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.

Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1284-1284
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
James Cormier ◽  
Junling Wang ◽  
Xuejun Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Myeloid Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Stem And Progenitor Cells ◽  
Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. 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.

Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 217-217
Author(s):  
Karin Golan ◽  
Aya Ludin ◽  
Tomer Itkin ◽  
Shiri Cohen-Gur ◽  
Orit Kollet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Daily Basis ◽  
Hematopoietic Stem ◽  
Activated Macrophage ◽  
Sphingosine 1 Phosphate ◽  
Stem And Progenitor Cells ◽  
Primitive State

Abstract Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Adedamola Elujoba-Bridenstine ◽  
Lijian Shao ◽  
Katherine Zink ◽  
Laura Sanchez ◽  
Kostandin V. Pajcini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Receptor Expression ◽  
Bone Marrow Niche ◽  
Transplant Recipients ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Null Mutants

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells which differentiate to maintain and replenish blood lineages throughout life. Due to these characteristics, HSPC transplants represent a cure for patients with a variety of hematological disorders. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. The nervous system has been shown to indirectly influence hematopoiesis by innervating the niche; however, we present a direct route of HSPC regulation via expression of neurotransmitter receptors on HSPC surface. We have identified Gamma Aminobutyric acid (GABA) receptor B subunit 1 (Gabbr1), a hitherto unknown hematopoietic player, as a regulator of HSPC function. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation remains unknown. Based on published RNA-seq data (Nestorowa et al., Blood 2016), we discovered that Gabbr1 is expressed on a subset of HSPCs. We confirmed this expression using RT-qPCR to assay hematopoietic populations in the bone marrow (BM). Surface receptor expression analysis showed that Gabbr1 protein is expressed on a subset of BM HSPCs. To detect GABA, the ligand for Gabbr1 in the BM microenvironment, we utilized imaging mass spectrometry (IMS). We detected regionally specific GABA signal in the endosteal region of the BM. We further identified B cells as a cellular source of GABA in the BM. To understand the role of Gabbr1 in hematopoiesis, we generated CRISPR-Cas9 Gabbr1 null mutants on a C57/BL6 background suitable for hematopoietic studies and studied their hematopoietic phenotype. We discovered a decrease in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but the long-term hematopoietic stem cells (LT-HSCs) remain unaffected. Further analysis of peripheral blood of Gabbr1 null mutants showed decreased white blood cells due to reduced B220+ cells. This differentiation defect was confirmed in an in vitro differentiation assay where Gabbr1 null HSPCs displayed an impaired ability to produce B cells. We show that Gabbr1 null HSCs show diminished reconstitution ability when transplanted in a competitive setting. Reduced Gabbr1 null HSC reconstitution persisted in secondary transplant recipients indicating a cell autonomous role for Gabbr1 in regulating reconstitution of HSCs in transplant recipients. Our results show a crucial role for Gabbr1 in HSPC regulation and may translate to human health as a rare human SNP within the GABBR1 locus that correlates with altered leukocyte counts has been reported (Astle et al., Cell 2016). Our studies indicate an important role for Gabbr1 in HSPC reconstitution and differentiation into B cell lineages. Disclosures No relevant conflicts of interest to declare.

The Bleeding Defect Exhibited by Aryl Hydrocarbon Receptor-Null Mice Is Due to Defective Collagen-Dependent Outside-in Signaling

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3294-3294
Author(s):  
Stephan C. Lindsey ◽  
Jinlin Jiang ◽  
Donna Woulfe ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Surface Area ◽  
Platelet Function ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Bone Marrow Niche ◽  
Inside Out

