Conditional Expression of Heterozygous or Homozygous Jak2V617F From Its Endogenous Promoter Induces a Polycythemia Vera-Like Disease.

Abstract Abstract 437 A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). However, the contribution of JAK2V617F in these three clinically distinct myeloproliferative disorders (MPDs) remained unclear. To investigate the actual role of JAK2V617F in the pathogenesis of MPDs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous Jak2V617F evoked all major features of human PV, which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum levels of erythropoietin (Epo) and Epo-independent erythroid colonies. Homozygous Jak2V617F expression resulted in a more severe form of PV associated with markedly elevated leukocytosis, neutrophilia and thrombocytosis, and a majority of these mice succumbed due to massive cardiac thrombosis. Activation of Stat5, Akt and Erk was significantly enhanced in erythroblast cells expressing homozygous Jak2V617F compared to heterozygous Jak2V617F, suggesting that the degree of activation of downstream signaling pathways would be affected by the Jak2V617F gene dosage. This may also partly explain the severe phenotype in mice expressing homozygous Jak2V617F. We conclude that heterozygous Jak2V617F is sufficient to cause PV, whereas homozygous Jak2V617F increases the severity of PV disease and the risk of thrombosis. Our results also provide strong evidence that Jak2V617F gene dosage does not play a defining role in determining PV versus ET phenotype in this mouse model of MPD. Disclosures: No relevant conflicts of interest to declare.

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Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera–like disease

Blood ◽

10.1182/blood-2009-04-215848 ◽

2010 ◽

Vol 115 (17) ◽

pp. 3589-3597 ◽

Cited By ~ 161

Author(s):

Hajime Akada ◽

Dongqing Yan ◽

Haiying Zou ◽

Steven Fiering ◽

Robert E. Hutchison ◽

...

Keyword(s):

Polycythemia Vera ◽

Gene Dosage ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Erythroid Progenitors ◽

Conditional Expression ◽

Biochemical Analyses ◽

Genetic Events ◽

Serum Erythropoietin

Abstract A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. However, contribution of the JAK2V617F mutation in these 3 clinically distinct myeloproliferative neoplasms (MPNs) remained unclear. To investigate the role of JAK2V617F in the pathogenesis of these MPNs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous mouse Jak2V617F evoked all major features of human polycythemia vera (PV), which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum erythropoietin (Epo) levels and Epo-independent erythroid colonies. Homozygous Jak2V617F expression also resulted in a PV-like disease associated with significantly greater reticulocytosis, leukocytosis, neutrophilia and thrombocytosis, marked expansion of erythroid progenitors and Epo-independent erythroid colonies, larger spleen size, and accelerated bone marrow fibrosis compared with heterozygous Jak2V617F expression. Biochemical analyses revealed Jak2V617F gene dosage-dependent activation of Stat5, Akt, and Erk signaling pathways. Our conditional Jak2V617F knock-in mice provide an excellent model that can be used to further understand the molecular pathogenesis of MPNs and to identify additional genetic events that cooperate with Jak2V617F in different MPNs.

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Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice

Blood ◽

10.1182/blood-2007-05-091579 ◽

2008 ◽

Vol 111 (10) ◽

pp. 5109-5117 ◽

Cited By ~ 155

Author(s):

Shu Xing ◽

Tina Ho Wanting ◽

Wanming Zhao ◽

Junfeng Ma ◽

Shaofeng Wang ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Gene Promoter ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Transgenic Expression ◽

Cell Counts

Abstract The JAK2V617F mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2V617F, showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2V617F expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2V617F can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2V617F and to develop treatment for MPDs.

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The possible role of Interleukin-6 as a regulator of insulin sensitivity in patients with neuromyelitis optica spectrum disorder

BMC Neurology ◽

10.1186/s12883-021-02198-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Zhila Maghbooli ◽

Abdorreza Naser Moghadasi ◽

Nasim Rezaeimanesh ◽

Abolfazl Omidifar ◽

Tarlan Varzandi ◽

...

