Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms

PubMed Central

Hinds, David A.; Barnholt, Kimberly E.; Mesa, Ruben A.; Kiefer, Amy K.; Do, Chuong B.; Eriksson, Nicholas; Mountain, Joanna L.; Francke, Uta; Tung, Joyce Y.; Nguyen, Huong (Marie); Zhang, Haiyu; Gojenola, Linda; Zehnder, James L.

2016-01-01

We conducted a genome-wide association study (GWAS) to identify novel predisposition alleles associated with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and JAK2 V617F clonal hematopoiesis in the general population. We recruited a web-based cohort of 726 individuals with polycythemia vera, essential thrombocythemia, and myelofibrosis and 252 637 population controls unselected for hematologic phenotypes. Using a single-nucleotide polymorphism (SNP) array platform with custom probes for the JAK2 V617F mutation (V617F), we identified 497 individuals (0.2%) among the population controls who were V617F carriers. We performed a combined GWAS of the MPN cases plus V617F carriers in the control population (n = 1223) vs the remaining controls who were noncarriers for V617F (n = 252 140). For these MPN cases plus V617F carriers, we replicated the germ line JAK2 46/1 haplotype (rs59384377: odds ratio [OR] = 2.4, P = 6.6 × 10−89), previously associated with V617F-positive MPN. We also identified genome-wide significant associations in the TERT gene (rs7705526: OR = 1.8, P = 1.1 × 10−32), in SH2B3 (rs7310615: OR = 1.4, P = 3.1 × 10−14), and upstream of TET2 (rs1548483: OR = 2.0, P = 2.0 × 10−9). These associations were confirmed in a separate replication cohort of 446 V617F carriers vs 169 021 noncarriers. In a joint analysis of the combined GWAS and replication results, we identified additional genome-wide significant predisposition alleles associated with CHEK2, ATM, PINT, and GFI1B. All SNP ORs were similar for MPN patients and controls who were V617F carriers. These data indicate that the same germ line variants endow individuals with a predisposition not only to MPN, but also to JAK2 V617F clonal hematopoiesis, a more common phenomenon that may foreshadow the development of an overt neoplasm. PMID:27365426

Increased risk of recurrent thrombosis in patients with essential thrombocythemia carrying the homozygous JAK2 V617F mutation.

PubMed

De Stefano, Valerio; Za, Tommaso; Rossi, Elena; Vannucchi, Alessandro M; Ruggeri, Marco; Elli, Elena; Micò, Caterina; Tieghi, Alessia; Cacciola, Rossella R; Santoro, Cristina; Vianelli, Nicola; Guglielmelli, Paola; Pieri, Lisa; Scognamiglio, Francesca; Cacciola, Emma; Rodeghiero, Francesco; Pogliani, Enrico M; Finazzi, Guido; Gugliotta, Luigi; Leone, Giuseppe; Barbui, Tiziano

2010-02-01

Evidence suggests that the JAK2 V617F mutation is associated with an increased risk of first thrombosis in patients with essential thrombocythemia (ET). Whether this mutation is also a risk factor for recurrent thrombosis is currently unknown. To investigate the impact of the JAK2 V617F mutation on the risk of recurrent thrombosis in patients with ET, we carried out a multicentre retrospective cohort study. We recruited 143 patients with previous arterial (64.4%) or venous major thrombosis (34.8%) or both (0.8%); 98 of them (68.5%) carried the mutation. Thrombosis recurred in 43 of the patients (30%); overall, after adjustment for sex, age, presence of vascular risk factors, and treatment after the first thrombosis, the presence of the JAK2 mutation did not predict recurrence (multivariable hazard ratio, HR, 0.88, 95% CI 0.46-1.68). Indeed, the individuals homozygous for the JAK2 V617F (allele burden >50%) mutation had an increased risk of recurrence in comparison with wild-type patients (HR 6.15, 95% CI 1.51-24.92). In conclusion, a homozygous JAK2 V617F mutation is an independent risk factor for recurrent thrombosis in patients with ET.

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations.

PubMed

Okabe, Masahiro; Yamaguchi, Hiroki; Usuki, Kensuke; Kobayashi, Yutaka; Kawata, Eri; Kuroda, Junya; Kimura, Shinya; Tajika, Kenji; Gomi, Seiji; Arima, Nobuyoshi; Mori, Sinichiro; Ito, Shigeki; Koizumi, Masayuki; Ito, Yoshikazu; Wakita, Satoshi; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Dan, Kazuo; Inokuchi, Koiti

2016-01-01

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells

PubMed Central

Wolf, Alexandra; Eulenfeld, René; Gäbler, Karoline; Rolvering, Catherine; Haan, Serge; Behrmann, Iris; Denecke, Bernd; Haan, Claude; Schaper, Fred

2013-01-01

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. PMID:24069558

Inferior vena cava thrombosis and its relationship with the JAK2V617F mutation and chronic myeloproliferative disease.

PubMed

Linnemann, Birgit; Kraft, Christiane; Roskos, Martin; Zgouras, Dimitrios; Lindhoff-Last, Edelgard

2012-06-01

Splanchnic vein thrombosis (SVT) is a typical manifestation of polycythaemia vera (PV) or essential thrombocythaemia (ET). The recently discovered JAK2V617F somatic mutation is closely associated with chronic myeloproliferative disease (CMD). We investigated whether thrombosis involving the inferior vena cava (IVC) is also related to the JAK2V617F mutation or CMD. Blood samples were obtained from 40 IVC thrombosis patients. Fifty-three patients with isolated lower extremity deep vein thrombosis (LE-DVT) and 20 SVT patients served as controls. The presence of the JAK2V617F mutation was assessed by real-time polymerase chain reaction (RT-PCR). The JAK2V617F allele was not detected in any of the IVC thrombosis patients but was detected in one patient (2%) with isolated LE-DVT. However, the mutation-carrying patient did not exhibit symptoms of CMD. Even after an observation period of 30months, the patient's complete blood cell count did not exhibit any pathology. In contrast, the JAK2V617F allele was detected in four patients with SVT (20%) and CMD. According to our data, there is no evidence that IVC thrombosis is associated with the JAK2V617F mutation or the presence of chronic myeloproliferative disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

JAK2 (V617F) mutation is not associated with thrombosis in Behcet syndrome.

PubMed

Ar, M Cem; Hatemi, Gülen; Ekizoğlu, Seda; Bilgen, Hülya; Saçli, Sevgi; Buyru, A Nur; Soysal, Teoman; Ülkü, Birsen; Yazici, Hasan

2012-07-01

The Janus kinase 2(V617F) (JAK2 (V617F)) mutation is an acquired genetic defect that is considered to enhance thrombosis in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Thrombosis is also a well-defined component of Behcet syndrome (BS). The aim of this study was to determine the frequency of JAK2 ( V617F ) mutation in BS-associated thrombosis. A total of 152 patients with BS (62 with thrombosis and 90 without thrombosis) were enrolled. An additional 186 patients with MPNs and 107 healthy blood donors were included to serve as diseased and healthy controls, respectively. None of the patients with BS and healthy controls carried the JAK2 (V617F) mutation, whereas 67% of patients with MPNs were positive for JAK2 ( V617F ). The frequency of thrombosis in patients with MPNs was not statistically different between carriers and non-carriers of JAK2 ( V617F ) mutation. Our data suggest that JAK2 (V617F) is not directly related to thrombosis in MPNs and in other thrombotic entities, such as BS.

