemp interacts with through the MBT repeats, what kind of genes are direct targets of Hemp, and how Hemp exerts its biological activity. Disclosures: No relevant conflicts of interest to declare.

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