Phenotypic Changes of Hematopoietic Stem Cells and Stromal Cells by Aberrant Expression of GATA−2; Possible Role for the Development of Aplastic Anemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1289-1289
Author(s):  
Yoko Okitsu ◽  
Hideo Harigae ◽  
Shinichiro Takahashi ◽  
Naoko Minegishi ◽  
Norio Suzuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Cdna Array ◽  
Specific Gene ◽  
Drop Formation ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Phenotypic Changes

Abstract (Introduction) Aplastic anemia (AA) is characterized by a fatty bone marrow and the reduced number of hematopoietic stem cell (HSC)s, resulting in peripheral pancytopenia. Several clinical studies have reported that the expression of transcription factor GATA−2 is significantly decreased in CD34 positive cells in AA, suggesting that downregulation of GATA−2 in HSCs may cause the reduction of number of HSCs in AA. Of note, GATA−2 has been shown to be essential for the maintenance of immaturity of preadipocytes as well as HSCs. If GATA−2 functions in the differentiation of stromal preadipocytes in bone marrow, GATA−2 may be involved in the formation of a fatty bone marrow in AA. In order to explore the role of GATA−2 for development of AA, phenotypic changes of HSCs and stromal preadipocytes by aberrant expression of GATA−2 were examined in this study. (Method) First, to analyze phenotypic changes of HSCs by decreased expression of GATA-2, lineage negative, sca-1 positive, c-kit positive (LSK) cells were sorted both from wild-type and GATA-2 haplodeficient mice (kindly provided Dr SH Orkin), and the gene expression profile was compared by cDNA array. The difference of expression level on cDNA array was confirmed by RQ-PCR. Next, in order to examine the role of GATA-2 in the differentiation of stromal preadipocytes, the GATA-2-GFP expression vector was transfected into TBR343, a mouse stromal preadipocyte cell line, and GFP positive cells were sorted. The differentiation capacity of these GATA-2 overexpressing TBR343 cells was compared to that of control cells. In addition, GATA-2 expression was suppressed by using siRNA in TBR343, and the phenotypic change was also examined. (Results) By comparing the gene expression profile, the expression levels of cyclin D1, D3, p21, E2F, c-Myc were found to be decreased in GATA-2 haplodeficient LSKs, suggesting GATA-2 down-regulation in HSCs may inhibit the cell cycle progression. When GATA-2 was overexpressed in TBR343, the oil drop formation and adipocyte-specific gene expression was significantly suppressed. Conversely, When GATA-2 was suppressed in TBR343 by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated, suggesting that decrease of GATA-2 expression accelerates the differentiation of stromal preadipocytes. (Conclusion) These results suggest that suppression of GATA-2 in the bone marrow leads to an inactive cell cycle of HSCs and the accumulation of mature adipocytes, which may contribute to the expression of the representative features of AA, a fatty bone marrow and the reduced number of HSCs.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 683-683
Author(s):  
Christopher Y. Park ◽  
Yoon-Chi Han ◽  
Govind Bhagat ◽  
Jian-Bing Fan ◽  
Irving L Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1505-1505
Author(s):  
Wendy W. Pang ◽  
Elizabeth A. Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Expression Profiles ◽  
Frequency Function ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Aberrant Expression Of miRNA and mRNA Of Cell Cycle and Adhesion-Related Genes In Bone Marrow Stroma Cells Derived From Patients With Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3149-3149 ◽  
Author(s):  
Rimma Berenstein ◽  
Blau Igor Wolfgang ◽  
Axel Nogai ◽  
Marlies Wächter ◽  
Antonio Pezzutto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genetic Alterations ◽  
Bone Marrow Stroma ◽  
Aberrant Expression ◽  
Healthy Donors ◽  
Marrow Stroma ◽  
Bone Marrow Stroma Cells

Abstract Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis. The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p<0,05). We investigated the expression of cell cycle and adhesion-associated genes (CCNE1, CCND1, CDKN1B, VCAM, ICAM, IKK-alpha) in BMSC (passage 4) using SYBR-Green Real-Time PCR and relative quantification by linear regression. A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression. Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns. Disclosures: No relevant conflicts of interest to declare.

