scholarly journals Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Camélia Benlabiod ◽  
Maira da Costa Cacemiro ◽  
Audrey Nédélec ◽  
Valérie Edmond ◽  
Delphine Muller ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Extramedullary Hematopoiesis ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Reading Frame ◽  
Reticulin Fibers ◽  
Stem Cell Compartment ◽  
Alternative Reading

Abstract Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.

scholarly journals Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 482-482
Author(s):  
Koki Ueda ◽  
Kazuhiko Ikeda ◽  
Kazuei Ogawa ◽  
Akiko Shichishima-Nakamura ◽  
Kotaro Shide ◽  
...  
Keyword(s):  
Disease Progression ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Epigenetic Modifiers ◽  
One Year

Abstract Myeloproliferative neoplasms (MPN) are characterized by chronic proliferation of myeloid cells, extramedullary hematopoiesis and occasional leukemic transformation. Mutations in JAK2, CALR and MPL have been established as drivers of myeloproliferative phenotype, but their roles in disease progression with clonal expansion remain unclear. In addition, studies have shown mutations in epigenetic modifiers including TET2, DNMT3A, ASXL1 and EZH2, and aberrant expressions of microRNAs in MPN, but downstream of these changes is also largely unknown. Recently, we showed high expression of HMGA2 mRNA partly correlated with reduced microRNA let-7 in granulocytes of patients with MPN, including 100% patients with primary myelofibrosis (MF) and 20% polycythemia vera and essential thrombocythemia (Harada-Shirado et al, Brit J Haematol, 2015). In mice, loss of epigenetic modifiers such as BMI1 and EZH2, along with the Arf/Ink4a knockout (Oguro et al, J Exp Med, 2012) or the JAK2 V617F (Sashida et al, ASH, 2013), leads to overexpression of HMGA2 with accelerating MPN. We have generated transgenic (Tg) mice of Hmga2 cDNA with truncated 3'UTR (ΔHmga2) lacking binding sites of let-7 thatrepresses expression of HMGA2 (Ikeda et al, Blood, 2011). Δ Hmga2 mice overexpress HMGA2 and develop MPN-like disease, and represent a clonal advantage in competitive repopulations with serial bone marrow (BM) transplants (BMT). Here, to clarify if HMGA2 affect JAK2 V617F+ hematopoiesis, we crossed Δ Hmga2+/- mice with JAK2 V617F+/- Tg mice (Shide et al, Leukemia, 2008). Δ Hmga2-/-JAK2 V617F-/- wild type (WT), Δ Hmga2+/-JAK2 V617F-/- (Δ Hmga2 -Tg), Δ Hmga2-/-JAK2 V617F+/- (JAK2 V617F-Tg) and Δ Hmga2+/-JAK2 V617F+/- (double-Tg) mice were born at expected Mendelian ratios and we could analyze 5 - 6 of each. At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly were most severe in double-Tg compared with JAK2 V617F-Tg or Δ Hmga2 -Tg mice. Relative to WT, peripheral leukocyte and platelet counts were nearly 16- and 4-fold higher in double-Tg, while 3- and 2-fold higher in JAK2 V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, Δ Hmga2 -Tg, JAK2 V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 107/femur, respectively. However, JAK2 V617F-Tg and double-Tg equally showed MF whereas no MF was detected in WT and DHmga2-Tg, suggesting that HMGA2 partly recovers cellularity in fibrotic BM. In the absence and presence of JAK2 V617F, HMGA2 augments lineage- Sca1+ Kit+ cells (WT: Δ Hmga2-Tg: JAK2 V617F-Tg: double-Tg= 0.17%: 0.19%: 0.17%: 0.27% in BM cells), endogenous erythroid colonies (1: 11: 13: 21 CFU-E/104 BM cells) and CD71+ Ter119+ erythroblasts (23%: 29%: 5.7%: 10% in BM and 2.0%: 4.4%: 7.9%: 16% in spleen cells), indicating HMGA2 contributes to expansion of hematopoietic stem/progenitor cells (HSPC) and erythroid commitment in JAK2 V617F+ hematopoiesis. Most Δ Hmga2-Tg and JAK2 V617F-Tg survived for over one year, but all double-Tg mice died within 4 months after birth due to severe splenomegaly and MF with no acute leukemia. To study the effect of HMGA2 on JAK2 V617F+ HSPC activity, we performed BMT with 0.25 x 106 Ly5.2+Δ Hmga2-Tg, JAK2 V617F-Tg or double-Tg cells with 0.75 x 106 Ly5.1+ competitor WT cells to lethally irradiated Ly5.1+ WT mice. Proportions of Ly5.2+ cells were higher in recipients of Δ Hmga2 -Tg than double-Tg cells, while JAK2 V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2 V617F+ hematopoiesis, we performed another BMT with 1 x 104 KIT+ cells of JAK2 V617F-Tg mice transduced with retroviral vector of Hmga2 with each let-7 -site-mutated full-length 3'UTR (Hmga2-m7) to sublethally irradiated WT mice. Recipients of JAK2 V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2 V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2 V617F by expanding HSPC, leading to disease progression. Disclosures No relevant conflicts of interest to declare.

