A Novel Strategy for Expanding Primitive Leukemic Cells from Chronic Phase CML Patients by Forced Overexpression of a NUP98-HOXA10 Homeodomain Fusion Gene.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1078-1078
Author(s):  
Ivan Sloma ◽  
Suzan Imren ◽  
Yun Zhao ◽  
Keith Humphries ◽  
Connie J. Eaves
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fusion Gene ◽  
Low Frequency ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Human Cord Blood ◽  
Hematopoietic Stem

Abstract Analysis of the leukemic stem cell compartment in CML patients with chronic phase disease remains a major challenge. This is due to the usually low frequency of these cells in the bone marrow and blood of most patients regardless of the WBC count and the fact that they are typically outnumbered by normal hematopoietic stem cells from which they cannot be currently separated. Moreover, thus far it has not been possible to identify conditions for their selective expansion in vitro or in vivo. To pursue this goal, we have begun to explore the effects of certain HOX gene-containing constructs on primitive chronic phase CML cells based on previous evidence that these genes markedly enhance the expansion of primitive normal murine and human cord blood cell numbers without inducing leukemia. Lineage-negative peripheral blood or bone marrow cells from 3 chronic phase CML patients (with >93%, <20% and <6% Ph+ LTC-ICs by G-banding karyotyping) were pre-stimulated overnight in a medium containing a serum substitute and 100 ng/ml hSteel Factor (SF), 100 ng/ml hFlt3-ligand and 20 ng/ml each of hIL-3, hIL-6 and hG-CSF. Cells were then exposed to a lenti-PGK-GFP virus with or without an upstream MDUS-NUP98-HOXA10 homeodomain (HD) element for 5 hours in the same medium. After removal of the virus, the cells were maintained in culture under the same conditions for 2 more days to allow full expression of the transduced genes. At this point, both cultures contained the same number of total cells, GFP+ cells and clonogenic progenitors (BFU-E + CFU-GM + CFU-GEMM); i.e., 2.2±0.5 x105 vs 2.2±0.6 x105 total cells, 1.0±0.2 x105 vs 1.3±0.3 x105 GFP+ cells, 3.6±1.7 x104 vs 3.4±1.7 x104 total CFCs and 1.7±0.9 x104 vs 2.4±1.3 x104 GFP+ CFCs per 105 starting lin- cells. However, after the 2-day post-transduction, cells had been maintained for 6 weeks in longterm cultures (LTCs) containing murine stromal cells producing hIL-3, hSF and hG-CSF, we noted a markedly higher (4 to 74-fold) output of CFCs from the NUP98-HOXA10HD-transduced cells. Moreover, whereas the proportion of GFP+ CFCs in the 2-day post-transduction cultures was on average only 31% and 48 % for the control and tested cells respectively, this increased to >98% in the 6-week LTCs initiated with cells that were overexpressing NUP98-HOXA10HD but remained constant at 39% in the control LTCs - suggesting a significant growth advantage conferred by the NUP98A10HD transgene. Importantly, RT-PCR genotyping of the colonies in these assays showed the majority of LTC-IC-derived CFCs from the NUP98-HOXA10HD-transduced cells to be BCR-ABL+, indicative of an even greater output of CFCs by the NUP98-HOXA10HD transduced BCR-ABL+ vs normal cells. These results highlight the potential of NUP98-HOXA10HD to selectively expand primitive CML cells isolated directly from chronic phase patients which will facilitate their further investigation and use to screen and validate new therapeutic agents.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

scholarly journals HIF-1α is a negative regulator of plasmacytoid DC development in vitro and in vivo

Blood ◽  
2012 ◽  
Vol 120 (15) ◽  
pp. 3001-3006 ◽  
Author(s):  
Andreas Weigert ◽  
Benjamin Weichand ◽  
Divya Sekar ◽  
Weixiao Sha ◽  
Christina Hahn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
Negative Regulator ◽  
Low Oxygen ◽  
Hematopoietic Stem ◽  
Lineage Differentiation ◽  
Marrow Cells

