Biological Characterization of the U2af1 S34F Mutation in the Pathogenesis of Myelodysplasia

Abstract Recent genetic studies have revealed frequent and specific pathway mutations involving multiple components of the RNA splicing machinery in myelodysplasia. Among these, U2AF1 mutations are more prevalent in MDS without increased ring sideroblasts and AML with myelodysplasia-related changes and are associated with a poor prognosis. Also found in approximately 4% of lung adenocarcinoma, U2AF1 mutations exclusively involved two highly conserved amino acid positions (S34 or Q157) within the amino- and the carboxyl-terminal zinc finger motifs flanking the U2AF homology motif (UHM) domain. Comprehensive analysis in a large cohort of MDS showed that U2AF1 mutations showed a significant trend to coexist with ASXL1. The molecular mechanism by which U2AF1 mutations lead to myelodysplasia have not fully been elucidated. To elucidate the role of U2AF1 mutations in the development of myelodysplasia, we generated heterozygous conditional knock-in mice for the U2af1 S34F mutation, which were crossed them with Vav1-Cre transgenic mice. Vav1-Cre mediated U2af1 S34F knock-in mice exhibited severe leukopenia and macrocytic anemia at 8-20 weeks after birth. Although there was no significant difference in blood cell morphology between wild-type and mutant mice in bone marrow (BM) and peripheral blood (PB) cells, there was strong myeloid skewing in lineage composition both in U2af1 mutant BM and PB cells compared to wild-type controls. Flow cytometry of U2af1 S34F BM cells showed a significant decrease in the number of hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs) and megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs), and an increase of the granulocyte/macrophage lineage-restricted progenitors (GMPs) compared to wild-type BM cells. These observations suggest that heterozygous U2af1 mutation leads to differentiation defects of HSCs, which however, is not sufficient for the development of MDS. We next assessed the phenotype of U2af1-mutated BM cells in transplantation settings to evaluate the effect of increasing replicative stress, which has been shown to substantially affect the behavior of normal and abnormal stem cells. In PB, progressive leukopenia, macrocytic anemia and decreased platelet counts were observed in mutant mice in transplantation settings. Surprisingly, all of the U2af1 mutant-transplanted mice died within two months after transplantation due to severe bone marrow failure. Cytological analysis of BM cells revealed morphological abnormalities in U2af1 mutant-transplanted mice, including hypersegmentation in neutrophils and erythroid dysplasia. Flow cytometrical analysis revealed decreased numbers of HSCs, CMPs and MEPs, and increased number of GMPs. These observations suggest that the U2af1 mutation leads to ineffective hematopoiesis and morphological abnormalities, which seems to recapitulate the phenotype of MDS in transplantation settings. Subsequently, we assessed the reconstitution capacity of whole BM cells from U2af1 mutant mice in competitive transplantation experiments. The donor chimerism of U2af1 S34F-derived cells in PB was remarkably reduced compared to that of wild-type cells. At four months post transplantation, the chimerism of U2af1 S34F-derived cells was markedly lower than that of wild-type cells in the fractions of HSCs, CMPs, MEPs, GMPs and common lymphoid progenitors (CLPs) in BM. RNA sequencing analysis of HSCs defined as Kit+Sca-1+Linlow (KSL) cells and CMPs from the mutant mice showed significant changes in alternative splicing and expression levels in many genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies. In summary, our results demonstrated that heterozygous U2af1 S34F mutation led to impaired HSC functions that was evident from reduced competitive repopulation and deregulated hematopoietic differentiation, which were augmented in transplantation settings. Our mice model provides a valuable tool to understand the molecular pathogenesis of U2af1-mutated myeloid neoplasms. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

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Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽

10.1182/blood.v120.21.1224.1224 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1224-1224

Author(s):

Junke Zheng ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Actin Binding ◽

Wild Type ◽

Cell Fates ◽

Hematopoietic Stem ◽

Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽

10.1182/blood.v110.11.1677.1677 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1677-1677

Author(s):

Zejin Sun ◽

Yanzhu Yang ◽

Yan Li ◽

Daisy Zeng ◽

Jingling Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Ex Vivo ◽

Bone Marrow Failure ◽

Wild Type ◽

Hematopoietic Stem ◽

Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

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Ott1(Rbm15)-Deficient Hematopoietic Stem Cells Are Unable to Maintain Quiescence During Replicative Stress and Display Features of Premature Aging,

Blood ◽

10.1182/blood.v118.21.3433.3433 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3433-3433

Author(s):

Nan Xiao ◽

Kaushal Jani ◽

Jonathan L Jesneck ◽

Glen D Raffel

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Gene Set Enrichment Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Replicative Stress