Abstract Abstract 3294 We previously identified AHR as a novel regulator of megakaryocytic (Mk) polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we propose that AHR influences multiple aspects of normal hematopoietic differentiation (Lindsey et al. Stem Cell Rev, 2012), including unpublished data suggesting a role for AHR in mediating platelet function. AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice. Abnormal Mk maturation played a role in this phenotype, as AhR-null mice had ca. 25% fewer high ploidy (= 32n) Mks residing within the murine bone marrow niche compared to WT mice. While investigating if AHR influenced platelet function, we found that AHR-null mice bleed 5.3 times longer (8 minutes for AHR-null mice compared to 1.5 minutes for WT mice) and lose 3 times as much blood as WT mice during bleeding time assays. Although significant, we felt that the decreased Mk polyploidization and resulting reduced platelet counts were not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). We previously showed that treatment of bone marrow-derived progenitor cells with AHR ligands such as TCDD (dioxin, a prototypic AHR ligand and activator) during ex vivo expansion could produce polyploid CD41-expressing cells in the absence of any cytokines. Here, we show that ex vivo expansion of murine progenitor cells with 10 mM of the AHR inhibitor 6',2',4'-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling. To examine the dramatic bleeding phenotype present in AHR-null mice, we next turned our attention toward platelet function, mediated by both outside-in and inside-out signaling. Defects in either or both of these signaling cascades could result in the bleeding defect present in AHR-null mice. In our initial experiments, we found that platelets from AHR-null mice bind fibrinogen equivalently to WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. As we investigated other measures of platelet activation, we found that although platelets from AHR-null mice efficiently aggregated in response to ADP and the PAR-4 agonist AYPGKF (n=3 p=0.897 and 0.914, respectively), only 20 percent of AHR-null murine platelets (compared to 60 percent for WT platelets) aggregated in response to collagen (n=3, p=0.013). Spreading assays further demonstrated defective collagen-dependent outside-in signaling in AHR-null mice. We found that 5 times as many AHR-null platelets remained round (lacking filipodia or lamellipodia) as WT platelets after resting on collagen-coated slides (100 ug/mL) for 5 minutes. Over 60 percent of WT platelets were fully spread after a 20 minute incubation on collagen, compared to only 35 percent of AHR-null platelets. Additionally, roughly 20 percent of AHR-null platelets failed to respond and maintained a round morphology, representing 8 times as many unresponsive platelets as WT mice. The extent of spreading also appeared altered in AHR-null platelets, as the surface area of AHR-null platelets spreading on collagen was reduced by 42% after 5 minutes and 39% after 20 minutes compared to WT (n=3; p<0.001 for both experiments). Similar responses were seen when AHR-null platelets were allowed to spread on fibrinogen (100 ug/mL) after activation by 1 ug/mL collagen, with a 39% and 28% reduction in the surface area of AHR-null platelets after 5 and 20 minutes, respectively (n=3; p<0.001 for both experiments). Based on these findings, we are now investigating the molecular mechanisms of the collagen signaling defects present in AHR-null platelets, beginning with known interactions between AHR and vav genes, critical mediators of collagen-dependant platelet outside-in signaling. Our work is significant in that it builds upon our previously reported data and provides evidence that AHR is a critical component of the physiologic response platelets undergo in response to collagen. This information may provide novel treatment options for patients with bleeding disorders. Disclosures: No relevant conflicts of interest to declare.

From the Bone Marrow to the Thymic Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5123-5123
Author(s):  
Sandrine Susini ◽  
Séverine Mouraud ◽  
Elodie Elkaim ◽  
Julien Roullier ◽  
Sonia Luce ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Epithelial Cells ◽  
Progenitor Cells ◽  
Chemokine Receptors ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Adult Life ◽  
Thymic Epithelial Cells