Abstract Hypoxia-inducible factors (HIFs) regulate hematopoiesis in the embryo and maintain hematopoietic stem cell function in the adult. How hypoxia and HIFs contribute to hematopoietic lineage differentiation in the adult is ill defined. Here we provide evidence that HIF-1 limits differentiation of precursors into plasmacytoid dendritic cells (pDCs). Low oxygen up-regulated inhibitor of DNA binding 2 (ID2) and suppressed Flt3-L–induced differentiation of bone marrow cells to pDCs in wild-type but not HIF-1αfl/fl LysM-Cre bone marrow cells. Moreover, pDC differentiated normally in hypoxic ID2−/− bone marrow cultures. Finally, we observed elevated pDC frequencies in bone marrow, blood, and spleen of HIF-1αfl/fl LysM-Cre and ID2−/−, but not HIF-2αfl/fl LysM-Cre mice. Our data indicate that the low oxygen content in the bone marrow might limit pDC development. This might be an environmental mechanism to restrict the numbers of these potentially autoreactive cells.

scholarly journals Expansion of senescent megakaryocyte-lineage cells maintains CML cell leukemogenesis

2020 ◽  
Vol 4 (24) ◽  
pp. 6175-6188
Author(s):  
Yamato Tanabe ◽  
Shimpei Kawamoto ◽  
Tomoiku Takaku ◽  
Soji Morishita ◽  
Atsushi Hirao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Fusion Gene ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Enhanced Production ◽  
Tgf Β1

Abstract BCR-ABL, an oncogenic fusion gene, plays a central role in the pathogenesis of chronic myeloid leukemia (CML). Oncogenic signaling induces oncogene-induced senescence and senescence-associated secretory phenotype (SASP), which is characterized by enhanced production of various cytokines. BCR-ABL gene transduction confers senescent phenotype in vitro; however, the in vivo relevance of senescence has not been explored in this context. Transplantation of BCR-ABL–expressing hematopoietic stem/progenitor cells caused CML in mice with an increase in bone marrow BCR-ABL+CD41+CD150+ leukemic megakaryocyte-lineage (MgkL) cells, which exhibited enhanced senescence-associated β-galactosidase staining and increased expression of p16 and p21, key molecules that are crucially involved in senescence. Moreover, knockout of p16 and p21 genes reduced both BCR-ABL–induced abnormal megakaryopoiesis and the maintenance of CML cell leukemogenic capacity, as evidenced by attenuated leukemogenic capacity at secondary transplantation. The expression of transforming growth factor-β1 (TGF-β1), a representative SASP molecule, was enhanced in the leukemic MgkL cells, and TGF-β1 inhibition attenuated CML cell leukemogenic capacity both in vitro and in vivo. Furthermore, BCR-ABL–expressing MgkL cells displayed enhanced autophagic activity, and autophagy inhibition reduced bone marrow MgkL cell number and prolonged the survival of CML mice, which had transiently received the tyrosine kinase inhibitor, imatinib, earlier. Thus, BCR-ABL induced the expansion of senescent leukemic MgkL cells, which supported CML leukemogenesis by providing TGF-β1.

scholarly journals Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 421-432 ◽  
Author(s):  
D DiGiusto ◽  
S Chen ◽  
J Combs ◽  
S Webb ◽  
R Namikawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Fetal Bone ◽  
Cd34 Antigen

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2237-2237
Author(s):  
Ravindra Majeti ◽  
Christopher Y. Park ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Abstract Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2440-2440
Author(s):  
Nils Heinrich Thoennissen ◽  
Tadayuki Akagi ◽  
Sam Abbassi ◽  
Daniel Nowak ◽  
Ann George ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Marrow Cells