Abstract Abstract 3433 With age, hematopoietic stem cells (HSCs) have numerical expansion, skewing towards myeloid development, loss of lymphoid potential, an underlying pro-inflammatory state and loss of self-renewal potential thus severely limiting responses to hematopoietic stress, ultimately leading to bone marrow failure. The mechanisms and pathways responsible for these changes in aged HSCs are incompletely understood. Using a conditional allele of Ott1, a gene originally isolated as the 5' fusion partner in t(1;22) acute megakaryocytic leukemia, we previously found a global regulatory role for the gene in hematopoiesis. Deletion of Ott1 in adult mice utilizing Mx1-cre recapitulated certain aspects of aging hematopoiesis including increased Lin−Sca1+c-Kit+ (LSK) population, myeloid expansion and decreased lymphopoiesis. The LSK compartment was further characterized using SLAM and CD34/Flk2 markers and demonstrated normal levels of LT-HSCs and increased ST-HSCs. Despite sufficient LT-HSC numbers, Ott1-deleted bone marrow was unable to competitively or non-competitively repopulate irradiated recipients. To exclude a homing or engraftment effect, Ott1flox/null Mx1-cre bone marrow was transplanted with competitor then excised post-engraftment. The rapid loss of the Ott1-deficient graft demonstrated Ott1 is required for maintenance under competitive stress. In contrast, primary mice undergoing Ott1 excision lived a normal lifespan and were able to maintain sufficient hematopoiesis although with a partial reduction in bone marrow clonagenicity showing loss of Ott1 is not limiting under steady state conditions. To test the HSC requirement for Ott1 under replicative stress, Ott1 knockout mice were challenged with 5-fluorouracil (5-FU). Ott1-deleted mice treated with 5-FU displayed delayed peripheral blood neutrophil recovery and showed accelerated bone marrow failure. Cell cycle analysis of steady state Ott1 knockout HSCs showed a similar profile to wild type controls, however, after 5-FU treatment, the G0 fraction was dramatically reduced. The G0 fraction is associated with the quiescent, self-renewing HSC population, therefore, Ott1 is required for maintaining HSC quiescence during replicative stress but not steady state hematopoiesis. To more specifically assess whether the functional hematopoietic changes seen after loss of Ott1 were accompanied by alterations in known aging-associated pathways, Gene Set Enrichment Analysis comparing Ott1-deleted HSCs in steady state to aged HSCs was performed and showed a highly enriched gene expression signature (NES 2.02 p<0.0001). Physiologic sequelae of HSC aging were observed after Ott1 excision including activation of NFκβ, elevation of reactive oxygen species (ROS), increase in DNA damage (γH2A.X levels) and activation of p38Mapk. Although ROS was elevated under steady state conditions, neither apoptosis, senescence or proliferation was significantly different from wild type control HSCs. Furthermore, anti-oxidant treatment with N-acetyl-cysteine was unable to rescue the HSC maintenance defect of the Ott1 knockout, signifying additional requirements in HSCs for Ott1 beyond regulation of ROS. An observed increase of mitochondrial mass in Ott1-deleted HSCs suggests an upstream function for Ott1 in metabolic control, potentially contributing to ROS generation or degradation. In summary, we have demonstrated an essential role for Ott1 in maintaining HSC quiescence during replicative stress and shown loss of Ott1 leads to the acquisition of key gene expression patterns and pathophysiologic changes associated with aging. These data suggest Ott1 functions in part to oppose specific consequences of aging in the hematopoietic compartment. Ott1 and Ott1-dependent pathways therefore represent a potential therapeutic target to prevent the morbidity and mortality arising from age-related defects in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v116.21.193.193 ◽

2010 ◽

Vol 116 (21) ◽

pp. 193-193

Author(s):

Pekka Jaako ◽

Johan Flygare ◽

Karin Olsson ◽

Ronan Quere ◽

Jonas Larsson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Flow Cytometric Analysis ◽

Wild Type ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Abstract 193 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

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The Obligate Role of TAK1 in the Survival of Hematopoietic Stem Cells and Progenitors in Mice.