Abstract To generate T cells throughout adult life, the thymus must import hematopoietic progenitor cells from the bone marrow via the blood. The cellular and molecular mechanisms governing the circulation of thymus-seeding progenitor cells are well characterized in mice but not in humans. The aim of the present study was to characterize the molecular mechanisms and cellular components involved in thymus colonization by lymphoid progenitors (CD34+/CD10+/CD7-/CD24-) and the early steps of thymopoiesis under physiological conditions in humans. Our results demonstrate that circulating lymphoid progenitor cells express CCR9 and CXCR4 chemokine receptors, VLA-4, VLA-5 and VLA-6 integrins and PSGL-1 and CD44 adhesion molecules. We used in vitro migration and adhesion assays to validate the functional status of these markers. As in the mouse, human circulating progenitor cells enter the thymus at the corticomedullary junction (CMJ). Once in the thymus, crosstalk with thymic epithelial cells causes the circulating progenitors to commit to the T-cell differentiation pathway. In order to characterize thymic niches and interactions between circulating progenitors and the thymic stroma, we undertook a chemokine/chemokine-receptor-focused gene expression analysis of sorted lymphoid progenitor cells and CMJ epithelial cells (based on the expression of EpCAM and Delta-like-4). We observed an unexpected gene expression profile for chemokines and chemokine regulators in thymus-seeding CD34+/CD10+/CD7-/CD24- cells and epithelial cells at the CMJ. The present results should help us to highlight candidate genes involved in the early steps of human thymopoiesis. Disclosures No relevant conflicts of interest to declare.

Loss of Foxo3 Reduces Erythroblast Apoptosis and Enhances RBC Production in Beta-Thalassemic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 756-756 ◽  
Author(s):  
Raymond Liang ◽  
Genís Campreciós ◽  
Carolina L. Bigarella ◽  
Saghi Ghaffari
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Mutant Mice ◽  
Mrna Levels ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Qrt Pcr

β-thalassemia arises as a result of mutations in the β-globin gene. As a consequence erythropoiesis, the process that insures the daily generation of billions of red blood cells (RBCs), becomes disrupted. Ineffective erythropoiesis is a major contributor to the β-thalassemic anemia and is partially due to aberrant apoptosis during late stages of erythroid maturation. Despite the importance of apoptosis, the underlying molecular mechanisms regulating this process in β-thalassemia erythroblasts are not fully elucidated. One potential mechanism involves the transcription factor Foxo3, which under specific contexts can act as a positive regulator of apoptosis, but is also an essential transcriptional regulator of terminal erythroblast maturation. Foxo3 has a range of outputs that it can execute from sustaining cellular integrity by mitigating oxidative stress to inducing apoptosis under conditions of overwhelming stress. Given these functions, we sought to determine if Foxo3 played a role in maintaining RBC maturation in β-thalassemic mice. To address this, we used Hbbth3/+ (th3/+) mice that display a phenotype similar to β-thalassemia intermedia, and produced double mutant Foxo3-/-/Th3/+ mice. The th3/+ mice display a mild erythroblast apoptotic phenotype. We hypothesized that loss of Foxo3 may exacerbate the β-thalassemic phenotype. On the contrary, we