Abstract CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

Cellular and Molecular Targets of MLL-AF9 in a Novel Conditional Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1280-1280
Author(s):  
Vaia Stavropoulou ◽  
Susanne Kaspar ◽  
Laurent Brault ◽  
Sabine Juge ◽  
Stefano Morettini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Mouse Model ◽  
Prognostic Markers ◽  
Bone Marrow Cells ◽  
Median Latency ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 1280 Previous studies have shown that the expression of several leukemia-associated mixed lineage leukemia (MLL) fusion genes transformed human and mouse bone marrow cells in vitro and in vivo. In order to dissect the molecular and cellular targets of the MLL-AF9 fusion, we generated a novel inducible doxycycline (DOX)-regulated transgenic mouse model. Conditional ex vivo activation of MLL-AF9 induced aberrant self-renewal and impaired differentiation of long-term or short-term hematopoietic stem (LT-HSC and ST-HSC), common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) cells in a fully reversible manner. Direct activation of the fusion in vivo or after transplantation of transgenic bone marrow cells into irradiated hosts induced an aggressive and transplantable disease after a median latency of 80days characterized as acute myelo-monocytic leukemia closely mimicking the human disease. Fusion gene expression and leukemia induction was DOX dosage dependent and reversible upon DOX removal. Activation of MLL-AF9 in isolated LT-HSC or GMP cells in vitro or in vivo resulted in the accumulation of immature blast-like cells with similar immunophenotypes. However, MLL-AF9-expressing stem and progenitor cells displayed distinct properties such as colony formation, differentiation and resistance to chemotherapeutic drugs. Turning-off the fusion resulted in multi-lineage differentiation of LT-HSC-derived cells, whereas GMP-derived cells were limited to mature macrophages and granulocytes suggesting partial maintenance of their original identity. In line with these in vitro observations, lower cell numbers of transplanted LT-HSCs induced a more aggressive leukemia with a significantly shorter latency as compared to ST-HSC, CMP or GMPs. Immunophenotypically 15% of the LT-HSC derived leukemias displayed a CMP–like phenotype and had a median latency of 37d (“early”) whereas the rest of the cases displayed a GMP-like phenotype with a median latency of 73d (“late”). In contrast, only GMP-like phenotypes and longer latencies were observed upon transplanting ST-HSCs (75d), CMPs (72d) or GMPs (100d). Transplantation of blasts from “early” LT-HSC- and GMP-derived leukemias into secondary recipients induced the disease after similar latency, however, cytarabine (Ara-C) treatment significantly delayed only the disease induced by GMP- but not by LT-HSC-derived blasts. Gene expression profiling in immortalized pre-leukemic cells revealed down-regulation of over 300 genes, including several well-known MLL targets such as Meis1, HoxA5, HoxA9 and HoxA10 upon reducing the levels of MLL-AF9 expression. Likewise, we observed a global decrease in histone H3 lysine 79 dimethylation consistent with a Dot1l function in MLL-AF9 driven leukemia. LT-HSC-derived (“early”) blasts displayed distinct genetic signatures with > 400 genes highly and > 1300 genes lowly expressed (p001 fc1.5), clearly separating them from the GMP-derived blasts. Evi-1 and Erg, two prognostic markers in patient-derived gene signatures, stood out among these genes. The aggressive “early” LT-derived murine leukemias showed high Evi-1 and Erg expression levels (Evi-1 high, Erg high) as compared to the “late” LT-derived (Evi-1 low, Erg high) or the GMP-derived leukemias (Evi-1 low, Erg low). These observations suggest that the previously reported poor prognosis associated with elevated EVI-1 and/or ERG expression might directly reflect the cell of origin of the disease. We are currently exploiting our highly informative MLL-AF9 disease model to evaluate the functional relevance of novel origin-dependent MLL-AF9 target genes and to identify novel prognostic markers and therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

