Fibrotic Tissues In Human Primary Myelofibrosis With JAK2 V617F Are Developed Clonally From Malignant Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4106-4106
Author(s):  
Takuji Yamauchi ◽  
Takahiro Shima ◽  
Katsuto Takenaka ◽  
Toshihiro Miyamoto ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Myeloid Cells ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Jak2 V617f Mutation ◽  
Fish Analysis ◽  
Hematopoietic Stem ◽  
V617f Mutation

Abstract Primary myelofibrosis (PMF) is a clonal stem cell disorder, characterized by deregulated proliferation of myelofibroblasts. Pathogenesis of PMF has been intensively investigated by analyzing clonality of the disease mainly by X-linked gene inactivation assays. These studies suggested that hematopoiesis in PMF is clonal, whereas fibroblasts are polyclonal not belonging to the PMF clone but they secondarily proliferate in response to cytokines produced by malignant megakaryocytes or monocytes. However, we have previously reported that PMF-initiating cells exist within the circulating CD34+CD38- fraction expressing CD45, whose phenotype is analogous to normal hematopoietic stem cells (HSCs), and this population can reconstitute myelofibrosis after xenotransplantation into NOD/SCID/IL2rg-null mouse newborns (Saito et al. ASH Annual Meeting 2007). In this assay, human PMF HSCs reconstitute PMF-like disease in mouse after transplantation, and strikingly the vast majority of myelofibroblasts were of human origin on FISH analysis, suggesting that myelofibroblasts in PMF can be generated directly from circulating PMF stem cells. We here tried to test this hypothesis by directly analyzing human PMF samples. We performed clonal analyses of fibrotic tissues in PMF patients at the single cell level by utilizing JAK2 V617F mutation as a clonal marker. Nine patients of PMF with JAK2 V617F mutation were enrolled in this study. The fibrotic bone marrow tissues were stained with vimentin, CD34 and KP1 to identify fibroblast-like cells, endothelial cells and myeloid cells, respectively. Single cells of fibrotic tissues were sampled by a laser micro-dissection system, and CD34+CD38-CD45+ HSCs were purified from the blood by FACS. Genomic DNA from single sampled cells was tested for JAK2 V617F mutation. This extensive analysis showed that ∼20-40% of single HSCs and granulocyte/monocyte progenitors (GMPs) had JAK2 V617F. Similar percentages of single vimentin+ fibroblast-like cells, CD34+ endothelial cells and KP-1+ myeloid cells had JAK2 V617F in each patient. Furthermore, each population also constituted similar percentages of heterozygous and homozygous JAK2 V617F as HSCs and GMPs did in each case. Because JAK2 V617F exists in each cell component of myelofibrotic tissues, whose frequency was almost equal to that of PMF HSCs, this study strongly suggests that the majority of myelofibrotic tissue cells belong to the PMF clone, and are differentiated directly from PMF HSCs. These data are compatible with our previous xenotransplantation data. Finally, it is also suggested that JAK2 V617F signaling is not necessary for PMF development. The new understanding of PMF pathophysiology shown here by our experiments might be useful to develop new treatment strategies for human PMF in future studies. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

scholarly journals Myeloid and endothelial cells cooperate to promote hematopoietic stem cells expansion in the fetal niche.

2021 ◽  
Author(s):  
Pietro Cacialli ◽  
Marie-Pierre Mailhe ◽  
Rachel Golub ◽  
Julien Y Bertrand
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Genetic Model ◽  
Fetal Liver ◽  
Myeloid Cells ◽  
Prostaglandin E ◽  
Hematopoietic Stem ◽  
Functional Changes ◽  
The Impact

During embryonic development, very few hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium, that will be expanded in a very specific niche. This fetal HSC niche comprises a complex and dynamic molecular network of interactions between multiple cell types, including endothelial cells (ECs) and mesenchymal stromal cells. It is known that functional changes in the hematopoietic niche, such as aging, vascular cell remodelling or inflammation can directly affect HSCs. Among all these inflammatory regulators, the eicosanoid prostaglandin E (PGE2) has been shown to be very important during embryonic life. However, the precise source of PGE2 in the embryo is still elusive. Here we show that all the genes involved in PGE2 synthesis and transport are expressed by distinct cells of the caudal hematopoietic tissue (CHT) in the zebrafish embryo and in the mouse fetal liver, suggesting that each cell type acts sequentially and collaboratively with the others to produce PGE2 and ultimately expand HSCs. Among these cells, we found myeloid cells (both neutrophils and macrophages) to be absolutely necessary, as they concur to the production of PGH2, the precursor of PGE2. To measure the impact of myeloid cells, we generated a genetic model of myeloid ablation, which caused a loss of HSCs in the CHT, that could be rescued by supplementing zebrafish embryos with PGE2 or PGH2. ECs expressed the slco2b1 transporter to import PGH2, and ptges3, the necessary enzyme to convert this latter into PGE2. Taken altogether, our data show that the triad composed of neutrophils, macrophages and ECs concurs to HSC expansion in the CHT.

Generation of Hematopoietic Stem Cells from Purified Embryonic Endothelial Cells by a Simple and Efficient Strategy

2013 ◽  
Vol 40 (11) ◽  
pp. 557-563 ◽  
Author(s):  
Zhuan Li ◽  
Fan Zhou ◽  
Dongbo Chen ◽  
Wenyan He ◽  
Yanli Ni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Efficient Strategy

Ifnα Attenuates the Disease Phenotype and Extends Survival in Mouse Models of MPN

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 53-53
Author(s):  
Harini Nivarthi ◽  
Andrea Majoros ◽  
Eva Hug ◽  
Ruochen Jia ◽  
Sarada Achyutuni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Models ◽  
Jak2 V617f ◽  
Type I ◽  
Type I Ifn ◽  
Hematopoietic Stem ◽  
Exon 9 ◽  
Ifn Treatment

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

2019 ◽  
Vol 44 (4) ◽  
pp. 492-498
Author(s):  
Gonca Gulbay ◽  
Elif Yesilada ◽  
Mehmet Ali Erkurt ◽  
Harika Gozukara Bag ◽  
Irfan Kuku ◽  
...  
Keyword(s):  
Blood Cell ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Hematological Parameters ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
V617f Mutation ◽  
The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1783-1783
Author(s):  
Mariela Sivina ◽  
Takeshi Yamada ◽  
Natalie Dang ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4885-4885
Author(s):  
Taghi Manshouri ◽  
Zeev Estrov ◽  
Alfonso Quintas-Cardama ◽  
Jorge Cortes ◽  
Francis Giles ◽  
...  
Keyword(s):  
Cell Line ◽  
Anticancer Agents ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
V617f Mutation

Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.

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