found that loss of Foxo3 in a β-thalassemic background improved RBC numbers and hemoglobin concentration (by 1g/dl, n=10 mice) in double mutant mice compared to th3/+ mice. Furthermore, double mutant mice had a statistically significant lower frequency of apoptosis (2 fold less) during bone marrow erythroblast maturation as measured by flow cytometry analysis of annexin V-binding and 7AAD staining in distinct erythroblast stages resolved by TER119, CD44 and cell size (n=3 mice per genotype). We predicted that high levels of oxidative stress may prematurely activate FOXO3 during erythroblast maturation in β-thalassemic mice. In turn, activated FOXO3 may potentially promote apoptosis in these cells. To evaluate this, we examined FOXO3 levels by qRT-PCR and immunofluorescence in FACS sorted populations of erythroblasts (TER119+,CD44,FSC) or erythroid progenitors (TER119-,c-KIT+,CD71HI) acquired from bone marrow of at least 3 mice per genotype. Our data show increased mRNA levels of Foxo3 in early erythroblasts, corresponding to increased FOXO3 protein expression in erythroid progenitors from β-thalassemic mice relative to wild-type mice. We also examined the activation status of p53, as it is also a major regulator of apoptosis that can be triggered by oxidative stress. Nuclear p53 levels were greater in β-thalassemic as compared to wild-type erythroid progenitors based on immunofluorescence analysis of sorted cells from bone marrow of 3 mice per genotype. These results suggest a higher level of active p53 in β-thalassemic erythroid progenitors. Our results provide evidence that FOXO3, a factor normally critical for erythroblast maturation, may cooperate with aberrantly active p53 to induce apoptosis in β-thalassemic erythroblasts. In support of this, downstream p53 targets including Gadd45a and p21 that are also Foxo3 targets were significantly upregulated in β-thalassemic erythroblasts relative to wild-type erythroblasts as determined by qRT-PCR of cDNA produced from 3 mice per genotype. To more closely examine the mechanism of decreased apoptosis in double mutant Foxo3-/-/Th3/+ erythroblasts, we compared the expression of multiple genes involved in apoptosis by qRT-PCR of sorted erythroblast populations from at least 3 mice per genotype. We found multiple pro-apoptotic genes including, Cycs, Tnfsf10, Puma, and Bim expressed at significantly lower levels at various erythroblast stages in double mutant compared to β-thalassemic erythroblasts. Together, our data suggests Foxo3 becomes inappropriately and prematurely activated in erythroid progenitors and early erythroblasts in the context of β-thalassemia and cooperates with p53 to promote apoptosis. These findings raise the possibility that cooperation of Foxo3 and p53 in β-thalassemic erythroblasts might contribute to the ineffective erythropoiesis of β-thalassemic mice. They also suggest the possibility that as a homeostatic maintaining factor, Foxo3 behaves differently in the context of disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential Effects of TLR1/2 and TLR2/6 Signaling on Normal and Premalignant Hematopoietic Stem and Progenitor Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1806-1806
Author(s):  
Darlene A. Monlish ◽  
Zev J. Greenberg ◽  
Sima T. Bhatt ◽  
Dagmar Ralphs ◽  
John L. Keller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
White Blood Cells ◽  
Hematopoietic Stem ◽  
Specific Effects ◽  
Stem And Progenitor Cells ◽  
Cell Cycling