PML-RARα Deregulates an Unexpectedly Small Number of Genes in Pre-Leukemic Promyelocytes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3526-3526
Author(s):  
Coline M Gaillard ◽  
Taku A Tokuyasu ◽  
Emmanuelle Passegué ◽  
Scott C. Kogan
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Leukemic Cells ◽  
Cytokine Signaling ◽  
Proliferation Capacity ◽  
Number Of Genes

Abstract Abstract 3526 Background: Acute Promyelocytic Leukemia (APL) is characterized by the accumulation in the blood and bone marrow of abnormal promyelocytes, which have the ability to transfer the disease to secondary recipients in animal models. The PML-RARα fusion protein is thought to be the primary abnormality implicated in the pathology, and is believed to prevent transcription of genes necessary for normal myeloid development and differentiation. Identifying PML-RARα targets is critical for understanding the road to leukemic transformation. However, such targets have so far been identified using cell line assays in vitro, murine cells differentiated into promyelocytes in vitro, or fully transformed murine or human leukemic cells. Focusing on the cell population in which the transforming potential is acquired, we describe here a novel strategy to identify the transcriptomic dysregulation induced by PML-RARα expression in maturing myeloid populations in vivo. Methods: We utilize a murine model of human APL in which the human PML-RARα fusion gene is expressed under the control of the MRP8 promoter, driving its expression in maturing myeloid populations. Those animals can be described as pre-leukemic since they eventually develop leukemia when additional mutations occur. Fresh bone marrows from normal (Fvb/n) or pre-leukemic (PML-RARα) animals were harvested. Using an improved cell surface antigen staining strategy and fluorescence-activated cell sorting, three populations of increasingly differentiated myeloid populations have been sorted (Granulocyte Macrophage Progenitor, Early promyelocyte and Late promyelocyte). RNA was extracted and submitted for whole-genome microarray analysis. In addition, we are using a variety of bioinformatics approaches to decipher the network of novel interactions driven by PML-RARα expression. Results: Markers used in our sorting strategy were validated in the dataset, including CD34 and Gr1. In the normal samples, markers of neutrophil maturation increased, largely as expected, and a number of early transcription factors decreased in an expected manner including Hoxa9 and Meis1. One remarkable finding was that despite the previously described ability of PML-RARα to regulate transcription from multiple sites in the genome, only a small number of genes were differentially impacted by the expression of this protein. Surprisingly, well-known regulators of myeloid differentiation that have been implicated in the retinoic acid responsiveness of APL including Sfpi1 (PU.1) and Cebpa were not differentially expressed. However, in pre-leukemic samples PML-RARα did cause decreased expression of multiple neutrophilic granule genes including Ltf, Mmp9 and Ngp. The gene most upregulated in the pre-leukemic samples was Spp1 which encodes the osteopontin phosphoprotein. Of interest, we identified the myeloid tumor suppressor Irf8 to be downregulated 5 fold in the presence of PML-RARα. To investigate the importance of IRF8 levels in APL initiation, we transplanted Irf8+/+ PML-RARα or Irf8+/− PML-RARα bone marrow into irradiated recipients. Despite the potential for decreased expression of IRF8 to contribute to APL, we observed no difference. This result does not confirm a role for IRF8 in APL pathogenesis, but further investigations are needed to exclude such a role. Bioinformatics studies highlighted enrichment in cell cycle-related genes upon PML-RARα expression, suggesting a possible difference in the proliferation capacity of the pre-leukemic cells, which is currently under investigation. Conclusions: We found that in vivo the transcriptome was only modestly dysregulated by the presence of PML-RARα. These observations open up new questions on the role of the fusion protein in pathogenesis: How does PML-RARα prime pre-leukemic cells for full transformation? How do secondary events allow an initiated cell to advance to a fully transformed state? Such questions are currently being investigated, with a special interest on looking at the cooperation between PML-RARα and activated cytokine signaling in leukemia initiation. Disclosures: No relevant conflicts of interest to declare.

