Generation of a Transgenic Mouse with Inducible Constitutively Active Stat5a,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3399-3399
Author(s):  
Harini Nivarthi ◽  
Andriy Tsyrulnyk ◽  
Wolfgang Warsch ◽  
Zhengqi Wang ◽  
Susanne Winkler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Molecular Mechanisms ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Fold Reduction ◽  
Transgenic Founder ◽  
Constitutively Active

Abstract Abstract 3399 The Jak/Stat signalling pathway is essential for survival and proliferation of haematopoietic cells. The Stat5 transcription factors (Stat5a and Stat5b) play a crucial role in the development of various lineages of the hematopoietic system. Stat5 is activated by many cytokines and growth factors that regulate hematopoiesis. Persistent Stat5 activity is frequently found in hematopoietic cancers due to aberrant tyrosine kinase signalling. We have previously characterized a constitutively activated version of Stat5 (called oncogenic Stat5; cS5F), which mimics persistent tyrosine kinase signalling and promotes multi-lineage leukaemia development in a bone marrow transplantation model. The aim of the study is to generate a novel inducible mouse model for oncogenic Stat5a using the BAC recombineering technology. A cassette expressing cS5F (with a C-terminal FLAG tag) and a reporter gene (truncated human CD2; hCD2) was cloned, sequenced and biochemically verified for persistent tyrosine phosphorylation in absence of cytokines or growth factors. The cassette is flanked by loxP sites in opposite orientation and was used to replace the original Stat5a gene in a BAC carrying the endogenous Stat3/5 locus using BAC recombineering. Therefore, the expression of the transgene is regulated by the endogenous Stat5a promoter. The transgene is in an inverted (off) orientation and can be switched into the transcribing (on) orientation by Cre activity. Pronuclear injections were performed with the linearized and purified BAC. We obtained 5 transgenic founder lines which showed germ line transmission of the transgene. The copy number of the BAC was estimated to be 2 by Southern blot analysis. The transgenic founder lines were bred with the Rosa CreERT2 mice, and the Cre activity was induced by treating the mice with 1 mg of tamoxifen every day, for 5 consecutive days. The recombination of the transgene into the ‘on' orientation in liver, lungs, kidney, spleen and bone marrow was demonstrated by Southern blotting. The expression of the hCD2 marker was detected in various hematopoietic lineages by FACS and the expression of the transgenic protein in liver was confirmed by Western blotting with anti-Stat5a and anti-FLAG antibodies. One week after induction of the transgene, all the mice induced with tamoxifen (n=6) developed atrophic thymii with nearly a 4-fold reduction in total thymocyte numbers (p=0.0003). However, the percentage of CD8+ cells was increased more than 3 fold in the thymus (p=0.0002). Moreover, there was a 2-fold reduction in the number of early hematopoietic progenitors; defined as lin−, Sca1+ and c-kit+, in the bone marrow (n=5, p=0.0217). We have established an inducible mouse model for expression of constitutively active Stat5a under the endogenous promoter. We are currently monitoring these mice for development of cancer as they provide a competent tool to study the molecular mechanisms triggered by persistent Stat5a activity, including identification of other cooperating signalling pathways that contribute to generation of cancer. Moreover, it would allow one to test interference strategies, which could lead to potential therapeutics. Disclosures: No relevant conflicts of interest to declare.

VEGFR-1 (Flt-1) Tyrosine Kinase Signaling Enhances Hematopoiesis, Proliferation/Differentiation and Immunity of Monocyte/Macrophage from Bone Marrow Hematopoietic Stem Cells, and Promotes Rheumatoid Arthritis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 778-778
Author(s):  
Masato Murakami ◽  
Shinobu Iwai ◽  
Sachie Hirasuka ◽  
Yoichiro Iwakura ◽  
Yoshiro Maru ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Vascular Tissue ◽  
Systemic Disease ◽  
Bone Destruction ◽  
Inflammatory Cells ◽  
Vegf Receptor ◽  
Hematopoietic Stem