The Role of TWIST1 Gene Expression and Hypoxic Microenvironment in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3839-3839
Author(s):  
Emilia Carolina Malafaia ◽  
A. Mario Marcondes ◽  
Ekapun Karoopongse ◽  
Daniele Serehi ◽  
Maria de Lourdes L. F. Chauffaille ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Twist1 Expression ◽  
Disease Biology ◽  
Acute Myeloid

Abstract TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML. Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals The PAF1c Subunit Cdc73 Is Essential for Hematopoiesis and Displays Differential Gene Regulation in MLL-AF9 Driven Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1280-1280
Author(s):  
Nirmalya SAHA ◽  
James Ropa ◽  
Lili Chen ◽  
Hsiang-Yu Hu ◽  
Maria Mysliwski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Flow Cytometry Analysis ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
A Cell

Abstract The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is a multi-protein complex composed of Paf1, Cdc73, Leo1, Ctr9, Rtf1 and WDR61, which have all been shown to play a role in disease progression and different types of cancer. Previous reports demonstrated that the PAF1c is required for MLL-fusion driven acute myeloid leukemia. This is due, in part, to a direct interaction between the PAF1c and wild type MLL or MLL fusion proteins. Importantly, targeted disruption of the PAF1c-MLL interaction impairs the growth of MLL-fusion leukemic cells but is tolerated by normal hematopoietic stem cells. These data point to differential functions for the PAF1c in normal and malignant hematopoietic cells that may be exploited for therapeutic purposes. However, a detailed exploration of the PAF1c in normal hematopoiesis is currently lacking. Here, we utilize a mouse genetic model to interrogate the role of the PAF1c subunit, Cdc73, in the development and sustenance of normal hematopoiesis. Using hematopoietic-specific constitutive and conditional drivers to express Cre recombinase, we efficiently excise floxed alleles of Cdc73 in hematopoietic cells. VavCre mediated excision of Cdc73 results in embryonic lethality due to hematopoietic failure. Characterization of the hematopoietic system demonstrated that cKit+ hematopoietic stem and progenitor cells (HSPC) are depleted due to Cdc73 knockout. We next investigated the role of Cdc73 in adult hematopoiesis using Mx1Cre mediated excision. Conditional knockout of Cdc73 in the adult hematopoietic system leads to lethality within 15 days of Cdc73 excision while no phenotype was observed in heterozygous Cdc73fl/wt controls. Pathological examination of bones in these mice showed extensive bone marrow failure. Flow cytometry analysis revealed that cKit+ HSPCs in adult mice are ablated following loss of Cdc73. Bone marrow transplantation assays demonstrated a cell autonomous requirement of Cdc73 for HSC function in vivo. To perform cellular characterization of HSPCs upon Cdc73 KO, we optimized excision conditions to capture cKit+ HSPCs with excised Cdc73 but before their exhaustion. Flow cytometry analysis demonstrated that Cdc73 KO leads to a cell cycle defect. Cdc73 excision leads to a 2.5 fold increase in the accumulation of HSPCs in the G0 phase of cell cycle with a reduction in the proliferative phases. This is accompanied with an increase in cellular death as indicated by Annexin V staining. Together, these data indicate that Cdc73 is required for cell cycle progression and HSPC survival. To understand the molecular function of Cdc73, we performed RNAseq analysis to identify genes regulated by Cdc73 in HSPCs. We observed 390 genes are upregulated and 433 genes are downregulated upon loss of Cdc73. Specifically, Cdc73 excision results in upregulation of cell cycle inhibitor genes such as p21 and p57, consistent with the cell cycle defect observed following Cdc73 excision. Further, when comparing our results to leukemic cells, we uncovered key differences in Cdc73 gene program regulation between ckit+ hematopoietic cells and MLL-AF9 AML cells. Loss of Cdc73 in leukemic cells leads to downregulation of genes associated with early hematopoietic progenitors and upregulation of myeloid differentiation genes consistent with previous studies. Interestingly, we observed a more even distribution of expression changes (non-directional) within these gene programs following Cdc73 inactivation in HSPCs. Most importantly, while loss of Cdc73 in MLL-AF9 AML cells leads to a profound downregulation of the Hoxa9/Meis1 gene program, excision of Cdc73 in HSPCs results in a modest non-directional change in expression of the Hoxa9/Meis1 gene program. This was attributed to no change in Hoxa9 and Meis1 expression in HSPCs following excision of Cdc73, in contrast to MLL-AF9 cells where these pro leukemic targets are significantly downregulated. Together, these data indicate an essential role for the PAF1c subunit Cdc73 in normal hematopoiesis but differential roles and context specific functions in normal and malignant hematopoiesis, which may be of therapeutic value for patients with AMLs expressing Hoxa9/Meis1 gene programs. Disclosures No relevant conflicts of interest to declare.