Abstract Prior studies from our lab and others have demonstrated a role for Toll-like receptor 2 (TLR2) in regulating both normal and premalignant hematopoietic stem and progenitor cells (HSPCs), however the contributions of its binding partners, TLR1 and TLR6, remain unknown. In CD34+ bone marrow cells of patients with myelodysplastic syndrome (MDS), increased TLR2 was associated with lower-risk disease, elevated rates of apoptosis associated with improved prognosis, and enhanced survival. Conversely, increased levels of TLR6, but not TLR1, was associated with higher-risk disease and an increased percentage of bone marrow blasts (Zeng et al., Exp Cell Res 2016 and Wei et al., Leukemia 2013). These data suggest that there may be heterodimer-specific effects of TLR2 signaling on HSPCs influencing disease progression. To elucidate the unique contributions of the heterodimer pairs in MDS pathogenesis and leukemogenesis, we utilized a well-established mouse model of MDS that expresses the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter. The "NHD13" mice recapitulate many of the salient features of human MDS and succumb to cytopenias or leukemia by 14 months of age (Lin et al., Blood 2005). Importantly, we observed significantly increased expression of TLRs 1, 2, and 6 on the c-Kit+, Sca-1+, Lineage- ("KSL") HSPCs of the NHD13 mice, similar to the increased expression of these TLRs on CD34+ cells of MDS patients. To begin to delineate the heterodimeric differences, NHD13 mice were treated chronically with either PAM2CSK4 (PAM2), a TLR2/6-specific agonist, or PAM3CSK4 (PAM3), a TLR1/2-specific agonist, to assess the effects on cytopenias and survival. After five months of treatment, a significant increase was observed in the total number of white blood cells in NHD13 mice treated with PAM2 (p=0.007), but not PAM3 (vs. vehicle (water)-treated controls), a finding that was not recapitulated in wild-type (WT) controls. On the contrary, a significant decrease in the total number of platelets in both NHD13 and WT mice treated with PAM3 was observed as compared to vehicle-treated controls (p=0.024 and p=0.011, respectively). Further supporting the existence of heterodimer-specific differences, death was expedited in NHD13 mice treated with PAM2 as compared to those treated with PAM3 (p=0.019), with a median survival of 243 days vs. 338 for the PAM3-treated cohort. The cause of death, as determined by a hematopathologist based on cytology and blast percentage, was most often due to leukemia. To investigate the potential mechanism through which enhanced TLR2/6 signaling accelerates leukemogenesis and death in NHD13 mice, the HSPCs of premalignant NHD13 mice treated with PAM2 or PAM3 were characterized by flow cytometry and evaluated for cell cycling and cell death. Both the total number and frequency of KSL cells were significantly increased in NHD13 mice treated with PAM2 (p=0.007 and p<0.0001, respectively), but not PAM3, vs. water-treated controls. No significant changes were noted in either cell cycling or apoptosis following agonist treatment. A microarray of bone marrow KSL cells revealed that stimulation of the TLR2/6 pathway is associated with an activated c-Myc signature, suggesting that enhanced signaling through this pathway, but not TLR1/2, may enhance leukemogenesis via Myc activation. Further, the expression levels of six downstream targets of c-Myc, including BAX, APEX1, ODC1, FKBP4, NCL, and HSPD1, were significantly increased in both WT and NHD13 mice following PAM2 treatment. Evaluation of serum cytokines also revealed heterodimer-specific alterations, including increased IL-6 levels in NHD13 mice treated with PAM2, but not PAM3. These data corroborate numerous previous reports linking IL-6 to MDS pathogenesis and transformation to acute myeloid leukemia. Ongoing studies involving mass cytometry, IL-6knockout mice, and pharmacological inhibitors of both IL-6 and c-Myc aim to further elucidate the mechanism through which TLR2/6-specific activation accelerates leukemogenesis and death in the NHD13 mouse model of MDS. These studies hope to inform more targeted therapeutics that could potentially delay MDS progression and reduce off-target effects. Disclosures No relevant conflicts of interest to declare.