Advanced Extramedullary Hematopoiesis In a Transplanted Liver Graft As Initial Presentation Of Primary Myelofibrosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5256-5256
Author(s):  
Abdulraheem Yacoub ◽  
Coralie Pickert ◽  
Wei Cui
Keyword(s):  
Liver Biopsy ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Graft Function ◽  
Primary Myelofibrosis ◽  
Extramedullary Hematopoiesis ◽  
Liver Graft ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
Work Up

Abstract Primary myelofibrosis (PMF) is one of the BCR-ABL-negative myeloproliferative neoplasms. The hallmarks of PMF include increased bone marrow (BM) fibrosis, megakaryocytic hyperplasia with hyperchromatic nuclei and abnormal lobation, leukoerythroblastosis, cytopenias, extramedullary hematopoiesis (EMH), and constitutional symptoms that can be debilitating. PMF is associated with the constitutive mobilization of CD34+ cells into the peripheral blood which characteristically occurs in the more clinically advanced phases of the disease. This dysregulation of hematopoietic stem cell (HSC) trafficking likely ultimately leads to the seeding of extramedullary sites with primitive hematopoietic capacity, resulting in EMH within the spleen and liver as well as a variety of other organs. We present a first report of a unique presentation of PMF in a liver transplant-recipient patient as EMH in the transplanted liver graft. Case A 76 year-old man with history of cryptogenic cirrhosis received cadaveric liver transplantation in 1996. He maintained a normal graft function and stable hematologic parameters until 2013 when he presented with anemia and progressive fatigue. Extensive work-up did not identify the etiology of the recent decline in his hemoglobin, but graft dysfunction and anti-rejection therapy were implicated. A liver biopsy was deemed necessary to determine the status of the liver and to further guide anti-rejection therapy. The liver biopsy showed findings of EMH within the sinusoids with increased megakaryocytes, some with atypical morphology. The liver parenchyma was unremarkable with no evidence of rejection or increased fibrosis [fig. 1]. The patient was referred to hematology for further evaluation. Additional work-up included a BM biopsy that revealed a hypercellular marrow (60 percent, normal appearing trilineage hematopoiesis, moderately increased reticulin fibrosis (grade 2/3) and less than 1% blasts. The number and morphology of megakaryocytes were not markedly abnormal [fig. 1]. Cytogenetic studies on the marrow aspirate showed abnormal karyotype: 47, XY, trisomy 8 and add (9) (q34). Polymerase chain reaction (PCR) analysis on the blood for JAK2 mutation was positive for V617F mutation in exon 14. Abdominal imaging showed a normal-size spleen and did not identify any sites of EMH outside of the liver. The diagnosis of intermediate-2 risk PMF was made. Discussion Extramedullary hematopoiesis is a feature of PMF, especially in advanced stages. Although it has been reported in many organs and tissues, there is tropism of the neoplastic HSC to seed in organs with hematopoietic potential, such as the spleen, liver, and lymph nodes. This case demonstrates predominant tropism to a transplanted liver graft with absence of EMH elsewhere. In fact, the megakaryocytic atypia were more pronounced in the liver compared to the BM, which might indicate that allograft microenvironment, and possible immune deregulation, facilitated the expansion of the malignant clone. We would like to emphasize that findings of EMH in subjects with no pre-existing hematologic neoplasm should warrant close follow up and assessment. Disclosures: No relevant conflicts of interest to declare.

Endothelial progenitor cells are clonal and exhibit the JAK2V617F mutation in a subset of thrombotic patients with Ph-negative myeloproliferative neoplasms

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2700-2707 ◽  
Author(s):  
Luciana Teofili ◽  
Maurizio Martini ◽  
Maria Grazia Iachininoto ◽  
Sara Capodimonti ◽  
Eugenia Rosa Nuzzolo ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Signal Transducer ◽  
Cell Compartment ◽  
Molecular Abnormalities ◽  
Endothelial Colony Forming Cells ◽  
Molecular Abnormality ◽  
Thrombotic Complications ◽  
Human Androgen Receptor