Keyword(s):

Insulin Sensitivity ◽

Neuromyelitis Optica ◽

Serum Levels ◽

Spectrum Disorder ◽

Control Group ◽

Neuromyelitis Optica Spectrum Disorder ◽

Downstream Signaling ◽

Reduce Insulin Sensitivity ◽

Circulating Levels

Abstract Background Neuromyelitis optica spectrum disorder (NMOSD) is associated with inflammatory mediators that may also trigger downstream signaling pathways leading to reduce insulin sensitivity. Methods We aimed to determine the risk association of hyperinsulinemia in NMOSD patients with seropositive AQP4-IgG and the serum levels of interleukin (IL)-6 and IL-17A compared with the control group. Serum levels of metabolic (Insulin, Fasting Blood Sugar (FBS), lipid profile) and inflammatory (IL-6 and IL-17) markers were assessed in 56 NMOSD patients and 100 controls. Results Hyperinsulinemia was more prevalent in NMOSD patients independent of age, sex and body mass index (BMI) (48.2% vs. 26%, p = 0.005) compared to control group. After adjusting age, sex and BMI, there was significant association between lower insulin sensitivity (IS) and NMOSD risk (95% CI: Beta = 0.73, 0.62 to 0.86, p = 0.0001). Circulating levels of IL-6 and IL-17 were higher in NMOSD patients, and only IL-6 had an effect modifier for the association between lower insulin sensitivity and NMOSD risk. Conclusions Our data suggests that inflammatory pathogenesis of NMOSD leads to hyperinsulinemia and increases the risk of insulin resistance.

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Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

Blood ◽

10.1182/blood-2005-09-3826 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3676-3682 ◽

Cited By ~ 164

Author(s):

Francesco Passamonti ◽

Elisa Rumi ◽

Daniela Pietra ◽

Matteo G. Della Porta ◽

Emanuela Boveri ◽

...

Keyword(s):

Polycythemia Vera ◽

Gene Dosage ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Disease Category ◽

Mutation Status ◽

Cell Counts ◽

Cd34 Cells ◽

V617f Mutation

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34+ cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34+ cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34+ cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34+ cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

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Thrombocytosis and Thrombosis

Hematology ◽

10.1182/asheducation-2007.1.363 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 363-370 ◽

Cited By ~ 38

Author(s):

Alessandro M. Vannucchi ◽

Tiziano Barbui

Keyword(s):

Management Strategies ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Platelet Counts ◽

Molecular Abnormalities ◽

Bone Marrow Histology ◽

Potential Risk Factors ◽

Diagnostic Approaches

Abstract The aim of this review is to discuss current diagnostic approaches to, and classification of, patients presenting with thrombocytosis, in light of novel information derived from the discovery of specific molecular abnormalities in chronic myeloproliferative disorders (CMPD), which represent the most common cause of primary thrombocytosis. The JAK2V617F and the MPLW515L/K mutations have been found in patients with essential thrombocythemia, polycythemia vera, and primary myelofibrosis, and less frequently in other myeloproliferative disorders complicated by thrombocytosis. However, neither mutation is disease specific nor is it universally present in patients with elevated platelet counts due to a CMPD; therefore, distinguishing between reactive and primary forms of thrombocytosis, as well as among the different clinical entities that constitute the CMPD, still requires a multifaceted diagnostic approach that includes as a key step the accurate evaluation of bone marrow histology. The role of elevated platelet counts in thrombosis, which represent the predominant complication of CMPD,significantly affecting prognosis and quality of life as well as, paradoxically, in the pathogenesis of the hemorrhagic manifestations, will be discussed. Established and novel potential risk factors for thrombosis, including the clinical relevance of the JAK2V617F mutation, and current management strategies for thrombocytosis are also briefly discussed.

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GATA-1 Mutation R216Q: Increased Bone Marrow Angiogenesis and Reticulin Fibrosis in X-Linked Thrombocytopenia with Thalassemia Trait (XLTT).