Abstract VEGF and its receptor family including VEGFR-1(Flt-1) are well known to be a crucial regulatory system for normal development and pathological angiogenesis. Rheumatoid arthritis(RA) is a chronic systemic disease characterized by an inflammatory erosive synovicitis, which show marked neovascularization, inflammatory cell infiltration and synovial hyperplasia, then produce a pannus of inflammatory vascular tissue and lead to irreversible cartilage and bone destruction. We have already shown VEGFR-1 is expressed not only in vascular endothelial cells but also in inflammatory cells, especially in monocyte/macrophage. A recent report suggests the involvement of VEGFR-1 in RA by using collagen induced RA mouse model. To examine whether the signaling from VEGFR-1 is important for the pathological process of RA, we used VEGFR-1 tyrosine kinase(−/−) mice which cannot generate the signaling from this receptor, and an arthritis mouse model system carrying Human T-cell leukemia virus(HTLV-1) pX transgene. VEGFR-1 TK(−/−) mice with pX gene clearly showed a reduction in the incidence and the degree of clinical symptom of arthritis. Furthermore, the heterozygote VEGFR-1 TK(+/−) with pX transgene showed a partial decrease in the degree of clinical as well as pathological scores. To explain the reason of reduction of clinical symptoms, we investigated involvement of VEGFR-1 TK signal in lineage of bone marrow hematopoietic stem cell(HSC) to monocyte/macrophage proliferation and differentiation and their immunity. VEGFR-1 TK activities are not associated in number of HSC in bone marrow. However, VEGFR-1 TK(−/−) HSC toward multi-lineage proliferation is suppressed in colony-formation. In addition, failures of monocyte/macrophage faculties are observed in immunological reaction, phagocytosis, cytokine secretion(IL-6, VEGF) and migration. Furthermore, expressions of hematopoiesis and inflammation related genes in VEGFR-1 TK(−/−) macrophage are downregulated by microarray analysis. Next we treated with small molecule inhibitors of VEGF receptor(VEGFR-1 and VEGFR-2) of tyrosine kinase, KRN951, in RA model(pX transgenic model and type II collagen Ab cocktail model) for treatment. Treatment with KRN951 strongly attenuated the disease symptom through inhibiting recruitment of BM hematopoietic cells into peripheral inflammatory cells. These observations indicate that VEGFR-1 signals play an important role in both RA mouse model. The tyrosine kinase activity and the signaling of VEGFR-1 enhances hematopoiesis, proliferation/differentiation and immunity of monocyte/macrophage from bone marrow HSC, and promotes rheumatoid arthritis, which may be a new possibilities for the treatment of RA in humans. Figure Figure

Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4683-4683
Author(s):  
Álvaro Cuesta-Domínguez ◽  
Mara Ortega ◽  
Cristina Ormazabal ◽  
Matilde Santos-Roncero ◽  
Marta Galán-Díez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nude Mice ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Chimeric Protein ◽  
Tyrosine Kinase Domain ◽  
Molecular Response

Abstract Abstract 4683 Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors. Disclosures: No relevant conflicts of interest to declare.

From Bench to Bedside: Hematopoietic Growth Factors (HGFs)- a Cause of Serious and Unanticipated Class Toxicities.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4555-4555
Author(s):  
Charles L. Bennett ◽  
Ari Polish ◽  
Athena T Samaras ◽  
Mi Zheng ◽  
Dennis P West ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Cancer Patients ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
European Medicines Agency ◽  
Severe Thrombocytopenia ◽  
Recombinant Erythropoietin ◽  
Breast Cancer Patients ◽  
Hematopoietic Growth Factors

Abstract Abstract 4555 Introduction HGFs have vastly improved the management of anemia, neutropenia, and thrombocytopenia, but are associated with unexpected toxicities. Methods Toxicities associated with granulocyte, erythroid, and thrombopoietic growth factors were reviewed. Data sources included systematic reviews, clinical trials, guidelines, and materials disseminated by the Food and Drug Administration (FDA), the European Medicines Agency (EMEA), and Canada Health and their advisory committees; product labels and safety notifications disseminated by manufacturers; and utilization data. Data abstracted were thromboembolic complications, immune-mediated cytopenias, bone marrow toxicities, systematic and synergistic toxicities, and regulatory responses. Results Granulopoiesis agents Pulmonary infiltrates were associated with administration of HGFs with bleomycin or chest radiation. Increased myelodysplasia (MDS) or acute myeloid leukemia (AML) risks among chemotherapy (chemo)-treated breast cancer patients were reported. ESAs Increased mortality and tumor progression were seen when cancer patients received ESAs versus placebo; increased mortality and cardiovascular events were reported when chronic kidney disease (CKD) patients received ESAs targeted to complete anemia correction. Between 1998 and 2003, 191 CKD patients developed neutralizing antibodies and pure red cell aplasia (PRCA) following recombinant erythropoietin administration. Thrombopoietins (TPO) TPO receptor agonist administration is associated with bone marrow reticulin and collagen deposition. 3% of healthy volunteers who received repeated rHuMGDF developed neutralizing autoantibodies and severe thrombocytopenia. Conclusions While ESAs, granulopoietic factors, and thrombopoietins have important clinical benefits, they also retain the potential to exhibit serious and unanticipated toxicities. Disclosures: No relevant conflicts of interest to declare.