Abstract In this study we investigated whether neoplastic transformation occurring in Philadelphia (Ph)–negative myeloproliferative neoplasms (MPNs) could involve also the endothelial cell compartment. We evaluated the level of endothelial colony-forming cells (E-CFCs) in 42 patients (15 with polycythemia vera, 12 with essential thrombocythemia, and 15 with primary myelofibrosis). All patients had 1 molecular abnormality (JAK2V617F or MPLW515K mutations, SOCS gene hypermethylation, clonal pattern of growth) detectable in their granulocytes. The growth of colonies was obtained in 22 patients and, among them, patients with primary myelofibrosis exhibited the highest level of E-CFCs. We found that E-CFCs exhibited no molecular abnormalities in12 patients, had SOCS gene hypermethylation, were polyclonal at human androgen receptor analysis in 5 patients, and resulted in JAK2V617F mutated and clonal in 5 additional patients, all experiencing thrombotic complications. On the whole, patients with altered E-CFCs required antiproliferative therapy more frequently than patients with normal E-CFCs. Moreover JAK2V617F-positive E-CFCs showed signal transducer and activator of transcription 5 and 3 phosphorylation rates higher than E-CFCs isolated from healthy persons and patients with MPN without molecular abnormalities. Finally, JAK2V617F-positive E-CFCs exhibited a high proficiency to adhere to normal mononuclear cells. This study highlights a novel mechanism underlying the thrombophilia observed in MPN.

scholarly journals JAK2V617F mutation in a patient with B-cell chronic lymphocytic leukemia and prefibrotic primary myelofibrosis

2015 ◽  
Vol 143 (11-12) ◽  
pp. 739-743 ◽  
Author(s):  
Slobodan Ristic ◽  
Milica Radojkovic ◽  
Tatjana Kostic ◽  
Vesna Spasovski ◽  
Sonja Pavlovic ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Lymphocytic Leukemia ◽  
Jak2v617f Mutation ◽  
Predisposing Factor ◽  
Lymphoid Leukemia ◽  
Hematopoietic Stem ◽  
Cell Clones

Introduction. Secondary malignancies, particularly solid tumors, are common in patients with chronic lymphocytic leukemia (CLL), but association of myeloproliferative neoplasms and chronic lymphocytic leukemia in the same patient is very rare. Case Outline. We report of a 67-year-old man with B-cell chronic lymphoid leukemia (B-CLL) who developed primary myelofibrosis (PMF) nine years after initial diagnosis. Patient received alkylation agents and purine analogue, which can be a predisposing factor for the development of myeloproliferative neoplasms. JAK2V617F mutation was not present initially at the time of CLL diagnosis, but was found after nine years when PMF occurred, which indicates that B-CLL and PMF represent two separate clonal origin neoplasms. Conclusion. Pathogenic mechanisms for the development of myeloproliferative and lymphoproliferative neoplasms in the same patient are unknown. Further research is needed to determine whether these malignancies originate from two different cell clones or arise from the same pluripotent hematopoietic stem cell.

scholarly journals In-Frame Exon 9 CALR Deletions Co-Occur with Other Alterations in the JAK-STAT Pathway in Myeloproliferative Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4588-4588 ◽  
Author(s):  
Yongbao Wang ◽  
Albert K Ho ◽  
Qiulu Pan ◽  
Frederick Karl Racke ◽  
Dan Jones
Keyword(s):  
Mutation Analysis ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Point Mutations ◽  
Jak2 V617f ◽  
Illumina Miseq ◽  
Primary Myelofibrosis ◽  
Ensembl Database ◽  
Stat Pathway