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories

PubMed Central

Perricone, Margherita; Palandri, Francesca; Ottaviani, Emanuela; Angelini, Mario; Bagli, Laura; Bellesia, Enrica; Donati, Meris; Gemmati, Donato; Zucchini, Patrizia; Mancini, Stefania; Marchica, Valentina; Trubini, Serena; Matteis, Giovanna De; Zacomo, Silvia Di; Favarato, Mosè; Fioroni, Annamaria; Bolzonella, Caterina; Maccari, Giorgia; Navaglia, Filippo; Gatti, Daniela; Toffolatti, Luisa; Orlandi, Linda; Laloux, Vèronique; Manfrini, Marco; Galieni, Piero; Giannini, Barbara; Tieghi, Alessia; Barulli, Sara; Serino, Maria Luisa; Maccaferri, Monica; Scortechini, Anna Rita; Giuliani, Nicola; Vallisa, Daniele; Bonifacio, Massimiliano; Accorsi, Patrizia; Salbe, Cristina; Fazio, Vinicio; Gusella, Milena; Toffoletti, Eleonora; Salvucci, Marzia; Svaldi, Mirija; Gherlinzoni, Filippo; Cassavia, Francesca; Orsini, Francesco; Martinelli, Giovanni

2017-01-01

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory. A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays. The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples. In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F. PMID:28427233

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

PubMed

Perricone, Margherita; Palandri, Francesca; Ottaviani, Emanuela; Angelini, Mario; Bagli, Laura; Bellesia, Enrica; Donati, Meris; Gemmati, Donato; Zucchini, Patrizia; Mancini, Stefania; Marchica, Valentina; Trubini, Serena; De Matteis, Giovanna; Di Zacomo, Silvia; Favarato, Mosè; Fioroni, Annamaria; Bolzonella, Caterina; Maccari, Giorgia; Navaglia, Filippo; Gatti, Daniela; Toffolatti, Luisa; Orlandi, Linda; Laloux, Vèronique; Manfrini, Marco; Galieni, Piero; Giannini, Barbara; Tieghi, Alessia; Barulli, Sara; Serino, Maria Luisa; Maccaferri, Monica; Scortechini, Anna Rita; Giuliani, Nicola; Vallisa, Daniele; Bonifacio, Massimiliano; Accorsi, Patrizia; Salbe, Cristina; Fazio, Vinicio; Gusella, Milena; Toffoletti, Eleonora; Salvucci, Marzia; Svaldi, Mirija; Gherlinzoni, Filippo; Cassavia, Francesca; Orsini, Francesco; Martinelli, Giovanni

2017-05-16

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

[Expression of JAK2V617F and MPLW515L/K mutation in 30 suspected cases of early myeloproliferative disorders].

PubMed

Fan, Zheng; Zhang, Ri; Shen, Yi-Min; Fei, Hai-Rong; Zhu, Zi-Ling; Cen, Jian-Nong

2008-09-01

To investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET). Genomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files. Of 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD. JAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Association of TNF polymorphisms with JAK2 (V617F) myeloproliferative neoplasms in Brazilian patients.

PubMed

Macedo, Luciana Conci; de Cesare Quintero, Fernanda; Pagliari-E-Silva, Sara; Pagnano, Katia Borgia Barbosa; Rodrigues, Camila; de Alencar, Josiane Bazzo; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2016-03-01

The classical chromosome Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of disorders that share clinical, hematological, and histological features. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) are elevated in patients with MPN. The aim of this study was to verify the association between the polymorphisms of TNF gene (-308G/A and -238 G/A) in BCR-ABL-negative MPN in our population. Blood samples obtained from MPN patients were genotyped for the JAK2V617F mutation and both TNF polymorphisms using PCR-RFLP. Thirty three (26.8%) patients with polycythemia vera (PV), 35 (28.7%) essential thrombocythemia (ET), 22 (17.7%) primary myelofibrosis (PMF), and 33 (26.8%) with unclassifiable MPN (MPNu) were included in the study. The JAK2 V617F mutation was detected in 94 (76.42%) patients. Were observed a significant increase on the frequency of the TNF-238 GA genotype in MPN patients compared to controls (OR=2.21, 95% CI=1.02-4.80, P<0.04). The distribution of the genotypes and allelic frequencies of TNF-308 was significantly different among the MPNs, JAK2V617F positive, PV and PMF, and controls. Our data has demonstrated that the polymorphisms on TNF-238 GA, TNF-308 GA were associated to MPN development in this population, triggered by JAK2 V617F mutation. Copyright © 2015. Published by Elsevier Inc.

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm

PubMed Central

Sangkhae, Veena; Etheridge, S. Leah; Kaushansky, Kenneth

2014-01-01

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F+ transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F+ mice, JAK2V617F+Mpl−/− mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl+/− mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F+Tpo−/− mice, although thrombocytosis was reduced compared with JAK2V617F+ mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F+ MPNs, highlighting an entirely novel target for therapeutic intervention. PMID:25339357

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm.

PubMed

Sangkhae, Veena; Etheridge, S Leah; Kaushansky, Kenneth; Hitchcock, Ian S

2014-12-18

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F(+) transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F(+) mice, JAK2V617F(+)Mpl(-/-) mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl(+/-) mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F(+)Tpo(-/-) mice, although thrombocytosis was reduced compared with JAK2V617F(+) mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F(+) MPNs, highlighting an entirely novel target for therapeutic intervention. © 2014 by The American Society of Hematology.

The Jak2 Inhibitor, G6, Alleviates Jak2-V617F-Mediated Myeloproliferative Neoplasia by Providing Significant Therapeutic Efficacy to the Bone Marrow1

PubMed Central

Kirabo, Annet; Park, Sung O; Majumder, Anurima; Gali, Meghanath; Reinhard, Mary K; Wamsley, Heather L; Zhao, Zhizhuang Joe; Cogle, Christopher R; Bisht, Kirpal S; Keserü, György M; Sayeski, Peter P

2011-01-01

We recently developed a Janus kinase 2 (Jak2) small-molecule inhibitor called G6 and found that it inhibits Jak2-V617F-mediated pathologic cell growth in vitro, ex vivo, and in vivo. However, its ability to inhibit Jak2-V617F-mediated myeloproliferative neoplasia, with particular emphasis in the bone marrow, has not previously been examined. Here, we investigated the efficacy of G6 in a transgenic mouse model of Jak2-V617F-mediated myeloproliferative neoplasia. We found that G6 provided therapeutic benefit to the peripheral blood as determined by elimination of leukocytosis, thrombocytosis, and erythrocytosis. G6 normalized the pathologically high plasma concentrations of interleukin 6 (IL-6). In the liver, G6 eliminated Jak2-V617F-driven extramedullary hematopoiesis. With respect to the spleen, G6 significantly reduced both the splenomegaly and megakaryocytic hyperplasia. In the critically important bone marrow, G6 normalized the pathologically high levels of phospho-Jak2 and phospho-signal transducer and activator of transcription 5 (STAT5). It significantly reduced the megakaryocytic hyperplasia in the marrow and completely normalized the M/E ratio. Most importantly, G6 selectively reduced the mutant Jak2 burden by 67%on average, with virtual elimination of mutant Jak2 cells in one third of all treated mice. Lastly, clonogenic assays using marrow stem cells from the myeloproliferative neoplasm mice revealed a time-dependent elimination of the clonogenic growth potential of these cells by G6. Collectively, these data indicate that G6 exhibits exceptional efficacy in the peripheral blood, liver, spleen, and, most importantly, in the bone marrow, thereby raising the possibility that this compound may alter the natural history of Jak2-V617F-mediated myeloproliferative neoplasia. PMID:22131881

[JAK2 V617F and exon 12 genetic variations in Korean patients with BCR/ABL1-negative myeloproliferative neoplasms].