Blood ◽

10.1182/blood.v114.22.5125.5125 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5125-5125

Author(s):

Eva Zetterberg ◽

Lovisa Olson ◽

Victoria Hahn-Strömberg ◽

Jan Palmblad ◽

Ian Jones ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Growth Factor Receptor ◽

Primary Myelofibrosis ◽

Pericyte Coverage ◽

Vessel Maturation ◽

Thalassemia Trait ◽

Chain Synthesis ◽

Underlying Mechanisms

Abstract Abstract 5125 We describe features of a previously unpublished 4-generation family with sex-linked thrombocytopenia and large platelets, first investigated in 1993 and followed since then. Whereas there is thrombocytopenia only in 2 males (patient I and III, patelet counts 43 and 29 ×109/l, respectively) a thalassemia trait with elevated HbA2 and HbF is also found in one female. Moderate hitherto non-progressive splenomegaly is present in III. DNA sequencing of exon 4 of the GATA-1 gene was performed in the affected males, and revealed a G to A mutation corresponding to the amino acid change R216Q. This mutation has earlier been shown in 4 other kindred as the cause of XLTT, characterized by thrombocytopenia, splenomegaly and imbalanced globin chain synthesis (Balduini et al Thromb Haemost 2004;91:129-40). Bone marrow findings in our affected males include increased megakaryocytes and morphological abnormalities in megakaryocytes and erythroblasts, confirming the role of GATA-1 for their maturation. A slight reticulin fibrosis (grade 1) is also noted, not hitherto described in other families with XLTT. In an attempt to further characterize this fibrosis and underlying mechanisms we performed CD34 staining which revealed a high microvessel density (MVD) and thus increased angiogenesis. We studied the pericyte coverage of vessels by performing double staining for CD34 and SMA-a, and expression of platelet derived growth factor receptor b (PDGFR-b). Comparisons were made to results in myelofibrosis patients and controls. MVD vessel/HPF Pericyte coverage % PDGFR-β+ pericytes PDGFR-β + cells/HPF BM biopsy from patient I 26.8 6 Yes 50 BM biopsy 1 from patient III 11.4 15.7 No 4.8 BM biopsy 2 from patient III 9.8 4.1 No 0.6 Myelofibrosis patients (n=20) 13±9 92±11 Yes Controls (n=9) 3.7±2 51±20 No Mice carrying the hypomorphic GATA-1low mutation have decreased expression of GATA-1 in megakaryoctes, defective megakaryocyte maturation and develop with age the typical traits of the human myeloproliferative disorder myelofibrosis, including reticulin fibrosis and increased angiogenesis (Vannucchi et al Semin Oncol 2005;32:365-72). In these mice as well as in the human disease myelofibrosis, immature megakaryocytes are retained in the bone marrow and pro-angiogenic and fibroblast stimulating factors are released because of pathologic emperipolesis. We show here that patients with the GATA-1 R216Q mutation also develop reticulin fibrosis and increased angiogenesis. It hasWe have previously been shown that the percentage of pericyte covered vessles is lower in the bone marrow of GATA-1low mice than in control mice, while on the contrary, patients with myelofibrosis have increased pericyte coverage as compared to controls (Zetterberg et al Haematologica 2007;92(5):597-604). We show above thatAs shown above, identical to mice with the GATA-1low mutation, patients with the GATA-1 R216Q mutation have lower pericyte coverage in their bone marrows than controls. Pericytes are cells of mesenchymal origin, important for vessel maturation and recruited to vessels by PDGF. Mice lacking either PDGF or its receptor have no pericyte coverage and die in utero because of hemorrhage from defective vessels. In contrast to malignant primary myelofibrosis, the reticulin fibrosis in our GATA-1 mutation R216Q patients appears non-progressive. We show here that the bone marrow stromal reactions are similar in patients with myelofibrosis and in patients with the GATA-1 R216Q mutation, with exception of pericyte coverage of vessels. Thus, GATA-1 dependent factors regulating pericyte recruitment might also be important for fibrosis progression in myelofibrosis and identifying these factors could forego therapeutic advances. Disclosures No relevant conflicts of interest to declare.

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Clinical Factors Predictive Of Myelofibrotic Evolutions In Patients With Essential Thrombocythemia and Polycythemia Vera

Blood ◽

10.1182/blood.v122.21.4082.4082 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4082-4082 ◽

Cited By ~ 1

Author(s):

Mario Tiribelli ◽

Federico De Marchi ◽

Daniela Barraco ◽

Luciana Marin ◽

Erika Codarin ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Clinical Feature ◽