Abstract Introduction: Mutations in the chaperone gene calreticulin (CALR) have been recently identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF), and are essentially mutually exclusive with JAK2 or MPL mutations. Normal and mutant CALR proteins may differentially affect the subcellular trafficking of JAK-STAT signaling components. CALR mutations previously reported in ET and PMF have been +1 frameshift (fs) mutations localized to exon (E) 9 that generate a novel C-terminal protein sequence with a shift from acidic to basic residues. CALR E9 in-frame (IF) deletions have been recently rarely reported as polymorphisms such as TMP_ESP_19_13054686_13054688 and TMP_ESP_19_13054650_13054658 (Ensembl database entries). We sought to determine the frequency and associated clinical features of CALR with E9 IF alterations in samples submitted for suspicion of a myeloproliferative neoplasm (sMPN). We also assessed whether CALR IF alterations are differentially associated with +1fs mutations or with JAK2 V617For other somatic mutations in MPN-associated genes. Materials and Methods: CALR mutation analysis of E9 was performed on genomic DNA extracted from blood, bone marrow (BM) aspirate or fixed BM biopsy sections using a Sanger sequencing assay with an analytic sensitivity of at least 15%. E9 IF cases were further assessed and mutations quantified by an Ion torrent sequencing panel assessing CALR, CSF3R, JAK2 and MPL, a second panel containing ASXL1, EZH2, IDH1, IDH2, KRAS, NRAS and TET2 and an Illumina MiSeq extended panel with 20 additional MPN-associated genes. These assays had a sensitivity of approximately 5%. JAK2 V617Fmutations were quantitated using a pyrosequencing assay with an analytic sensitivity of 1%. Results: We assessed CALR E9 mutation status in 733 sMPN samples that were negative for JAK2 V617F mutation. 148 (20.1%) had typical +1fs mutations (95 type 1 and variants, 53 type 2 and variants); 2 (0.3%) had point mutations (E381A and D7373M); 7 (1.0%) had IF deletions including E381_A382>A, D397_D400>D (n =4), D400_K401>D and E405_V409>V. All E9 IF deletions were present at ~50% of reads. Clinical diagnoses were cytopenia/BM fibrosis, ET, thrombocytosis/anemia, and sMPN unspecified. Mutation analysis for 27 additional MPN-associated genes revealed mutations in 5/7 (71.4%) IF deletion cases including in MPL (W515L,40%; D163Y,12%), CSF3R (A470T 46%), ASXL1 (D954fs*26, 45%) and ZRSR2 (S449_R450dup, 27%). No additional mutations were found in the 2 cases with non-synonymous CALR point mutations/SNPs. In a parallel set of 76 MPN samples that had JAK2 V617F at varying levels, we noted 1 E9 IF deletion (D397_D400>D) in a sMPN case with 21.6% JAK2 V617F, and a typical +1fs mutation (K385fs*47) in a case with low (4.2%) JAK2 V617F. All other JAK2 V617F cases had no E9 CALR alterations. Conclusions: CALR E9 in-frame deletions occur in up to 1% of sMPN samples and involve a variety of codons in the acidic domain. Therefore, sizing assays without DNA sequencing are not sufficient to unequivocally distinguish IF deletions from the characteristic +1 frameshift somatic mutations associated with ET and PMF. Given their level, these CALR IF deletions are likely germline sequence variants but are associated with a high frequency of somatic mutations in other MPN-associated genes but not with CALR +1fs mutations. Their co-occurrence with pathogenic somatic mutations in JAK2, MPL and CSF3R affecting the JAK-STAT pathway raises the possibility for a contributory role of altered CALR proteins produced by these E9 deletions in the pathogenesis of MPN. Disclosures Wang: Quest Diagnostics: Employment. Ho:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.

scholarly journals The Effect of Postirradiation Bleeding or Endotoxin on Proliferation and Differentiation of Hematopoietic Stem Cells

Blood ◽  
1972 ◽  
Vol 40 (3) ◽  
pp. 375-389 ◽  
Author(s):  
Sallie S. Boggs ◽  
Paul A. Chervenick ◽  
Dane R. Boggs
Keyword(s):  
Stem Cell ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Split Dose ◽  
Stem Cell Compartment ◽  
Proliferation And Differentiation ◽  
Early Rise ◽  
Short Time ◽  
And Control ◽  
Self Replication

Abstract Bleeding after irradiation failed to affect the time of onset or rate of regeneration of the hematopoietic stem cell compartment as measured by a split-dose irradiation method. Bleeding after a single 600 rad exposure hastened the time of onset of erythropoiesis as measured by spleen colonies and 59Fe uptake, but the early rise was abortive and the second increase began at the same time as the first increase seen in irradiated controls. The largest abortive increase was seen when bleeding occurred within 4 hr of irradiation. Lesser effects were seen when bleeding occurred 12 or 24 hr after and no effect 48 hr after irradiation. A generation time of 6.4 hr for a colony-forming cell that forms a colony in 4 days was calculated from the estimated number of cells in one such colony. Injection of cytosine arabinoside at the time of bleeding reduced colony numbers and 59Fe uptake equally in bled and control mice. Injection of 25 µg Salmonella typhosa endotoxin just after irradiation produced a similar early abortive rise in microscopic granulocytic colonies. These results are compatible with a model for pluripotential stem cell compartment in which: (1) differentiation and self-replication occur concomitantly unless the compartment is reduced below approximately 1O% of normal size; then self-replication occurs without appreciable differentiation; (2) for a short time (∼ 24 hr) after depletion below the threshold, differentiation can occur in response to strong stimuli.

scholarly journals CXCR4 is required for the quiescence of primitive hematopoietic cells

2008 ◽  
Vol 205 (4) ◽  
pp. 777-783 ◽  
Author(s):  
Yuchun Nie ◽  
Yoon-Chi Han ◽  
Yong-Rui Zou
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Regulatory Factor ◽  
Cell Compartment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Stem Cell Compartment

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4−/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

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