scholarly journals Exploring the Mechanisms of Thalassemic Erythropoiesis Improvement Caused By Bone Marrow Tfr2 Deletion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3624-3624
Author(s):  
Antonella Nai ◽  
Irene Artuso ◽  
Maria Rosa Lidonnici ◽  
Sandro Altamura ◽  
Giacomo Mandelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Globin Gene ◽  
Proteasome Activity ◽  
Mutant Mice ◽  
Erythroid Cells ◽  
Ineffective Erythropoiesis ◽  
Rbc Count

Abstract Transferrin receptor 2 (TFR2), the type 3 hemochromatosis gene, is an activator of the iron hormone hepcidin in the liver and a partner of erythropoietin (EPO) receptor in erythroid cells. The loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity inducing erythrocytosis in mice (Nai et al, Blood 2015). We explored whether deletion of BM Tfr2 improves anemia and ineffective erythropoiesis in β-thalassemias, iron-loading anemias due to recessive β-globin gene mutations. We generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through BM transplantation (BMT). Deletion of BM Tfr2 ameliorates RBC morphology with consistent and persistent increase of RBC count and Hb levels in thalassemic mice, accompanied by reduced iron accumulation. Around 22 weeks after BMT the improvement fades in double mutantanimals: Hb levels return comparable to those of Hbbth3/+mice, while RBC count persists higher. Anemia improvement in double mutant mice reduces serum EPO levels and improves erythropoiesis, in particular 22 weeks after BMT. Overall these data prove that the loss of the beneficial effect of deleting Tfr2 is not accounted for by erythropoiesis failure, but likely by exhaustion of splenic iron consumed by the enhanced erythropoiesis. In order to elucidate the molecular mechanisms of the phenotype improvement, we investigated whether the EPO-EPOR signaling pathway is overactive, as occurs in Tfr2 null erythroid cells (Nai et al, Blood 2015). Taking into account that Tfr2BMKO/Hbbth3/+ mice have lower serum EPO than Hbbth3/+, the expression levels of target genes of the EPOR-JAK2-STAT5 (Erfe and Bcl-xl) and of EPOR-PI3K-AKT pathway (Fasl, Epor and Ccng2) is consistent with the signaling being inappropriately active in double mutant mice. To start unraveling the global molecular/cellular processes underlying the remarkable phenotype amelioration, we performed RNAseq analysis on spleen samples from double mutant and Hbbth3/+ control mice at the time point of maximal erythropoiesis improvement (22 weeks post BMT). Spleens are enlarged in both genotypes with about 80% Ter119+ (erythroid) cells in both. In total we identified 2796 genes (1997 protein coding) differentially regulated between the two genotypes. The analysis of iron-related genes reveals a strong reduction of the expression of the iron exporter Fpn, Hmox1 and Alas2, suggestive of decreased hemolysis and/or of free heme accumulation in double mutants. Gene ontology analysis reveals enrichment of genes involved in cell cycle and proliferation, mitochondrial function, as well as proteasome activity and of most of the antioxidant targets (Sod1, Sod2, Fth1, Txn1, Txn2, Gstpi) of the canonical NF-kB pathway. Underrepresented genes are those involved in lipid handling, leukocyte/lymphocyte differentiation and coagulation. In summary, the RNAseq patterns indicate an increased spleen erythroid commitment and a mitochondrial metabolic shift, similar to the shift occurring during hematopoietic development to sustain erythroid proliferation and differentiation. We speculate that this effect is mediated by the enhanced EPO sensitivity. Interestingly EPO directly stimulates mitochondrial genes expression in adipocytes. Also the increased proteasome activity may significantly contribute to the improved erythropoiesis, since proteasomal degradation is required in the process of erythroblast enucleation. Finally, the activation of the NF-kB antioxidant response may contrast ROS increase and limit ineffective erythropoiesis. In conclusion, targeting erythroid TFR2 might become a novel erythropoiesis stimulating approach, worth to be tested in other forms of anemia. Disclosures Muckenthaler: Novartis: Research Funding. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Lack of the Transcription Factor NFAT (Nuclear Factor of Activated T cells) c2 in Hematopoietic Progenitor Cells Results in Profound Hematological Abnormalities in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1296-1296
Author(s):  
Laleh S. Arabanian ◽  
Michael Haase ◽  
Ivonne Habermann ◽  
Malte von Bonin ◽  
Claudia Waskow ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Hematological Abnormalities

Abstract Abstract 1296 Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow microenvironment in vivo is of considerable interest. We have previously shown that aged mice lacking the transcription factor NFATc2 develop bone marrow hypoplasia, anemia, and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic progenitor cells (HPC) ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the bone marrow hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immunomagnetically purified NFATc2-deficient (ko) HPCs to lethally irradiated wildtype (wt) mice, and vice versa. After a posttransplantation period of 6–8 months, peripheral blood, bone marrow as well as spleen and liver of the transplanted animals were analyzed and compared to wt and ko mice transplanted with control cells. Transplantation of NFATc2-deficient HPCs into wt recipients (ko → wt) induced similar hematological abnormalities as those occurring in non-transplanted ko mice or in ko mice transplanted with ko cells (ko → ko). Compared to wt mice transplanted with wt cells (wt → wt), ko → wt mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their bone marrow. Likewise, ko → wt mice developped clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in wt → wt control animals. Our data demonstrate for the first time, that NFAT transcription factors directly regulate the intrinsic function of hematopoietic progenitor cells in vivo. The transcriptional targets for NFAT in these cells are yet unknown and are the focus of further investigations. Disclosures: No relevant conflicts of interest to declare.

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