Baseline Characteristics ◽

Wbc Count ◽

Prognostic Risk Factors

Abstract Introduction Evolution to myelofibrosis (MF) represents a relatively rare but always severe event in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Few reports have focused on the clinical and biological features at diagnosis of ET and PV that correlate with progression to MF. Aims and Methods We retrospectively studied a series of patients with post-ET and post-PV MF and compared with a group of ET and PV patients with a long follow-up without myelofibrotic evolution, with the aim to identify prognostic factors for MF. Forty-three patients with post-ET (n=29) and post-PV (n=14) MF followed at our institution were compared with 125 ET and 75 PV patients with at least 9 years of follow-up without evolution. Diagnosis of ET and PV was confirmed according to WHO criteria (including JAK2 analysis, performed since 2006 and study), evolution to MF was defined according to IWG-MRT proposed criteria. The following parameters, available for all patients at diagnosis of ET or ET, were taken into consideration to find prognostic risk factors for myelofibrosis: age, platelet (PLT) count, hemoglobin (Hb) and hematocrit (Hct) levels, white blood cell (WBC) count. Statistical analyses were conducted using Student t test. Results Median time from diagnosis of ET/PV and progression to MF was 156 months (range: 29-314). Comparing baseline characteristics of patients who evolved to MF and those who did not, we did not found any significant correlation. Mean data at diagnosis for patients with (n = 43) or without (n=200) subsequent evolution to MF were as follow: age 52.1 vs 53.1 years (p=0.79), Hb 15.4 vs 15.7 g/dl (p=0.59), Hct 47.2 vs 47.1% (p=0.67), WBC 9.8 vs 9.1 x 109/l (p=0.11), PLT 713 vs 689 x 109/l (p=0.87). Also when considering only the 29 post-ET MF and the 125 ET patients, there was no clinical feature present at diagnosis that could foresee a future myelofibrotic evolution. Conversely, in the 14 post-PV MF and 75 PV patients, progression to MF was predicted only by higher WBC count (11.4 vs 9.3 x 109/l, p=0.046), while no correlation was found with age, Hb, Hct or PLT [Table 1]. Conclusions Concordant with some previous reports, our data suggest a possible role of leucocytosis as an adverse risk factor for progression to MF in patients with PV, though not in ET. Other clinical characteristics present at diagnosis, such as advanced age, anemia or polycythemia and thrombocytosis do not seem to be associated with higher risk of fibrotic evolution in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

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The Relationship Between the Serum Level of Vitamin D and Hypocalcemia After Total Thyroidectomy

ACTA MEDICA IRANICA ◽

10.18502/acta.v59i12.8057 ◽

2021 ◽

Author(s):

Behrang Motamed ◽

Hossein Hemmati ◽

Mehdi Pursafar ◽

Mohaya Farzin ◽

Zakiyeh Jafaryparvar ◽

...

Keyword(s):

Vitamin D ◽

Total Thyroidectomy ◽

Vitamin D Deficiency ◽

Significant Relationship ◽

Serum Levels ◽

Vitamin D Supplementation ◽

Severe Form ◽

Benign Goiter ◽

Male Patients

Vitamin D plays a crucial role in calcium metabolism through the parathormone-dependent process. The deficiency of this important nutrient may be associated with hypocalcemia after thyroidectomy. To evaluate the role of vitamin D in predicting hypocalcemia following total thyroidectomy. In this study, sixty-two patients who underwent total thyroidectomy for benign or malignant thyroid disease were included in this prospective study. Preoperative vitamin D serum levels and parathormone (PTH) levels were determined. The association between preoperative vitamin D status and the development of hypocalcemia was investigated. In this study, 62 patients were evaluated. The mean age of the subjects was 47 years. Of the 62 patients studied, of which 9 were male patients (14.5%), and 53 were female (85.5%), the results of our study showed. In both groups with and without vitamin D deficiency, calcium levels decreased significantly (P=0.01). In our study, it was found that there was no significant relationship between postoperative hypocalcemia and vitamin D deficiency. (P=0.441). After reviewing the data and according to Spearman correlation statistical test, no significant relationship was observed between serum parathyroid hormone (PTH) and calcium after thyroidectomy (P=0.340). Vitamin D deficiency is a risk factor of hypocalcemia after total thyroidectomy for benign goiter. Although post thyroidectomy hypocalcemia is multifactorial, vitamin D deficiency, particularly severe form, is significantly associated with the development of biochemical and clinical hypocalcemia. Vitamin D supplementation can prevent this unwanted complication in such patients.