CD81 Is Essential for HSC Self-Renewal through Suppressing Proliferation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 76-76 ◽  
Author(s):  
Kuanyin Karen Lin ◽  
Lara Rossi ◽  
Margaret A. Goodell
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Background Level ◽  
Quiescent State ◽  
Self Renewal ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Transplantation Assay

Abstract Hematopoietic stem cells (HSCs) comprise only ~0.02% of the whole bone marrow cells but possess the capacity to extensively proliferate in order to restore hematopoietic homeostasis. Under homeostasis, HSCs are relatively quiescent with a slow cell cycle progression rate. However, upon stimulation, HSCs are able to promptly proliferate and undergo self-renewal to regenerate HSCs as daughter cells. While regulatory mechanisms involved in cell cycle progression are well characterized to be essential for HSC self-renewal, the mechanisms that facilitate the return of proliferating HSC to their quiescent state have been largely overlooked. The expression of CD81 (also called TAPA-1), a transmembrane protein that belongs to the Tetraspanin family, has been found associated with HSC proliferation. While CD81 is normally absent on HSC, it becomes markedly upregulated during HSC proliferation (Figure 1). To understand the function of CD81 in regenerating HSCs, we utilized a murine stem cell retroviral vector to deliver genes into 5-FU treated bone marrow progenitors to test the effect of enforced CD81 overexpression on HSC. The CD81-transduced proliferating progenitors were found to give rise to an increased number of phenotypically-defined HSC (SP-KLS) without significantly affecting the homeostasis in peripheral organs. In addition, we also characterized the HSCs from CD81 knock-out mice. We discovered that CD81-null HSC failed to engraft in peripheral blood of secondary recipients in serial transplantation assays (Figure 2), suggesting a role of CD81 in preserving a functional HSC compartment during proliferation-induced stress. When investigating further, we discovered that CD81 is a cell cycle suppressor for HSC, as the CD81KO HSCs are delayed in returning quiescence. In addition, clustering of CD81 on the HSC cell membrane using a monoclonal antibody rapidly induced a quiescent phenotype. This was found to be associated with an altered phosphorylation level of AKT, an inhibitor of the transcription factor FOXO1a and FOXO3a, which have been reported to be essential for HSC self-renewal through suppressing HSC proliferation. Taken together, these results demonstrate an essential role of CD81 in HSC self-renewal, and a novel mechanism that advances quiescence from a proliferating state. Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01). Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01).

Nucleophosmin-1 Interacts with CCAAT Enhancer Binding Protein Alpha (C/EBPα) to Facilitate Granulocyte Maturation: Implications in MDS and AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2768-2768
Author(s):  
Arati Khanna-Gupta ◽  
Jian Chen ◽  
Matthew Silver ◽  
Hong Sun ◽  
Nirmalee Abayasekara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Molecular Basis ◽  
Conflicts Of Interest ◽  
Chromatin Condensation ◽  
Normal Karyotype ◽  
Specific Gene ◽  
Mouse Bone Marrow ◽  
Chromosome 5Q