PubMed

Kim, Jeong Tae; Cho, Yong Gon; Choi, Sam Im; Lee, Young Jin; Kim, Hye Ran; Jang, Sook Jin; Moon, Dae Soo; Park, Young Jin; Park, Geon

2010-12-01

JAK2 genetic variations have been described in a high proportion of patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN). This study was designed to analyze the frequencies of JAK2 V617F and exon 12 variations, and their correlations with clinical characteristics of Korean patients with BCR/ABL1-negative MPN. We examined a total of 154 patients with BCR/ABL1-negative MPN that included 24, 26, 89, and 15 patients with polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), and unclassified myeloproliferative neoplasms (MPNU), respectively. We performed allele-specific PCR to detect V617F in all BCR/ABL1-negative patients, and performed direct sequencing to detect exon 12 variations in 47 V617F-negative MPN patients. JAK2 c.1641+179_183del5 variation was detected by restriction fragment length polymorphism assay in 176 healthy subjects. JAK2 V617F was detected in 91 patients (59.1%): PV (91.6%), PMF (46.2%), ET (52.8%), and MPNU (66.7%). In V617F-negative MPN patients, no mutations were found in exon 12. The c.1641+179_183del5 was detected in 68.1% of V617F-negative MPN patients and 45.4% of healthy subjects (P=0.008). JAK2 V617F was closely correlated with age and leukocytosis in BCR/ABL1-negative MPN patients (P<0.05). However, c.1641+179_183del5 was not related to age, sex, or complete blood cell count parameters in V617F-negative MPN patients and healthy subjects. The c.1641+179_183del5 was associated with an increased odds ratio for MPN (odds ratio, 2.6; 95% confidences interval, 1.3-5.1; P=0.007). Frequencies of V617F are similar to reported results. JAK2 exon 12 mutations may be rare and c.1641+179_183del5 may influence the occurrence of MPN in Korean patients with V6 17F-negative MPN.

AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2

PubMed Central

Wang, Wei; Schwemmers, Sven; Hexner, Elizabeth O.

2010-01-01

The transcription factor NF-E2 is overexpressed in the majority of patients with polycythemia vera (PV). Concomitantly, 95% of these patients carry the JAK2V617F mutation. Although NF-E2 levels correlate with JAK2V671F allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression has not been described. Here we show that NF-E2 expression is also increased in patients with essential thrombocythemia and primary myelofibrosis independent of the presence of the JAK2V617F mutation. Characterization of the NF-E2 promoter revealed multiple functional binding sites for AML1/RUNX-1. Chromatin immunoprecipitation demonstrated AML1 binding to the NF-E2 promoter in vivo. Moreover, AML1 binding to the NF-E2 promoter was significantly increased in granulocytes from PV patients compared with healthy controls. AML1 mRNA expression was elevated in patients with PV, essential thrombocythemia, and primary myelofibrosis both in the presence and absence of JAK2V617F. In addition, AML1 and NF-E2 expression were highly correlated. RNAi-mediated suppression of either AML1 or of its binding partner CBF-β significantly decreased NF-E2 expression. Moreover, expression of the leukemic fusion protein AML/ETO drastically decreased NF-E2 protein levels. Our data identify NF-E2 as a novel AML1 target gene and delineate a role for aberrant AML1 expression in mediating elevated NF-E2 expression in MPN patients. PMID:20339092

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

PubMed

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

[High resolution melting analysis for detecting of JAK2V617F mutation in patients with myeloproliferative neoplasms].

PubMed

Chen, Hai-Hua; Yang, Ji-Long; Lu, Hui-Fang; Zhou, Wei-Jun; Yao, Fei; Deng, Lan

2014-02-01

This study was purposed to investigate the feasibility of high resolution melting (HRM) in the detection of JAK2V617F mutation in patients with myeloproliferative neoplasm (MPN). The 29 marrow samples randomly selected from patients with clinically diagnosed MPN from January 2008 to January 2011 were detected by HRM method. The results of HRM analysis were compared with that detected by allele specific polymerase chain reaction (AS-PCR) and DNA direct sequencing. The results showed that the JAK2V617F mutations were detected in 11 (37.9%, 11/29) cases by HRM, and its comparability with the direct sequencing result was 100%. While the consistency of AS-PCR with the direct sequencing was moderate (Kappa = 0.179, P = 0.316). It is concluded that the HRM analysis may be an optimal method for clinical screening of JAK2V617F mutation due to its simplicity and promptness with a high specificity.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan

PubMed Central

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Background and Objective: Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph +) CML patients in Pakistan. Methods: The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region – Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. Results: All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. Conclusion: It is concluded that the co-existence of Ph +CML and JAK2 V617F mutation is possible. PMID:24639858

Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis

PubMed Central

Nienhold, Ronny; Zmajkovic, Jakub; Hao-Shen, Hui; Geier, Florian; Dirnhofer, Stephan; Feenstra, Jelena D. Milosevic

2016-01-01

Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2. In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2−/− mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2+/− mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)–quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F–expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN. PMID:27401344

IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in JAK2V617F-positive myeloproliferative neoplasms

PubMed Central

de Melo Campos, Paula; Machado-Neto, João A.; Eide, Christopher A.; Savage, Samantha L.; Scopim-Ribeiro, Renata; da Silva Souza Duarte, Adriana; Favaro, Patricia; Lorand-Metze, Irene; Costa, Fernando F.; Tognon, Cristina E.; Druker, Brian J.; Saad, Sara T. Olalla; Traina, Fabiola

2016-01-01

The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2V617F HEL cells, but not in the JAK2WT U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2V617F-positive but not JAK2WT specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34+ cells from essential thrombocythemia patients compared to healthy donors, and in JAK2V617F MPN patients when compared to JAK2WT. Our data indicate that IRS2 is a binding partner of JAK2V617F in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN. PMID:26755644

The association of JAK2V617F mutation and leukocytosis with thrombotic events in essential thrombocythemia.

PubMed

Hsiao, Hui-Hua; Yang, Ming-Yu; Liu, Yi-Chang; Lee, Ching-Ping; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Lin, Sheng-Fung

2007-11-01

The Janus kinase 2 mutation, JAK2 (V617F), and megakaryocytic mutations, MPL (W515L/K), have been identified and correlated with a subtype of essential thrombocythemia (ET) patients. We investigated the frequency of mutations in ET patients and analyzed the relationship with their clinical features. Fifty-three ET patients were enrolled in the study. The amplification refractory mutation system was applied for the mutation survey of the JAK2V617F, while the polymerase chain reaction with sequencing was used for the mutation survey of MPLW515L/K. Thirty-five (66%) patients harboring the JAK2 (V617F) mutation, including 3 homozygous and 32 heterozygous changes, but no MPLW515L/K mutation, were found. During follow-up, 17 (32.1%) patients suffered from documented thrombotic events, with 15 having JAK2V617F mutations. Statistical analysis showed that patients with the JAK2 mutation had significantly higher leukocytes, hemoglobin level, and thrombotic event (p = 0.043, p = 0.001, and p = 0.029, respectively). Thrombotic events were also significantly correlated with leukocytosis and older age. The JAK2V617F mutation was noted in a certain population of ET patients and correlated with leukocytosis, high hemoglobin level, and thrombosis. Therefore, detection of the JAK2V617F mutation can affect not only the diagnosis, but also the management of ET patients.

Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera.

PubMed

Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

2017-02-22

The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia.

PubMed

Nakatani, Takuya; Imamura, Toshihiko; Ishida, Hiroyuki; Wakaizumi, Katsuji; Yamamoto, Tohru; Otabe, Osamu; Ishigami, Tsuyoshi; Adachi, Souichi; Morimoto, Akira

2008-12-01

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations.

PubMed

Pardanani, A; Hood, J; Lasho, T; Levine, R L; Martin, M B; Noronha, G; Finke, C; Mak, C C; Mesa, R; Zhu, H; Soll, R; Gilliland, D G; Tefferi, A

2007-08-01

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM), FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.

Oncogenic JAK2V617F requires an intact SH2-like domain for constitutive activation and induction of a myeloproliferative disease in mice.

PubMed

Gorantla, Sivahari P; Dechow, Tobias N; Grundler, Rebekka; Illert, Anna Lena; Zum Büschenfelde, Christian Meyer; Kremer, Marcus; Peschel, Christian; Duyster, Justus

2010-11-25

The oncogenic JAK2V617F mutation is found in myeloproliferative neoplasms (MPNs) and is believed to be critical for leukemogenesis. Here we show that JAK2V617F requires an intact SH2 domain for constitutive activation of downstream signaling pathways. In addition, there is a strict requirement of cytokine receptor expression for the activation of this oncogene. Further analysis showed that the SH2 domain mutation did not interfere with JAK2 membrane distribution. However, coimmunoprecipitated experiments revealed a role for the SH2 domain in the aggregation and cross-phosphorylation of JAK2V617F at the cell membrane. Forced overexpression of cytokine receptors could rescue the JAK2V617F SH2 mutant supporting a critical role of JAK2V617F abundance for constitutive activation. However, under physiologic cytokine receptor expression the SH2 domain is absolutely necessary for oncogenic JAK2V617F activation. This is demonstrated in a bone marrow transplantation model, in which an intact SH2 domain in JAK2V617F is required for the induction of an MPN-like disease. Thus, our results points to an indispensable role of the SH2 domain in JAK2V617F-induced MPNs.

Detection of JAK2 V617F mutation increases the diagnosis of myeloproliferative neoplasms

PubMed Central

ZHANG, SHU-PENG; LI, HUI; LAI, REN-SHENG

2015-01-01

The Janus kinase (JAK)2 gene, which is located on chromosome 9p24, is involved in the signaling transduction pathways of the hematopoietic and immune system. Mutations in the JAK2 gene have served as disease markers for myeloproliferative neoplasms (MPNs). The aim of the present study was to investigate the occurrence of the JAK2 gene mutation in 140 clinical samples, and to evaluate its clinical significance in MPNs and other hematological diseases. Genomic DNA was extracted from the peripheral blood leukocytes or bone marrow karyocytes of 140 clinical samples, which included 130 patients with various types of hematological disease and 10 control patients. In addition, exons 12 and 14 of the JAK2 gene were analyzed by direct sequencing and the mutation rates of various MPN subtypes were evaluated. Of the 140 samples, exons 12 and 14 were tested in 74 samples, however, exon 14 only was tested in 66 samples. No mutations were identified in exon 12. The V617F mutation rate in polycythemia vera was 82.1% (23/28), and the mutation rates in essential thrombocythemia histiocytosis, primary myelofibrosis and other MPNs were 53.1% (17/32), 40.0% (4/10) and 60.0% (6/10), respectively. Therefore, the total mutation rate of the JAK2 gene in MPN was 62.5% (50/80). For non-MPN hematological diseases, four V617F mutations were detected in samples of leukocytosis of unknown origin (4/12), however, no JAK2 V617F mutations were identified in the 10 controls. Therefore, JAK2 V617F mutations may present a novel marker for diagnosis of MPNs. Furthermore, the direct sequencing method appeared to be satisfactory for the clinical gene testing of hematological samples. PMID:25624900

Detection of MPL exon10 mutations in 103 Chinese patients with JAK2V617F-negative myeloproliferative neoplasms.

PubMed

Chen, Xiuhua; Qi, Xiling; Tan, Yanhong; Xu, Zhifang; Xu, Aining; Zhang, Linlin; Wang, Hongwei

2011-06-15

JAK2V617F mutation has been reported in 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thromobocythemia (ET) and primary myelofibrosis (PMF). Recently, acquired mutations in the transmembrane-juxtamembrane region of MPL (MPLW515 mutations) have been reported in approximately 5% of JAK2V617F-negative PMF and about 1% of all cases of ET. MPL is the receptor for thrombopoietin that regulates the production of platelets by bone marrow. It is likely that some mutations more closely related to ET in MPL exon10 may have been missed by current assays. We inferred that there might be other mutations in MPL exon10 for MPN patients in addition to MPLW515 mutations. To investigate its mutation types and prevalence in Chinese patients with myeloproliferative neoplasms (MPN), we performed mutation detection on MPL exon10 in 103 JAK2V617F-negative MPN patients by single strand conformation polymorphism (SSCP) and allele-specific PCR (AS-PCR) combined with sequencing. As a result, one previously unrecognized MPL mutation (12-bp in-frame insertion) was identified in one patient with ET in addition to an MPLW515K mutation identified in one PMF patient. This confirms our hypothesis that BCR/ABL negative and JAK2V617F-negative MPN patients have other mutations besides W515 mutation in MPL exon10 and mutations other than single nucleotide exchange also exist. In addition, MPL mutation was associated with Chinese MPN patients. Copyright © 2011 Elsevier Inc. All rights reserved.

Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-β1 and Cxcl12 pathways.

PubMed

Dutta, Avik; Hutchison, Robert E; Mohi, Golam

2017-08-17

Myelofibrosis (MF) is a devastating blood disorder. The JAK2V617F mutation has been detected in ∼50% cases of MF. Elevated expression of high-mobility group AT hook 2 (HMGA2) has also been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2 V617F knockin mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. Expression of Hmga2 enhanced megakaryopoiesis, increased extramedullary hematopoiesis, and accelerated the development of MF in mice expressing Jak2 V617F Mechanistically, the data show that expression of Hmga2 enhances the activation of transforming growth factor-β1 (TGF-β1) and Cxcl12 pathways in mice expressing Jak2 V617F In addition, expression of Hmga2 causes upregulation of Fzd2, Ifi27l2a, and TGF-β receptor 2. Forced expression of Cxcl12, Fzd2, or Ifi27l2a increases megakaryocytic differentiation and proliferation in the bone marrow of Jak2 V617F mice, whereas TGF-β1 or Cxcl12 stimulation induces collagen deposition in the bone marrow mesenchymal stromal cells. Together, these findings demonstrate that expression of Hmga2 cooperates with Jak2 V617F in the pathogenesis of MF. © 2017 by The American Society of Hematology.

JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

PubMed

Hasan, Salma; Lacout, Catherine; Marty, Caroline; Cuingnet, Marie; Solary, Eric; Vainchenker, William; Villeval, Jean-Luc

2013-08-22

The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.

Low frequency of V617F mutation in JAK2 gene in Indian patients with hepatic venous outflow obstruction and extrahepatic portal venous obstruction.

PubMed

Rai, Praveer; Kumar, Pankaj; Mishra, Swapnil; Aggarwal, Rakesh

2016-09-01

Hepatic venous outflow tract obstruction (HVOTO) and extrahepatic portal venous obstruction (EHPVO) are important causes of portal hypertension and related complications in India. Both these conditions result from splanchnic venous thrombosis. In recent years, a V617F somatic mutation in Janus kinase 2 (JAK2) gene which is highly specific for myeloproliferative disorders has been detected in 40 % to 50 % and 30 % to 35 % of Western patients with HVOTO and EHPVO, respectively. However, data on this mutation in these conditions from Asian countries are limited. We looked for JAK2 V617F mutation in Indian patients with HVOTO (n = 40, median age 31 [range 17-51] years, 21 female) and EHPVO (n = 50, median age 23 [15-70] years, 25 female) by using two separate methods. Both the methods involved polymerase chain reaction using allele-specific primers. Positive results on one or both of these techniques were confirmed using DNA sequencing. None of the 40 patients with HVOTO and only 1 of 50 patients with EHPVO was found to have JAK2 V617F mutation. In the one patient who was found to have this mutation, both the PCR methods and DNA sequencing showed positive results. Hypercoagulability associated with JAK2 V617F mutation and associated chronic myeloproliferative disorders was not a major cause of HVOTO and EHPVO in this population.

Estimation of diagnosis and prognosis in ET by assessment of CALR and JAK2V617F mutations and laboratory findings: a meta-analysis.

PubMed

Saki, N; Shirzad, R; Rahim, F; Saki Malehi, A

2017-07-01

Essential thrombocythemia (ET) is a benign disease with slow progress in which thrombosis is a cause of mortality. JAK2 V617F and calreticulin (CALR) are the most frequent mutations in this disease. In this systematic review and meta-analysis, we compared the prevalence of JAK2 V617F and CALR mutations in ET and examined the incidence of thrombosis and other hematologic indices. After choosing MeSH keywords, including essential thrombocythemia, JAK2 V617F , calreticulin, prognosis, and diagnosis, as well as searching Medline/PubMed and Scopus, 12 papers were selected. Data were pooled, and summary prevalence and OR were estimated using either a random-effects model or a fixed-effects model. The frequency of JAK2 V617F and CALR shows heterogeneity in Caucasian population [JAK2 V617 I 2 % = 84.3, P < 0.001, 95% CI 0.56 (0.51-0.61)], [CALR I 2 % = 96.1, P < 0.001, 95% CI 0.23 (0.15-0.31)]. The prevalence of JAK2 V617F and CALR was 0.57 (95% CI 0.53-0.61), I 2 % = 79.3 and 0.22 (95% CI 0.16-0.27), I 2 % = 94, respectively. JAK2 V617F positive ET was associated with increasing odds of thrombosis [OR 2.35 (95% CI 1.83-3.02), P < 0.001]. The incidence of splenomegaly was not statistically different between these two mutations. Hemoglobin, platelet, and WBC count did not affect the risk of thrombosis. Detection of CALR mutation is helpful for molecular diagnosis of ET patients as well as JAK2 V617F . Due to reduction of thrombosis in CALR-positive patients, it can be stated that such patients have less thrombotic disorders and better prognosis relative to patients bearing JAK2 V617F mutation. Therefore, detection of mutation in CALR and JAK2 V617F may contribute to diagnosis and prognosis of ET patients.

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

PubMed

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-03-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×10 9 /l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

After 10years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera.

PubMed

Grinfeld, Jacob; Godfrey, Anna L

2017-05-01

The JAK2V617F mutation accounts for the vast majority of patients with polycythaemia vera (PV) and around half of those with other Philadelphia-negative myeloproliferative neoplasms. Since its discovery in 2005, numerous insights have been gained into the pathways by which JAK2V617F causes myeloproliferation, including activation of JAK-STAT signalling but also through other canonical and non-canonical pathways. A variety of mechanisms explain how this one mutation can be associated with distinct clinical disorders, demonstrating how constitutional and acquired factors may interact in the presence of a single mutation to determine disease phenotype. Important biological questions remain unanswered in PV, in particular how JAK2V617F affects stem cell function and what mechanisms drive myelofibrotic and leukaemic transformation. Whilst current management is largely centred on prevention of cardiovascular events, future therapies must aim to target the JAK2-mutant clone, to reverse the underlying marrow pathology and to address the risk of transformation events. Copyright © 2016 Elsevier Ltd. All rights reserved.

Renovascular hypertension associated with JAK2 V617F positive myeloproliferative neoplasms treated with angioplasty: 2 cases and literature review.

PubMed

Mishima, Eikan; Suzuki, Takehiro; Takeuchi, Yoichi; Seiji, Kazumasa; Fukuhara, Noriko; Takase, Kei; Harigae, Hideo; Abe, Takaaki; Ito, Sadayoshi

2018-04-01

Myeloproliferative neoplasms (MPNs) with Janus kinase 2 (JAK2) mutation are associated with a high risk for occlusive vascular diseases. We report 2 cases of renovascular hypertension associated with JAK2 V617F mutation-positive MPNs and provide a literature review. In Case 1, a 63-year-old woman had resistant hypertension, massive proteinuria, and erythrocytosis. Evaluations revealed right renal artery stenosis causing renovascular hypertension and polycythemia vera with JAK2 V617F mutation. Renin-angiotensin system inhibitors and subsequent angioplasty controlled the blood pressure and the proteinuria resolved. In Case 2, a 74-year-old woman had resistant hypertension and thrombocytosis. Evaluations confirmed left renal artery stenosis and essential thrombocythemia with JAK2 V617F. Angioplasty cured the hypertension. A literature review of 18 cases revealed the following as the most common characteristics of MPN-associated renovascular hypertension: manifests primarily in women; is associated with untreated polycythemia vera and essential thrombocythemia, concomitant leukocytosis, and JAK2 mutation positivity; and is responsive to angioplasty. This report demonstrates that JAK2 mutation-positive MPNs are a less common but important underlying cause of adult renovascular hypertension. ©2018 Wiley Periodicals, Inc.

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia

PubMed Central

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-01-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×109/l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR. PMID:28450924

The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population

PubMed Central

2012-01-01

Background Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the JAK2 locus on MPNs in a Japanese population. Methods We sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression. Results A novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count. Conclusions Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the

Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations

PubMed Central

Lee, Tai-Sung; Ma, Wanlong; Zhang, Xi; Kantarjian, Hagop; Albitar, Maher

2009-01-01

Background The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K). Results Simulation results are consistent with all currently available clinical/experimental evidence. The simulation-derived putative interface, not possibly obtained from static models, between the kinase (JH1) and pseudokinase (JH2) domains of JAK2 provides a platform able to explain the mutational effect for all mutants, including presumably benign control mutants, at the atomic level. Conclusion The results and analysis provide structural bases for mutational mechanisms of JAK2, may advance the understanding of JAK2 auto-regulation, and have the potential to lead to therapeutic approaches. Together with recent mutation profiling results demonstrating the breadth of clinically observed JAK2 mutations, our findings suggest that molecular testing/diagnostics of JAK2 should extend beyond V617F and exon 12 mutations, and perhaps should encompass most of the pseudo-kinase domain-coding region. PMID:19744331

JAK2 V617F, MPL, and CALR mutations in essential thrombocythaemia and major thrombotic complications: a single-institute retrospective analysis.