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Microenvironment Induced Myelophthisis Caused By CYP26 Deficiency

Blood ◽

10.1182/blood-2018-99-115331 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1297-1297

Author(s):

Laura Palau ◽

Jessica M. Synder ◽

Traci Beth Topping ◽

Cathryn Hogarth ◽

Nina Isoherranen ◽

...

Keyword(s):

Histological Analysis ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Myeloproliferative Neoplasm ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. Retinoic acid (RA) is a powerful morphogen that controls stem cell behavior across a variety of systems. In bone marrow (BM), mesenchymal stroma cells (MSCs) express cytochrome P450 (CYP)26 enzymes and can inactivate endogenous and pharmacological retinoids (Ghiaur G et al PNAS 2013, Su M et al. PlosOne 2015, Alonso S et al. 2016). Stromal CYP26 activity is required to maintain human HSCs ex vivo (Ghiaur G et al PNAS 2013). Here we set to study the role of CYP26 in HSC homeostasis in vivo. For this, we induced CYP26 knockout via injection of Tamoxifen in ROSA26CreERT CYP26A1loxP/loxPCYP26B1loxP/loxPmice (CYP26KO) and ROSA26CreERT wildtype mice (CTR). After 5 daily tamoxifen injections, the knockout was confirmed at DNA and RNA level in multiple tissues of CYP26KO mice. Within 4 weeks, CYP26KO mice showed profound leukocytosis (5.97 ±0.37 vs. 21.12 ±3.81 k/mm3, n=4, p<0.01 CTR vs. CYP26KO) with neutrophilia and monocytosis compared to CTR mice. By 6 weeks, they experience profound weight loss, became moribund and had to be sacrificed. At that time, they had massive splenomegaly and lymphadenopathy as well as brittle/pale bones compared to CTR (Figure A). Histological analysis revealed presence of extramedullary hematopoiesis with a predominantly myeloid infiltrate in the spleen (confirmed by flow cytometry analysis) and lymph nodes but also multiple clusters megakaryocytes (Figure B). The mice also displayed decreased BM cellularity (11.25 ±1.01 vs. 5.62 ±0.36 x106/femur, n=5, p<0.01, CTR vs. CYP26KO), increased frequency of CFU-C (44.33 ±3.72 vs. 69.83 ±8.10 CFU/25000 BM mononuclear cells (MNCs), n=10, p=0.02, CTR vs. CYP26KO) with a myeloid bias (26.76±3.89 vs. 44.57 ±3.21 %CFU-GM/G/M, n=10, p<0.01, CTR vs. CYP26KO). When cellularity was taken into account, total CFU-C per femur was comparable between the two groups. The mice also had increased frequency of LSK cells in the BM (0.26 ±0.12 vs 0.73 ±0.18 % LSK of MNCs, n=2, CTR vs. CYP26KO) and an increased frequency of circulating CFU-C in the peripheral blood (37 ±4.04 vs. 179.5 ±43.06 per 200 ml of blood, n=4, p=0.01, CTR vs. CYP26KO). On histological analysis, the BM is dominated by a myeloid infiltrate and shows a striking decrease in radial bone diameter (Figure C,D). MSCs derived from CYP26KO mice have lower levels of CXCL12 and SCF and have impaired osteoblast differentiation potential compared to MSCs derived from control mice (Figure E). These findings were confirmed with ex vivo generated CYP26KO MSCs via retroviral mediated Cre recombination of CYP26A1loxP/loxPCYP26B1loxP/loxPstroma cells. More so, preliminary studies suggest that the hematopoietic phenotype observed depends on cell extrinsic presence of CYP26 activity as transplantation of CYP26A1loxP/loxPCYP26B1loxP/loxP BM cells into wildtype BoyJ recipients did not reproduce the phenotype upon injection with Tamoxifen of the recipient mice in spite of 100% donor derived hematopoiesis. Alternatively, transplant of wildtype cells into CYP26A1loxP/loxPCYP26B1loxP/loxPrecipients, did reproduce the phenotype after injection of Tamoxifen. In conclusion, we show here that dysregulated RA homeostasis in the BM impairs MSCs function and result in egress of hematopoiesis to extramedullary sites. These results come to complement data from RARγKO (Walkley CR et al Cell 2007) and SMRTmRIDmice models (Hong S-H et al PNAS 2013) and suggest a pivotal role of RA signaling in pathogenesis of myeloproliferative neoplasm and myelofibrosis. To what extent these findings correlate with human primary myelofibrosis is currently under investigation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Abnormal Regulation of Intracellular Calcium in Human Megakaryocytes Contributes to the Pathophysiology of Calr-Mutant Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-116166 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1782-1782 ◽

Cited By ~ 1

Author(s):

Christian Andrea Di Buduo ◽

Vittorio Abbonante ◽

Elisa Rumi ◽

Daniela Pietra ◽

Francesco Moccia ◽

...