Abstract Abstract 2768 Poster Board II-744 NPM-1, a gene that resides on chromosome 5q, is a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones. NPM-1 is a multifunctional protein and has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestation (E11.5) due to severe anemia, underscoring the role of this gene in embryonic development. NPM-1 has been shown to be deleted or involved with chromosomal translocations in several hematologic malignancies. Mutant NPM-1 (NPMc+) has been found to be aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Additionally, mutations in the NPM1 gene have been described in 50% of normal karyotype AML patients. This makes NPM-1 one of the most frequently mutated genes in AML. The NPM-1 gene maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans, and is deleted in 50% of MDS associated with 5q deletions. NPM-1+/− mice develop a hematological syndrome similar to that observed in MDS patients. NPM-1 thus appears to act as a haploinsufficient tumor suppressor in the hematological compartment, the molecular basis of which remains unexplored. The NPM-1 deficient model provides an excellent platform to interrogate not only the molecular basis of MDS but also to study AML progression. To further explore the role of NPM1 in myeloid malignancy, we have generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. We demonstrate compromised neutrophil function and neutrophil-specific gene expression in the NPM1+/− cells similar to that observed in Npm1+/− mouse bone marrow, and attribute these observations to decreased expression of the master myeloid regulator C/EBPα in the Npm1+/− cells. We have demonstrated that overexpression of wildtype NPM1 but not mutant NPM1 (NPMc+) upregulates C/EBPα expression, and that overexpression of C/EBPα in Npm1 deficient cells rescues the myeloid phenotype. We further demonstrate by co-immunoprecipitation and oligonucleotide pulldown assays that NPM1 and C/EBPα physically interact. Our observations suggest that as a result of protein-protein interactions at specific cis elements in the promoters of C/EBPα target genes, C/EBPα and NPM1 act in concert to facilitate normal neutrophil gene expression and function. Interestingly, mutations in the C/EBPα gene have also been implicated in normal karyotype AML. We demonstrate for the first time a connection between these two components of the myeloid compartment, dysregulation of which can lead to disruption of the myeloid maturation program. Taken together, our observations may explain the defective myeloid phenotype observed when NPM1 expression is compromised in MDS and AML. We are now poised to examine the effects of knocking down genes such as RPS14 (the pathogenic gene in the 5q- syndrome) or overexpressing ones such as FLT3-ITD (associated with MDS/AML harboring NPM1 mutations) in our factor-dependent NPM1+/− cell line, to determine if these genes cooperate with NPM1 deficiency to render the cells factor independent. This cell system should be a useful model that will allow us to further dissect the role of NPM-1 in MDS and AML at the molecular level. Disclosures: No relevant conflicts of interest to declare.

Coordinating Self-Renewal and Proliferation via MLL1-HOX Pathways and Beyond

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-28-SCI-28
Author(s):  
Patricia Ernst ◽  
Erika L. Artinger ◽  
Bibhu P. Mishra ◽  
Kristin M. Zaffuto ◽  
Bin E. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Transcriptional Regulators ◽  
Conditional Knockout ◽  
Specific Gene ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Specific Gene Expression ◽  
Lineage Specific Gene

Abstract Abstract SCI-28 Epigenetic regulation of gene expression plays a central role in normal hematopoietic stem cell (HSC) maintenance and leukemogenesis. The histone methyltransferase, MLL1, is essential for the maintenance of HSCs and is a common target of chromosomal translocations that result in acute leukemia. To discover genetic networks regulated by MLL1 in HSCs, we identified genes that were acutely deregulated upon Mll1 loss in HSCs, using a conditional knockout approach and lineage-negative, c-Kit+, Sca-1+, CD48-negative (LSK/CD48neg) cells. The majority of genes that changed were proliferation-associated genes, upregulated in Mll1−/− LSK/CD48neg cells. This reflected the fact that Mll1-deficient HSCs exhibit increased proliferation in vivo, a phenotype previously documented using the Mx1-cre inducible model. To determine whether the increased proliferation was cell-intrinsic, we performed single cell proliferation studies in serum-free medium containing SCF, IL-11, and Flt3L. We found that Mll1−/− LSK/CD48neg single cells entered the cell cycle earlier and that each cell cycle was shorter than wild-type controls. Evidence for failure to suppress lineage-specific gene expression was also observed; specifically, five percent of the upregulated genes encoded erythroid-specific proteins. These included erythroid transcriptional regulators such as GATA1 and KLF1, but also structural proteins such as spectrin, KEL, and EpoR. The relationship between erythroid-lineage genes and Mll1 was unique, since no other lineage-specific programs were upregulated in Mll1−/− LSK/CD48neg cells. Among the genes downregulated upon Mll1 loss, the largest category was comprised of transcriptional regulators, including Mecom, Pbx1, and Prdm16, which are known to control HSC self-renewal and quiescence. As observed in many other tissues, Mll1−/− LSK/CD48neg cells also exhibited reduced Hoxa9 expression. Interestingly, not all identified MLL1 target genes required menin, a cofactor thought to participate in directing MLL1 to particular genomic loci in vivo, and not all targets were Mll1-dependent in nonhematopoietic tissues. Chromatin immunoprecipitation and functional studies suggest that the identified genes act within a series of parallel pathways as direct transcriptional targets of MLL1. Interestingly, reexpression of Prdm16 alone could rescue Mll1-deficient cells from rapid attrition in bone marrow chimeras. Furthermore, Prdm16 corrected the hyperproliferation phenotype of Mll1−/− LSK/CD48neg cells. These data demonstrate that MLL1 coordinately regulates proliferation, lineage-specific gene expression programs, and self-renewal. By elucidating the normal MLL1-dependent transcriptional network within HSCs, we show that this pathway is overlapping but distinguishable from the leukemogenic pathway, suggesting that targeted therapy with minimal side effects on hematopoiesis will be feasible. Disclosures: No relevant conflicts of interest to declare.