PubMed

Pósfai, Éva; Marton, Imelda; Király, Péter Attila; Kotosz, Balázs; Kiss-László, Zsuzsanna; Széll, Márta; Borbényi, Zita

2015-07-01

Thrombo-haemorrhagic events are the main cause of morbidity and mortality in essential thrombocythemia. The aim of this study was to estimate the incidence of thrombotic events and the impact of the JAK2V617F, MPL (W515L, W515K, W515R, W515A and S505N) and CALR (type-1, type-2) mutations on 101 essential thrombocythaemia patients (72 females and 29 males with a mean age of 61 years) diagnosed in a Southern Hungarian regional academic centre. The incidence of major thrombosis was 13.86 %. Sixty percent of the patients carried the JAK2V617F mutation. The MPL mutations were analysed by sequencing and the W515L was the only one we could identify with an incidence of 3.96 %. Type-2 CALR mutation could be identified in 3 cases among the patients who had JAK2/MPL-unmutated ET. Statistical analyses revealed that the JAK2V617F mutation was associated with significantly increased levels of platelet (p = 0.042), haemoglobin (p = 0.000), red blood cell (p = 0.000) and haematocrit (p = 0.000) and hepatomegaly (p = 0.045) at diagnosis compared to JAK2V617F negative counterparts, however there was no significant association between the JAK2V617F mutation status (relative risk: 1.297, 95 % CI 0.395-4.258; p = 0.668) and subsequent thrombotic complications. The impact of JAK2V617F, MPL W515L and CALR mutations on the clinical findings at the diagnosis of ET was obvious, but their statistically significant role in the prediction of thrombotic events could not be proven in this study. Our results indirectly support the concept that, besides the quantitative and qualitative changes in the platelets, the mechanisms leading to thrombosis are more complex and multifactorial.

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

PubMed Central

Kent, David G.; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C.; Silber, Yvonne; Hamilton, Tina L.; Sneade, Rachel; Simons, Benjamin D.; Green, Anthony R.

2013-01-01

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F

The Stilbenoid Tyrosine Kinase Inhibitor, G6, Suppresses Jak2-V617F-mediated Human Pathological Cell Growth in Vitro and in Vivo*

PubMed Central

Kirabo, Annet; Embury, Jennifer; Kiss, Róbert; Polgár, Tímea; Gali, Meghanath; Majumder, Anurima; Bisht, Kirpal S.; Cogle, Christopher R.; Keserű, György M.; Sayeski, Peter P.

2011-01-01

Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease. PMID:21127060

JAK2V617F-mutant megakaryocytes contribute to hematopoietic stem/progenitor cell expansion in a model of murine myeloproliferation

PubMed Central

Zhan, H; Ma, Y; Lin, CHS; Kaushansky, K

2016-01-01

The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs, but the mechanism(s) responsible for MPN stem cell expansion remain incomplete. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. We report here that mice bearing a human JAK2V617F gene restricted exclusively to the MK lineage develop many of the features of a MPN. Specifically, these mice exhibit thrombocytosis, splenomegaly, increased numbers of marrow and splenic hematopoietic progenitors and a substantial expansion of HSPCs. In addition, wild-type mice transplanted with cells from JAK2V617F-bearing MK marrow develop a myeloproliferative syndrome with thrombocytosis and erythrocytosis as well as pan-hematopoietic progenitor and stem cell expansion. As marrow histology in this murine model of myeloproliferation reveals a preferentially perivascular localization of JAK2V617F-mutant MKs and an increased marrow sinusoid vascular density, it adds to accumulating data that MKs are an important component of the marrow HSPC niche, and that MK expansion might indirectly contribute to the critical role of the thrombopoietin/c-Mpl signaling pathway in HSPC maintenance and expansion. PMID:27133820

[The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

PubMed

Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A

2014-11-01

The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

Extending Jak2V617F and MplW515 mutation analysis to single hematopoietic colonies and B and T lymphocytes.

PubMed

Pardanani, Animesh; Lasho, Terra L; Finke, Christy; Mesa, Ruben A; Hogan, William J; Ketterling, Rhett P; Gilliland, Dwight Gary; Tefferi, Ayalew

2007-09-01

JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell.

The development of gastric cancer in a patient with polycythemia Vera, 3P deletion, and JAK2 V617F mutation.

PubMed

Ayvaz, Ozlem; Yavasoglu, Irfan; Kadikoylu, Gurhan; Meydan, Nezih; Barutca, Sabri; Bolaman, Zahit

2010-12-01

3p deletion which is frequently associated with solitary tumors and hematological malignancies is a chromosomal abnormality. Recently, Janus kinase-2 (JAK2) V617F mutation has an important role in the diagnosis of myeloproliferative disorders, especially in polycythemia vera (PV). We reported the development of gastric cancer in a 75-year-old patient with PV, 3p 12-14 deletion and JAK2 V617F mutation. PV was diagnosed according to the classification of World Health Organization. JAK2 V617F mutation with polymerase chain reaction and 3p12-14 deletion with cytogenetic examination of the bone marrow were detected. We investigated solitary tumors in the patient using computed tomographies of thorax, neck, ear, nose, and throat. However, they were normal. After 2 years, gastric cancer appeared in the patient. In conclusion, cytogenetic examination may be important in both the development and the diagnosis of hematological malignancies and solitary tumors. So the patients should be followed closely.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

PubMed

Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

2014-10-01

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms.

PubMed

dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-02-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms

PubMed Central

dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-01-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN. PMID:23986553

Influence of the JAK2 V617F mutation and inherited thrombophilia on the thrombotic risk among patients with essential thrombocythemia

PubMed Central

De Stefano, Valerio; Za, Tommaso; Rossi, Elena; Fiorini, Alessia; Ciminello, Angela; Luzzi, Claudia; Chiusolo, Patrizia; Sica, Simona; Leone, Giuseppe

2009-01-01

It is uncertain whether the JAK2 V617F mutation increases the thrombotic risk in patients with essential thrombocythemia, and it is unknown whether inherited thrombophilia is an additive risk factor in mutated subjects. We studied 132 patients with essential thrombocythemia, 38 of them (29%) with a history of thrombosis. The JAK2 mutation was present in 83 (63%), and inherited thrombophilia in 7. The mutated patients <60 years had a relative risk (RR) for thrombosis at any time of 3.83 (95%CI 1.27–11.49) in comparison with wild-type patients; in those with both the mutation and thrombophilia the RR was 2.23 (95%CI 1.57–3.18) and 7.66 (95%CI 2.66–22.03) in comparison with mutated or wild-type patients without thrombophilia, respectively. During the follow-up, only the homozygotes for JAK2 V617F were more prone to thrombosis (RR 17.25, 95%CI 2.33–127.4). Among the patients >60 years, no increase in RR was associated with the JAK2 mutation. In conclusion, in the younger patients with ET the thrombotic risk is higher in the JAK2 V617F-mutated and is further increased by the presence of inherited thrombophilia. PMID:19336736

Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells.