Hematologic Manifestations and Changes of Cytokines or Hematopoietic Growth Factors in Mice with Iron Overlaod.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5103-5103
Author(s):  
Dae-Chul Jeong ◽  
Hui Seung Hwang ◽  
Nack Gyun Chung ◽  
Bin Cho ◽  
Hyun Jung Shin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Iron Overload ◽  
Conflicts Of Interest ◽  
Iron Concentration ◽  
Iron Dextran ◽  
Hematopoietic Growth Factors ◽  
Liver Iron ◽  
Platelet Counts ◽  
Gm Csf

Abstract Abstract 5103 Background Iron overload by repeated transfusions induced organ toxicity including liver, heart. We investigated hematologic manifestations and cytokines or hematopoietic growth factors in murine secondary hemochromatosis. Materials and methods We established murine secondary hemochromatosis model using 6 week-old male C57/BL6 (H-2b) with iron dextran. Mice (n=10∼12) were intraperitoneally injected with 10 mg of iron dextran for 2 or 4 weeks. We divided five groups: control (PBS injection), iron 100mg, iron 200mg, iron 200mg with deferasirox (DFX) 300mg, and only DFX 300mg. We examined hematocrit, platelet counts and plasma iron concentration (PIC) in peripheral blood, and liver iron contents (LIC) by atomic absorption spectrophotometer. We evaluated colony forming capacity from bone marrow according to experimental group. For cytokines and hematopoietic growth factors, we performed real-time PCR for IL-1b, iNOS, IFN-g, TNF-a, TGF-b, SCF, TPO, GM-CSF, and IL-11 in bone marrow. We compared each values of relative ratio with b-actin. Results There was no difference of hematocrit among experimental groups. The platelet counts were significantly decreased in iron 200mg among groups (P<0.05), and showed increased trends after administration of DFX. The levels of LIC and PIC were dependent on cumulative dose of iron loaded, and decreased by DFX (P<0.01). This findings showed positive correlation between PIC and LIC (P<0.01, R2=0.726). The CFU-GEMM and CFU-GM decreased in iron 200mg, iron 200mg+DFX300mg, and DFX300mg compared with control and iron 100mg (P<0.01). Most colonies in DFX300mg were not observed except CFU-GM. In cytokines, there was shown no difference for IL-1b, iNOS, IFN-g, TNF-a, TGF-b according to experiments (P>0.05). However, SCF was shown diminished expressions for treated mice compared with control (P=0.02). The levels of TPO were increased in hemochromatosis, and decreased after administration of DFX (P=0.05). The GM-CSF was observed significantly lower in iron 200mg, iron 200mg plus DFX, DFX than control and iron 100mg (P<0.01). Conclusions Our results suggested that iron overload might affect hematopiesis and these findings were due to effects of hematopoietic growth factors including SCF, TPO, GM-CSF, not inhibitory cytokines. Also, we need further study for DFX in hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1234-1234
Author(s):  
Robert S Welner ◽  
Giovanni Amabile ◽  
Deepak Bararia ◽  
Philipp B. Staber ◽  
Akos G. Czibere ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cells ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

PU.1 and p53 Double Mutant Mice Develop Aggressive AML with Dysplastic Features

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 769-769
Author(s):  
Petra Vlckova ◽  
Libor Stanek ◽  
Pavel Burda ◽  
Karin Vargova ◽  
Filipp Savvulidi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Regulatory Element ◽  
Tumour Suppressor ◽  
Mutant Mice ◽  
Stem And Progenitor Cells