FIP1L1/PDGFRα synergizes with SCF to induce systemic mastocytosis in a murine model of chronic eosinophilic leukemia/hypereosinophilic syndrome

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2500-2507 ◽  
Author(s):  
Yoshiyuki Yamada ◽  
Abel Sanchez-Aguilera ◽  
Eric B. Brandt ◽  
Melissa McBride ◽  
Nabeel J. H. Al-Moamen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fusion Gene ◽  
Systemic Mastocytosis ◽  
Hypereosinophilic Syndrome ◽  
Growth Factor Receptor ◽  
Hematopoietic Stem ◽  
Chronic Eosinophilic Leukemia ◽  
Factor Receptor Alpha

Abstract Expression of the fusion gene FIP1-like 1/platelet-derived growth factor receptor alpha (FIP1L1/PDGFRα, F/P) and dysregulated c-kit tyrosine kinase activity are associated with systemic mastocytosis (SM) and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES). We analyzed SM development and pathogenesis in a murine CEL model induced by F/P in hematopoietic stem cells and progenitors (HSCs/Ps) and T-cell overexpression of IL-5 (F/P-positive CEL mice). These mice had more mast cell (MC) infiltration in the bone marrow (BM), spleen, skin, and small intestine than control mice that received a transplant of IL-5 transgenic HSCs/Ps. Moreover, intestinal MC infiltration induced by F/P expression was severely diminished, but not abolished, in mice injected with neutralizing anti–c-kit antibody, suggesting that endogenous stem cell factor (SCF)/c-kit interaction synergizes with F/P expression to induce SM. F/P-expressing BM HSCs/Ps showed proliferation and MC differentiation in vitro in the absence of cytokines. SCF stimulated greater migration of F/P-expressing MCs than mock vector–transduced MCs. F/P-expressing bone marrow–derived mast cells (BMMCs) survived longer than mock vector control BMMCs in cytokine-deprived conditions. The increased proliferation and survival correlated with increased SCF-induced Akt activation. In summary, F/P synergistically promotes MC development, activation, and survival in vivo and in vitro in response to SCF.

scholarly journals Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow

2015 ◽  
Vol 212 (3) ◽  
pp. 385-399 ◽  
Author(s):  
Jaeyop Lee ◽  
Gaëlle Breton ◽  
Thiago Yukio Kikuchi Oliveira ◽  
Yu Jerry Zhou ◽  
Arafat Aljoufi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cell ◽  
Cord Blood ◽  
Culture System ◽  
Human Monocyte ◽  
Lymphoid Tissues ◽  
Human Cord Blood ◽  
Hematopoietic Stem

In mice, two restricted dendritic cell (DC) progenitors, macrophage/dendritic progenitors (MDPs) and common dendritic progenitors (CDPs), demonstrate increasing commitment to the DC lineage, as they sequentially lose granulocyte and monocyte potential, respectively. Identifying these progenitors has enabled us to understand the role of DCs and monocytes in immunity and tolerance in mice. In humans, however, restricted monocyte and DC progenitors remain unknown. Progress in studying human DC development has been hampered by lack of an in vitro culture system that recapitulates in vivo DC hematopoiesis. Here we report a culture system that supports development of CD34+ hematopoietic stem cell progenitors into the three major human DC subsets, monocytes, granulocytes, and NK and B cells. Using this culture system, we defined the pathway for human DC development and revealed the sequential origin of human DCs from increasingly restricted progenitors: a human granulocyte-monocyte-DC progenitor (hGMDP) that develops into a human monocyte-dendritic progenitor (hMDP), which in turn develops into monocytes, and a human CDP (hCDP) that is restricted to produce the three major DC subsets. The phenotype of the DC progenitors partially overlaps with granulocyte-macrophage progenitors (GMPs). These progenitors reside in human cord blood and bone marrow but not in the blood or lymphoid tissues.

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