PubMed

Mullally, Ann; Lane, Steven W; Ball, Brian; Megerdichian, Christine; Okabe, Rachel; Al-Shahrour, Fatima; Paktinat, Mahnaz; Haydu, J Erika; Housman, Elizabeth; Lord, Allegra M; Wernig, Gerlinde; Kharas, Michael G; Mercher, Thomas; Kutok, Jeffery L; Gilliland, D Gary; Ebert, Benjamin L

2010-06-15

We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN. Copyright 2010 Elsevier Inc. All rights reserved.

Acute Myeloid Leukemia with MYC Rearrangement and JAK2 V617F Mutation

PubMed Central

Ohanian, Maro; Bueso-Ramos, Carlos; Ok, Chi Young; Lin, Pei; Patel, Keyur; Alattar, Mona Lisa; Khoury, Joseph D.; Rozovski, Uri; Estrov, Zeev; Huh, Yang O.; Cortes, Jorge; Abruzzo, Lynne V.

2016-01-01

Little is known about MYC dysregulation in myeloid malignancies, and we can find no published studies that have evaluated MYC protein expression in primary cases of myelodysplastic syndromes (MDS) or acute myeloid leukemias (AML). We describe the clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic findings in two MDS/AML cases that contained both MYC rearrangement and JAK2-V617F mutation. We demonstrate MYC protein expression by immunohistochemistry in both patients. PMID:26382622

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2V617F in mice

PubMed Central

Walz, Christoph; Ahmed, Wesam; Lazarides, Katherine; Betancur, Monica; Patel, Nihal; Hennighausen, Lothar; Zaleskas, Virginia M.

2012-01-01

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2V617F in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1–induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2V617F failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2V617F, and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs. PMID:22234689

CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

PubMed Central

Tyner, Jeffrey W.; Bumm, Thomas G.; Deininger, Jutta; Wood, Lisa; Aichberger, Karl J.; Loriaux, Marc M.; Druker, Brian J.; Burns, Christopher J.; Fantino, Emmanuelle

2010-01-01

Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells. PMID:20385788

Myeloproliferative neoplasms with concurrent BCR-ABL1 translocation and JAK2 V617F mutation: a multi-institutional study from the bone marrow pathology group.

PubMed

Soderquist, Craig R; Ewalt, Mark D; Czuchlewski, David R; Geyer, Julia T; Rogers, Heesun J; Hsi, Eric D; Wang, Sa A; Bueso-Ramos, Carlos E; Orazi, Attilio; Arber, Daniel A; Hexner, Elizabeth O; Babushok, Daria V; Bagg, Adam

2018-05-01

Myeloproliferative neoplasms arise from hematopoietic stem cells with somatically altered tyrosine kinase signaling. Classification of myeloproliferative neoplasms is based on hematologic, histopathologic and molecular characteristics including the presence of the BCR-ABL1 and JAK2 V617F. Although thought to be mutually exclusive, a number of cases with co-occurring BCR-ABL1 and JAK2 V617F have been identified. To characterize the clinicopathologic features of myeloproliferative neoplasms with concomitant BCR-ABL1 and JAK2 V617F, and define the frequency of co-occurrence, we conducted a retrospective multi-institutional study. Cases were identified using a search of electronic databases over a decade at six major institutions. Of 1570 patients who were tested for both BCR-ABL1 and JAK2 V617F, six were positive for both. An additional five patients were identified via clinical records providing a total of 11 cases for detailed evaluation. For each case, clinical variables, hematologic and genetic data, and bone marrow histomorphologic features were analyzed. The sequence of identification of the genetic abnormalities varied: five patients were initially diagnosed with a JAK2 V617F+ myeloproliferative neoplasm, one patient initially had BCR-ABL1+ chronic myeloid leukemia, while both alterations were identified simultaneously in five patients. Classification of the BCR-ABL1-negative myeloproliferative neoplasms varied, and in some cases, features only became apparent following tyrosine kinase inhibitor therapy. Seven of the 11 patients showed myelofibrosis, in some cases before identification of the second genetic alteration. Our data, reflecting the largest reported study comprehensively detailing clinicopathologic features and response to therapy, show that the co-occurrence of BCR-ABL1 and JAK2 V617F is rare, with an estimated frequency of 0.4%, and most often reflects two distinct ('composite') myeloproliferative neoplasms. Although uncommon, it is important to be

Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells

PubMed Central

Wang, Xiaoli; Zhang, Wei; Tripodi, Joseph; Lu, Min; Xu, Mingjiang; Najfeld, Vesna; Li, Yan

2010-01-01

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC), we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34+ cells. Treatment of PMF CD34+ cells with chromatin-modifying agents (CMAs) but not hydroxyurea, Janus kinase 2 (JAK2) inhibitors, or low doses of interferon-α led to the generation of greater numbers of CD34+ chemokine (C-X-C motif) receptor (CXCR)4+ cells, which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F+. Furthermore, sequential treatment of PMF CD34+ cells but not normal CD34+ cells with decitabine (5-aza-2′-deoxycytidine [5azaD]), followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA), or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F+ PMF CD34+ cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγnull mice, the percentage of JAK2V617F/JAK2total in human CD45+ marrow cells was dramatically reduced. These findings suggest that both PMF HPCs, short-term and long-term SCID repopulating cells (SRCs), are JAK2V617F+ and that JAK2V617F+ HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs, therefore, represents a possible effective means of treating PMF at the level of the malignant SRC. PMID:20858855

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies

PubMed Central

Oaks, Joshua J.; Santhanam, Ramasamy; Walker, Christopher J.; Roof, Steve; Harb, Jason G.; Ferenchak, Greg; Eisfeld, Ann-Kathrin; Van Brocklyn, James R.; Briesewitz, Roger; Saddoughi, Sahar A.; Nagata, Kyosuke; Bittman, Robert; Caligiuri, Michael A.; Abdel-Wahab, Omar; Levine, Ross; Arlinghaus, Ralph B.; Quintas-Cardama, Alfonso; Goldman, John M.; Apperley, Jane; Reid, Alistair; Milojkovic, Dragana; Ziolo, Mark T.; Marcucci, Guido; Ogretmen, Besim; Neviani, Paolo

2013-01-01

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)–activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase-dependent manner through the PI-3Kγ-PKC–induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2V617F also utilizes an alternative sphingosine kinase-1–mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET–mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2V617F MPNs. PMID:23926298

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

PubMed

Jovanovic, J V; Ivey, A; Vannucchi, A M; Lippert, E; Oppliger Leibundgut, E; Cassinat, B; Pallisgaard, N; Maroc, N; Hermouet, S; Nickless, G; Guglielmelli, P; van der Reijden, B A; Jansen, J H; Alpermann, T; Schnittger, S; Bench, A; Tobal, K; Wilkins, B; Cuthill, K; McLornan, D; Yeoman, K; Akiki, S; Bryon, J; Jeffries, S; Jones, A; Percy, M J; Schwemmers, S; Gruender, A; Kelley, T W; Reading, S; Pancrazzi, A; McMullin, M F; Pahl, H L; Cross, N C P; Harrison, C N; Prchal, J T; Chomienne, C; Kiladjian, J J; Barbui, T; Grimwade, D

2013-10-01

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Resolution of bone marrow fibrosis in a patient receiving JAK1/JAK2 inhibitor treatment with ruxolitinib.

