Differential expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic stem cells and leukemia

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3945-3950 ◽  
Author(s):  
Yunfang Jiang ◽  
Hong Liang ◽  
Wei Guo ◽  
Lazar V. Kottickal ◽  
Lalitha Nagarajan
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Receptor Activation ◽  
Myelogenous Leukemia ◽  
Blood Leukocytes ◽  
Hematopoietic Stem ◽  
Acid Protein ◽  
Acute Myelogenous ◽  
Inactivating Mutations

Abstract SMADs are evolutionarily conserved transducers of the differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands. Upon receptor activation, the ligand-restricted SMADs1–35 are phosphorylated in the C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene to the nucleus to mediate target gene expression. Frequent inactivating mutations of SMAD4, or less common somatic mutations ofSMAD2 seen in solid tumors, suggest that these genes have a suppressor function. However, there have been no identified mutations of SMAD5, although the gene localizes to the critical region of loss in chromosome 5q31.1 (chromosome 5, long arm, region 3, band 1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia (AML). A ubiquitously expressed novel isoform,SMAD5β, encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations. The levels of SMAD5β transcripts are higher in the undifferentiated CD34+ hematopoietic stem cells than in the terminally differentiated peripheral blood leukocytes, thereby implicating the β form in stem cell homeostasis. Yeast 2-hybrid interaction assays reveal the lack of physical interactions between SMAD5β and SMAD5 or SMAD4. The expression ofSMAD5β may represent a novel mechanism to protect pluripotent stem cells and malignant cells from the growth inhibitory and differentiation signals of BMPs.

Differential expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic stem cells and leukemia

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3945-3950 ◽  
Author(s):  
Yunfang Jiang ◽  
Hong Liang ◽  
Wei Guo ◽  
Lazar V. Kottickal ◽  
Lalitha Nagarajan
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Receptor Activation ◽  
Myelogenous Leukemia ◽  
Blood Leukocytes ◽  
Hematopoietic Stem ◽  
Acid Protein ◽  
Acute Myelogenous ◽  
Inactivating Mutations

SMADs are evolutionarily conserved transducers of the differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands. Upon receptor activation, the ligand-restricted SMADs1–35 are phosphorylated in the C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene to the nucleus to mediate target gene expression. Frequent inactivating mutations of SMAD4, or less common somatic mutations ofSMAD2 seen in solid tumors, suggest that these genes have a suppressor function. However, there have been no identified mutations of SMAD5, although the gene localizes to the critical region of loss in chromosome 5q31.1 (chromosome 5, long arm, region 3, band 1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia (AML). A ubiquitously expressed novel isoform,SMAD5β, encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations. The levels of SMAD5β transcripts are higher in the undifferentiated CD34+ hematopoietic stem cells than in the terminally differentiated peripheral blood leukocytes, thereby implicating the β form in stem cell homeostasis. Yeast 2-hybrid interaction assays reveal the lack of physical interactions between SMAD5β and SMAD5 or SMAD4. The expression ofSMAD5β may represent a novel mechanism to protect pluripotent stem cells and malignant cells from the growth inhibitory and differentiation signals of BMPs.

Tribbles Homolog 2 (Trib2) Inactivates C/EBPalpha and Causes Acute Myelogenous Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 776-776 ◽  
Author(s):  
Karen Keeshan ◽  
Yiping He ◽  
Bas J. Wouters ◽  
Olga Shestova ◽  
Lanwei Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Acute Myelogenous Leukemia ◽  
Function Analysis ◽  
Myelogenous Leukemia ◽  
Serine Threonine Kinase ◽  
Hematopoietic Stem ◽  
Aggressive Cancer ◽  
Myeloid Development ◽  
Acute Myelogenous

Abstract Trib2 is a member of the Trib family of serine/threonine kinase-like proteins (Trib1, Trib2, Trib3), whose function in hematopoiesis is not documented. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML), with a median survival of 179 days. Retroviral Trib2 expression in hematopoietic stem cells perturbed myeloid development, enhanced progenitor proliferation, and directly inhibited the function of C/EBPα, a critical transcription factor that is frequently dysregulated in AML. Furthermore, an analysis of microarray data generated from 285 AML patient samples identified elevated Trib2 expression in a distinct subset of patients in a cluster with a high frequency of C/EBPα mutations. In mechanistic studies, we found that Trib2 associated with and led to the proteasomal-dependent degradation of C/EBPα. To determine the structural requirements for Trib2 to inhibit C/EBPα, and to induce AML, we have initiated a structure/function analysis of Trib2. Together, our data identify Trib2 as a novel oncogene that induces AML through a mechanism involving inactivation of C/EBPα. The identification of Trib2 as potent leukemogen points to new pathogenic mechanisms and possible therapeutic opportunities in this aggressive cancer, which is not currently curable in the majority of patients.

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4185-4193 ◽  
Author(s):  
Hanno Glimm ◽  
IL-Hoan Oh ◽  
Connie J. Eaves
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Cell Activity ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
A Cell

Abstract An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G1fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G0/G1and S/G2/M. Interestingly, the G0 cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle–associated change in in vivo stem cell homing, the cultured G0/G1 and S/G2/M CD34+ CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor β1 that increased the G0/G1 fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle–associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo–manipulated grafts.

scholarly journals Bone Morphogenetic Proteins Regulate the Developmental Program of Human Hematopoietic Stem Cells

1999 ◽  
Vol 189 (7) ◽  
pp. 1139-1148 ◽  
Author(s):  
Mickie Bhatia ◽  
Dominique Bonnet ◽  
Dongmei Wu ◽  
Barbara Murdoch ◽  
Jeff Wrana ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Morphogenetic Proteins ◽  
Transforming Growth Factor ◽  
Critical Role ◽  
Type I ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34+CD38−Lin− stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor β, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34+CD38− phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34+ CD38−Lin− cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.

Stability of Self-Renewal in Hematopoietic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Norman N. Iscove
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Short Term ◽  
Blood Leukocytes ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58 Disclosures: No relevant conflicts of interest to declare.

Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3584-3584
Author(s):  
Takahiro Shima ◽  
Yoshikane Kikushige ◽  
Toshihiro Miyamoto ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Acute Myelogenous Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Myelogenous Leukemia ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Kit Mutation ◽  
Acute Myelogenous

Abstract Abstract 3584 The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow (BM) Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated with Nilotinib

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1570-1570
Author(s):  
Santa Errichiello ◽  
Simona Caruso ◽  
Concetta Quintarelli ◽  
Biagio De Angelis ◽  
Novella Pugliese ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitor Cell ◽  
Newly Diagnosed ◽  
Hematopoietic Stem ◽  
Optimal Response

Abstract Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4185-4193 ◽  
Author(s):  
Hanno Glimm ◽  
IL-Hoan Oh ◽  
Connie J. Eaves
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Cell Activity ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
A Cell

An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G1fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G0/G1and S/G2/M. Interestingly, the G0 cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle–associated change in in vivo stem cell homing, the cultured G0/G1 and S/G2/M CD34+ CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor β1 that increased the G0/G1 fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle–associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo–manipulated grafts.

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