Abstract Abstract 769 Introduction: Downregulation of tumour suppressor transcription factor PU.1 in haematopoietic stem and progenitor cells represents primary underlying mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. Human AML often display differences in aggressiveness that are associated with mutations of a well known tumour suppressor p53. We produced murine model carrying mutations of p53 and URE that develops highly aggressive AML and focused on molecular mechanisms that are responsible for AML aggressiveness. Mouse models: PU.1ure/ure (Rosenbauer F, et al. 2004) and p53−/− (Jacks T, et al. 1994) mice were used. Conditional deletion of the URE leads to downregulation of PU.1 and is marked by clonal accumulation of myeloid c-Kit+Mac-1low Gr-1low blast cells within bone marrow, spleen, and peripheral blood mirrored by lower numbers of lymphoid and erythroid cells. AML development in PU.1ure/ure mice involves a preleukaemic phase (at 2–3 months) marked by proliferation of myeloid c-Kit+Gr-1+ cells and splenomegaly. Interestingly, p53−/−mice do not develop AML, instead loss of p53 predisposes mice to solid tumours, mostly lymphomas, by 6 months of age. Results: Deletion of TP53 in the PU.1ure/ure mice (PU.1ure/ure p53−/−) results in more aggressive AML with significantly shortened overall survival, prominent hepatosplenomegaly and cachexia (wasting syndrome). Mild differences in cell surface phenotype of bone marrow derived cells were observed between PU.1ure/ure and PU.1ure/ure p53−/− mice by flow cytometry (these included: blasts expansion and lymphopenia). Next, the PU.1 expression was determined in all genotypes at progenitor and stem cell levels. PU.1 mRNA level in more aggressive PU.1ure/ure p53−/− murine AML is decreased in the entire c-Kit+tumour cell population compared to AML in PU.1ure/ure mice including haematopoietic stem and progenitor cells (HSPCs). Correspondingly to RNA level, in the PU.1ure/ure progenitors the PU.1 protein was decreased compared to p53−/− progenitors and is yet further reduced in the PU.1ure/ure p53−/− c-Kit+ Mac1+progenitors. p53−/− progenitors express similar level of PU.1 as wild type progenitors indicating that despite p53 can bind DNA as a transcription factor, it does not regulate PU.1 level directly. In addition to URE deletion we searched for other mechanisms that control PU.1 levels and found that PU.1-inhibiting microRNA miR-155 gene display altered chromatin structure and expression of both pri-miR-155 as well as its spliced mature form in the AML of PU.1ure/ure and (to higher extent in) PU.1ure/ure p53−/− murine progenitors. Upregulation of miR-155 coincides with upregulation of the Mir155hg activators: Myc and Myb. Finally, upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth. Conclusion: In summary, PU.1 and p53 double mutant mice develop aggressive AML with dysplastic features. Defective control of PU.1 levels in PU.1ure/ure and PU.1ure/ure p53−/−AML involves miR-155. Lastly, restored PU.1 level and cell differentiation capacity are achieved by inhibiting either Myb or miR-155 in the PU.1ure/ure p53−/− progenitors. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, P301/12/P380. MK was sponsored by GAUK 251070 45410, 251135 82210) Disclosures: No relevant conflicts of interest to declare.

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2653-2653 ◽  
Author(s):  
Yuichiro Nakata ◽  
Norimasa Yamasaki ◽  
Takeshi Ueda ◽  
Kenichiro Ikeda ◽  
Akiko Nagamachi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Epigenetic Mark ◽  
M2 Macrophage ◽  
Leukemic Stem Cells ◽  
Cell Potential ◽  
Hematopoietic Stem

Abstract Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive. To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs. We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. Disclosures No relevant conflicts of interest to declare.

Determining the Signal Source in Magnetic Resonance Imaging of Myelofibrotic Bone Marrow

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3140-3140
Author(s):  
Shinobu Matsuura ◽  
Shenia Patterson ◽  
Hector Lucero ◽  
Aaron Grant ◽  
Victoria L. M. Herrera ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Bone Marrow ◽  
Magnetic Resonance ◽  
Mouse Model ◽  
Conflicts Of Interest ◽  
Integrative Approach ◽  
Histological Findings ◽  
Resonance Imaging ◽  
Non Invasive ◽  
Lipid Signal

Abstract Magnetic resonance imaging (MRI) is a promising diagnostic method for evaluation of bone marrow myelofibrosis for offering non-invasive, wider-area coverage over conventional histology. Although the most striking feature in histology of myelofibrotic bone marrow is the proliferation of reticulin fibers, the spectrum of findings involves abnormalities in cell number and composition, vessel proliferation, as well as possible pre-fibrotic abnormalities in matrix such as inflammation/edema. Correlation of MRI observations with such histological findings to accurately define the pathogenic phases of myelofibrosis has been challenging. Here, we present the first MRI analysis of myelofibrosis in a mouse model (Gata-1low mice) in an attempt to correlate MRI signal observations with histological findings. T2-weighted MRI images of femur bone marrow of Gata-1low animals at different ages (12 to 46 weeks) showed high MR signal intensity, absent in age-matched wild-type controls, that is detected as early as 12 weeks of age, before onset of histologically-defined fibrosis in this mouse model. MRI acquisitions employing fat suppression, a technique that suppresses signals from lipids, together with the absence of chemical shift artifacts in the images, excluded the possibility of lipids as the source of the prominent MR signal. To determine the source of this prominent non-lipid signal in Gata-1low bone marrow, cellular composition, vascularization and vascular permeability were tested. Our findings demonstrate a multi-source nature of MRI signals in bone marrow, which evolves from young (12 weeks) to old (40 weeks) Gata-1low bone marrow consistent with the pathologic progression of myelofibrosis. Data support the importance of an integrative approach to further the development of this non-invasive modality for following the dynamics and progression of this multifactorial pathology in humans. Disclosures No relevant conflicts of interest to declare.

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