Keyword(s):

Peripheral Blood ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Binding Activity ◽

Type I ◽

Ip3 Receptor ◽

Human Cord Blood ◽

Mutant Proteins ◽

Downstream Signaling

Abstract The BCR-ABL-negative Myeloproliferative Neoplasms (MPNs), are a group of clonal hematopoietic malignancies, that consist of three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Approximately one quarter of patients with ET or PMF carry a somatic mutation of CALR, the gene encoding for the endoplasmic reticulum (ER) chaperone calreticulin. A 52-bp deletion (Type I) and a 5-bp insertion (Type II mutation) are the most frequent lesions. Both generate a new carboxy-terminus of the mutant proteins, in which the ER retention signal KDEL is lost, causing abnormal interaction of the mutants with the thrombopoietin (TPO) receptor (MPL), thus constitutively activating its downstream signaling in megakaryocytes. CALR Type I mutation causes important changes in the charge of the carboxyl-terminal tail, which is responsible for calcium (Ca2+) binding activity. As a consequence, megakaryocytes from CALR Type I patients present enhanced activation of Store-Operated Ca2+ Entry (SOCE), the mechanisms that trigger extracellular Ca2+ inflow upon agonist-induced intracellular Ca2+ mobilization from the ER. A fine control of SOCE and overall Ca2+ homeostasis is fundamental to ensure proper megakaryocyte function. Despite this knowledge, the role of a crosstalk among MPL, CALR and SOCE in regulating megakaryopoiesis has never been hypothesised. Our research analysed the activation of MPL downstream pathways and Ca2+ signalling in human cord blood and peripheral blood-cultured megakaryocytes, upon stimulation with recombinant human TPO (rhTPO). Additionally, we cultured megakaryocytes from the peripheral blood of patients carrying the CALR Type I mutation to investigate whether CALR and SOCE alterations determine abnormal activation of the MPL downstream pathway in MPNs. We demonstrated that binding of rhTPO to MPL induces a series of downstream signaling events that lead to the activation of STAT5, AKT and ERK1/2 in human megakaryocytes. This activation relies on significant accumulation of inositol triphosphate (IP3), and consequent intracellular Ca2+ release from the ER followed by extracellular Ca2+ entry, indicative of activated SOCE. Pharmacologic inhibition of the IP3 receptor or SOCE, with 2-APB or BTP-2 respectively, resulted in the complete absence of rhTPO-induced MPL signaling activation. SOCE activation is thus dependent on the dissociation of CALR and STIM1, a protein of the SOCE machinery, which normally forms a complex in un-stimulated megakaryocytes. Importantly, we verified that in human megakaryocytes in rhTPO-starved conditions, CALR forms a molecular complex with its co-chaperone, ERp57, and STIM1, while they dissociate upon rhTPO stimulation. Conversely, we observed that CALR, ERp57 and STIM1 are constitutively dissociated in megakaryocytes from CALR Type I patients, even in complete absence of rhTPO. This dissociation resulted in markedly increased TPO-induced cytosolic Ca2+ flows with a consequent abnormal megakaryocyte proliferation that could be counteracted by pharmacological inhibition of SOCE with BTP-2. Our findings provide the first evidence that CARL Type I, in addition to MPL activation, concurs to the alteration of SOCE, which sustains the phosphorylation of STAT5, AKT and ERK1/2, and induces megakaryocyte proliferation. Further investigation is needed to understand whether this altered megakaryocyte function is determined by the decrease in CALR levels or by the presence of CALR mutants in the ER. Nevertheless, the abnormal regulation of Ca2+ flows may represent a potentially actionable target. Disclosures No relevant conflicts of interest to declare.

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