Blood ◽

10.1182/blood.v110.11.642.642 ◽

2007 ◽

Vol 110 (11) ◽

pp. 642-642

Author(s):

Minghui Tang ◽

Zhenbiao Xia ◽

Shubin Zhang ◽

Shanshan Zhang ◽

Xudong Wei ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

B Cell ◽

Knockout Mice ◽

Mutant Mice ◽

Wild Type ◽

Hematopoietic Stem ◽

Expression And Activity

Abstract TGFβ1-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of stress and proinflammatory signals. TAK1 can be activated by inflammation-mediating cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1b (IL-1β), as well as by T- and B- cell receptors (TCR/BCR), and Toll-like receptors (TLRs) signals. Activated TAK1 induces the nuclear localization of NF-kB and the activation of JNK/AP1 by stimulating IKKβ and MKK3/MKK6 phosphorylation respectively. TAK1 has been found to play an important role in inflammation, immunity, T- and B-cell activation, and epithelial cell survival. The TAK1−/ − phenotype is lethal in mice at the early embryonic stage. We found higher levels of TAK1 expression and activity in hematopoietic stem cells and progenitors (HSC/Ps), and reduced expression and activity in differentiated mature hematopoietic cells. To study the role of TAK1 in bone marrow hematopoiesis, we generated inducible-TAK1 knockout mice by crossing TAK1loxp mice with Mx1Cre mice, the latter being an interferon-inducible Cre mouse line. After injection of polyI:C to induce the knockout, we found that all the TAK1 knockout mice died within 8 to 10 days after the first polyI:C injection, showing severe hematopoietic and other defects; heterozygotes were phenotypically comparable to wild-type control animals. The TAK1 deletion in these mice resulted in ablation of bone marrow hematopoiesis due to the loss of C-Kit+ HSC/Ps. Annexin-V staining showed a 3-fold increase in apoptosis in the C-Kit+ HSC/Ps from TAK1 mutant mice compared to those from littermate control mice. Almost all of the mutant animals showed intestinal bleeding as well as other hemorrhaging due to the significant reductions in platelet counts. In reciprocal bone marrow transplantation experiments, we found that the TAK1-mutant bone marrow microenvironment was able to support the growth and function of wild-type HSC/Ps, while HSC/Ps from TAK1−/ − mice failed to grow within the wild-type bone marrow microenvironment. These observations suggest that the bone marrow ablation phenotype which develops in TAK1-mutant mice is the result of intrinsic defects in HSC/P’s. We propose that TAK1-mutant HSC/Ps might mediate a survival signal for HSC/Ps stimulated by hematopoietic growth factors and cytokines, such as stem cell factor (SCF). The details of possible mechanisms by which this phenomenon might occur is currently under active investigation by our group.

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Erg is required for self-renewal of hematopoietic stem cells during stress hematopoiesis in mice

Blood ◽

10.1182/blood-2011-03-344739 ◽

2011 ◽

Vol 118 (9) ◽

pp. 2454-2461 ◽

Cited By ~ 31

Author(s):

Ashley P. Ng ◽

Stephen J. Loughran ◽

Donald Metcalf ◽

Craig D. Hyland ◽

Carolyn A. de Graaf ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Bone Marrow Failure ◽

Mutant Mice ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are rare residents of the bone marrow responsible for the lifelong production of blood cells. Regulation of the balance between HSC self-renewal and differentiation is central to hematopoiesis, allowing precisely regulated generation of mature blood cells at steady state and expanded production at times of rapid need, as well as maintaining ongoing stem cell capacity. Erg, a member of the Ets family of transcription factors, is deregulated in cancers; and although Erg is known to be required for regulation of adult HSCs, its precise role has not been defined. We show here that, although heterozygosity for functional Erg is sufficient for adequate steady-state HSC maintenance, Erg+/Mld2 mutant mice exhibit impaired HSC self-renewal after bone marrow transplantation or during recovery from myelotoxic stress. Moreover, although mice functionally compromised for either Erg or Mpl, the receptor for thrombopoietin, a key regulator of HSC quiescence, maintained sufficient HSC activity to sustain hematopoiesis, Mpl−/−Erg+/Mld2 compound mutant mice displayed exacerbated stem cell deficiencies and bone marrow failure. Thus, Erg is a critical regulator of adult HSCs, essential for maintaining self-renewal at times of high HSC cycling.

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Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.4381.4381 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4381-4381

Author(s):

Takuya Kamio ◽

Bai-wei Gu ◽

Timothy S. Olson ◽

Yanping Zhang ◽

Philip J Mason ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Ribosomal Proteins ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Negative Regulator ◽