Critical Role Of Jak2 In The Maintenance and Function Of Adult Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1180-1180
Author(s):  
Hajime Akada ◽  
Saeko Akada ◽  
Golam Mohi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Receptor Protein ◽  
Specific Gene ◽  
Hematopoietic Stem ◽  
And Function

Abstract Hematopoietic stem cells (HSCs) play an essential role in the long-term maintenance of hematopoiesis. Various intracellular signaling proteins, transcription factors and extracellular matrix proteins contribute to the maintenance and function of HSCs. Jak2, a member of the Janus family of non-receptor protein tyrosine kinases, is activated in response to a variety of cytokines. It has been shown that germ-line deletion of Jak2 results in embryonic lethality whereas post-natal or adult stage deletion of Jak2 results in anemia and thrombocytopenia in mice. However, the role of Jak2 in the maintenance and function of adult HSCs has remained elusive. Understanding the normal function of Jak2 in adult HSC/progenitors is of considerable significance since mutations in Jak2 have been associated with several myeloproliferative neoplasms (MPNs), and most patients treated with Jak2 inhibitors exhibit significant hematopoietic toxicities. To assess the role of Jak2 in adult HSCs, we have utilized a conditional Jak2 knock-out (Jak2 floxed) allele and an inducible MxCre line that can efficiently express Cre recombinase in adult HSC/progenitors after injections with polyinosine-polycytosine (pI-pC). We have found that deletion of Jak2 in adult mice results in pancytopenia, bone marrow aplasia and 100% lethality within 25 to 42 days after pI-pC induction. Analysis of the HSC/progenitor compartments revealed that Jak2-deficiency causes marked decrease in long-term HSCs, short-term HSCs, multipotent progenitors and early progenitors of all hematopoietic lineages, indicating a defect at the earliest stage of adult hematopoietic development. We have found that deletion of Jak2 leads to increased HSC cell cycle entry, suggesting that Jak2-deficiency results in loss of quiescence in HSCs. Jak2-deficiency also resulted in significant apoptosis in HSCs. Furthermore Jak2-deficient bone marrow cells were severely defective in reconstituting hematopoiesis in lethally-irradiated recipient animals. Competitive repopulations experiments also show that Jak2 is essential for HSC functional activity. We also have confirmed that the requirement for Jak2 in HSCs is cell-autonomous. To gain insight into the mechanism by which Jak2 controls HSC maintenance and function, we have performed phospho flow analysis on HSC-enriched LSK (lin-Sca-1+c-kit+) cells. TPO and SCF-evoked Akt and Erk activation was significantly reduced in Jak2-deficient LSK compared with control LSK. Stat5 phosphorylation in response to TPO was also completely inhibited in Jak2-deficient LSK cells. In addition, we observed significantly increased intracellular reactive oxygen species (ROS) levels and enhanced activation of p38 MAPK in Jak2-deficient LSK cells, consistent with the loss of quiescence observed in Jak2-deficient HSCs. Treatment with ROS scavenger N-acetyl cysteine partially rescued the defects in Jak2-deficient HSCs in reconstituting hematopoiesis in lethally irradiated recipient animals. Gene expression analysis revealed significant downregulation of HSC-specific gene sets in Jak2-deficient LSK cells. Taken together, our data strongly suggest that Jak2 plays a critical role in the maintenance of quiescence, survival and self-renewal of adult HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology

2021 ◽  
Vol 22 (8) ◽  
pp. 4099
Author(s):  
Ioanna E. Stergiou ◽  
Konstantinos Kambas ◽  
Aikaterini Poulaki ◽  
Stavroula Giannouli ◽  
Theodora Katsila ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hypoxia Inducible Factor ◽  
Aberrant Expression ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Transcription Activity ◽  
Hypoxia Inducible Factor 1 ◽  
Abnormal Mitochondria

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

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