Mutant Mice ◽

Mdm2 Gene ◽

Hematopoietic Stem ◽

P53 Response

Abstract MDM2, an E3 ubiquitin ligase, is an important negative regulator of p53. In turn the MDM2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited by the binding of some ribosomal proteins, including RPL5 and RPL11, and that the resulting increase in p53 levels may be important in the red cell aplasia seen in DBA and in 5q- MDS. DBA and 5q- MDS are associated with inherited (DBA) and acquired (5q-MDS) haploinsufficiency of ribosomal proteins. A mutation in MDM2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation, retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we inadvertently noticed that Mdm2 C305F/ C305F mice had perturbed hematopoiesis. These mice had a mild macrocytic anemia with reticulocytosis and high erythropoietin levels. In the bone marrow, these mice showed a significant decrease compared to wildtype (WT) littermates in Ter119+ cells, while no decrease in the number of immature erythroid cells (Ter119+CD71+) was found in the spleen, which showed significantly increased hematopoiesis. In methyl cellulose cultures BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls(p < 0.01). Further investigation revealed that there was a decrease in Lin-Sca-1+c-Kit+(LSK)cells, accompanied by significant decreases in short-term hematopoietic stem cells(ST-HSC) (p < 0.05) and multipotent progenitor(MPP) cells(p < 0.05). These results suggest that control of the cell cycle through interactions between ribosomal proteins and MDM2 is important in the regulation of normal hematopoiesis as well as in the pathogenesis of 5q- MDS and DBA. Disclosures No relevant conflicts of interest to declare.

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Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2011-08-371963 ◽

2011 ◽

Vol 118 (23) ◽

pp. 6087-6096 ◽

Cited By ~ 82

Author(s):

Pekka Jaako ◽

Johan Flygare ◽

Karin Olsson ◽

Ronan Quere ◽

Mats Ehinger ◽

...

Keyword(s):

Bone Marrow ◽

Rna Interference ◽

Ribosomal Protein ◽

Mouse Models ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Down Regulation ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Ribosomal Protein S19

Abstract Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

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New Role of the Regulatory Gene SOX2 in Hematopoiesis.

Blood ◽

10.1182/blood.v104.11.4195.4195 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4195-4195

Author(s):

Elena Levantini ◽

Francesca Bertolotti ◽

Francesco Cerisoli ◽

Anna L. Ferri ◽

Elisa Brescia ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Blood Cell ◽

Molecular Mechanisms ◽

Bone Marrow Cells ◽

Regulatory Gene ◽

Embryonic Stem ◽

Mutant Mice ◽

Wild Type ◽

Cell Production

Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

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A Novel hTERC Deletion Manifesting with Features of Dyskeratosis Congenita and Genetic Anticipation.

Blood ◽

10.1182/blood.v104.11.4157.4157 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4157-4157

Author(s):

Stan Benke ◽

D. S. Houston ◽

Inderjeet Dokal ◽

Tom Vulliamy

Keyword(s):

Bone Marrow ◽

Telomere Length ◽

Peripheral Blood ◽

Autosomal Dominant ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Phenotypic Expression ◽

Dyskeratosis Congenita ◽

Sequencing Analysis ◽

Genetic Anticipation

Abstract The gene encoding the RNA component of human telomerase (hTERC) is mutated in families with the autosomal dominant form of dyskeratosis congenita (DC). The phenomenon of genetic anticipation has recently been reported to accompany this form of DC, with disease severity increasing in offspring of affected individuals. It has been postulated that anticipation in these families relates to the adverse impact of hTERC mutations on inherited telomere length, with progressive telomere shortening seen in succeeding generations (Nat Gen2004; 36:447). We describe here a novel hTERC mutation, with affected individuals presenting in adulthood with mild mucocutaneous abnormalities, bone marrow failure and a pattern of penetrance supporting the presence of disease anticipation. The proband in the family studied presented at age 49 with squamous cell carcinoma of the tongue and a history of oral leukoplakia which he had developed at age 30. Peripheral blood on presentation was remarkable only for a mild macrocytic anemia. During treatment of his malignancy, severe and irreversible bone marrow hypoplasia was precipitated by a single cycle of cisplatinum chemotherapy. The patient’s brother at age 25 had been previously diagnosed with severe aplastic anemia; this was refractory to standard immunosuppression with cyclosporine and antithymocyte globulin. No somatic abnormailites were identified in this patient. Testing for Fanconi anemia in both siblings was negative. Direct sequencing analysis of hTERC in these patients revealed both to be heterozygous for a novel hTERC mutation (79 deletion C). Further studies among family members documented heterozygosity for the mutation in the mother of these two siblings. At age 77, she displayed none of the mucocutaneous signs associated with DC, while the only abnormality seen in her peripheral blood was an elevated mean corpuscular volume. The hTERC mutation seen in this family most likely exerts its effects through disruption of the pseudoknot domain. The findings of an individual with normal longevity, minimal phenotypic expression and affected offspring are further evidence of genetic anticipation being an important feature of autosomal dominant DC. Correlation with determination of telomere length has been initiated.

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