PubMed

Wilkins, Bridget S; Radia, Deepti; Woodley, Claire; Farhi, Sarah El; Keohane, Clodagh; Harrison, Claire N

2013-12-01

Ruxolitinib, a JAK1/JAK2 inhibitor, is currently the only pharmacological agent approved for the treatment of myelofibrosis. Approval was based on findings from two phase 3 trials comparing ruxolitinib with placebo (COMFORT-I) and with best available therapy (COMFORT-II) for the treatment of primary or secondary myelofibrosis. In those pivotal trials, ruxolitinib rapidly improved splenomegaly, disease-related symptoms, and quality of life and prolonged survival compared with both placebo and conventional treatments. However, for reasons that are currently unclear, there were only modest histomorphological changes in the bone marrow, and only a subset of patients had significant reductions in JAK2 V617F clonal burden. Here we describe a patient with post-polycythemia vera myelofibrosis who received ruxolitinib at our institution (Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom) as part of the COMFORT-II study. While on treatment, the patient had dramatic improvements in splenomegaly and symptoms shortly after starting ruxolitinib. With longer treatment, the patient had marked reductions in JAK2 V617F allele burden, and fibrosis of the bone marrow resolved after approximately 3 years of ruxolitinib treatment. To our knowledge, this is the first detailed case report of resolution of fibrosis with a JAK1/JAK2 inhibitor. ClinicalTrials.gov Identifier: NCT00934544.

Mutational subtypes of JAK2 and CALR correlate with different clinical features in Japanese patients with myeloproliferative neoplasms.

PubMed

Misawa, Kyohei; Yasuda, Hajime; Araki, Marito; Ochiai, Tomonori; Morishita, Soji; Shirane, Shuichi; Edahiro, Yoko; Gotoh, Akihiko; Ohsaka, Akimichi; Komatsu, Norio

2018-06-01

The majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) harbor JAK2, CALR, or MPL mutations. We compared clinical manifestations of different subtypes of JAK2 and CALR mutations in Japanese patients with MPNs. Within our cohort, we diagnosed 166 patients as polycythemia vera (PV), 212 patients as essential thrombocythemia (ET), 23 patients as pre-primary myelofibrosis (PMF), 65 patients as overt PMF, and 27 patients as secondary myelofibrosis following the 2016 WHO criteria. Compared to patients with JAK2V617F-mutated PV, JAK2 exon 12-mutated PV patients were younger, showed lower white blood cell (WBC) counts, lower platelet counts, higher red blood cell counts, and higher frequency of thrombotic events. Compared to JAK2-mutated ET patients, CALR-mutated ET patients were younger, showed lower WBC counts, lower hemoglobin levels, higher platelet counts, and fewer thrombotic events. CALR type 1-like mutation was the dominant subtype in CALR-mutated overt PMF patients. Compared with JAK2V617F-mutated ET patients, JAK2V617F-mutated pre-PMF patients showed higher LDH levels, lower hemoglobin levels, higher JAK2V617F allele burden, and higher frequency of splenomegaly. In conclusion, Japanese patients with MPNs grouped by different mutation subtypes exhibit characteristics similar to those of their Western counterparts. In addition, ET and pre-PMF patients show different characteristics, even when restricted to JAK2V617F-mutated patients.

Role of JAK-STAT signaling in the pathogenesis of myeloproliferative disorders.

PubMed

Levine, Ross L; Wernig, Gerlinde

2006-01-01

The identification of JAK2V617F mutations in polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF) represents an important advance in our understanding of these myeloproliferative disorders (MPD). Most, if not all, patients with PV and a significant number of patients with ET and MF are JAK2V617F positive, and the mutation likely arises in the hematopoietic stem cell compartment. JAK2V617F is a constitutively active tyrosine kinase that is able to activate JAK-STAT signaling most efficiently when co-expressed with the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte colony-stimulating factor receptor (GCSFR). Data from murine models supports the central role of JAK2V617F in the pathogenesis of MPD, as expression of JAK2V617F in a bone marrow transplantation assay results in polycythemia and myelofibrosis in recipient mice. Activation of JAK-STAT signaling by JAK2V617F in some, but not all MPD patients with ET and MF led to the identification of the constitutively active MPLW515L allele in ET and MF. Small molecule inhibitors of JAK-STAT signaling are currently being developed, which offer potential for molecularly targeted therapy for patients with PV, ET, and MF. Despite these advances, many questions remain regarding the role of a single disease allele in three phenotypically distinct MPD, the potential clinical efficacy of JAK2 inhibitors, and the identity of oncogenic alleles in JAK2V617F/MPLW515-negative MPD.

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

PubMed

Pardanani, A; Lasho, T; Smith, G; Burns, C J; Fantino, E; Tefferi, A

2009-08-01

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Concomitant presence of JAK2V617F mutation and BCR‑ABL translocation in two patients: A new entity or a variant of myeloproliferative neoplasms (Case report).

PubMed

Mousinho, Filipa; Azevedo, Ana P; Mendes, Tatiana; Santos, Paula Sousa E; Cerqueira, Rita; Matos, Sónia; Santos, Sónia; Ramos, Sância; Viana, João Faro; Lima, Fernando

2018-05-17

Myeloproliferative neoplasms (MPNs) are classically divided into BCR RhoGEF and GTPase activating protein (BCR)-ABL proto‑oncogene 1 non‑receptor tyrosine kinase (ABL) positive chronic myeloid leukemia (CML) and BCR‑ABL negative MPNs, including essential thrombocythemia (ET). One of the major diagnostic criteria for ET is the absence of the philadelphia chromosome, thus when present it is almost indicative of CML. ET and CML are considered to be mutually exclusive; however, there are rare situations in which patients with ET present positive BCR‑ABL without the features of CML. Although from the literature review, the frequency of JAK2V617F mutation and BCR‑ABL translocation coexistence in MPNs is low, it may be higher than expected. The current study reported cases of two patients with an initial diagnosis of ET in the presence of JAK2V617F mutation and BCR‑ABL translocation by fluorescent in situ hybridization. Both patients presented with a heterozygous BCR‑ABL translocation, and absence of p190 and p210 transcripts, seemingly a der(9) in the background of an ET JAK2V617F mutation.

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms.

PubMed

Yao, Qiu-Mei; Zhou, Jiao; Gale, Robert Peter; Li, Jin-Lan; Li, Ling-Di; Li, Ning; Chen, Shan-Shan; Ruan, Guo-Rui

2015-10-01

Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2(V617F). Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2(V617F) negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

Vascular events in Korean patients with myeloproliferative neoplasms and their relationship to JAK2 mutation.

PubMed

Bang, Soo-Mee; Lee, Jong-Seok; Ahn, Jeong Yeal; Lee, Jae Hoon; Hyun, Myung Soo; Kim, Bong Seog; Park, Moo Rim; Chi, Hyun-Sook; Kim, Ho Young; Kim, Hyo Jung; Lee, Moon Hee; Kim, Hwak; Won, Jong Ho; Yoon, Hwi Joong; Oh, Do-Yeun; Nam, Eun-Mi; Bae, Sung Hwa; Kim, Byoung-Kook

2009-03-01

Evaluation of the Janus kinase 2 (JAK2) V617F mutation has been widely used for the diagnosis of myeloproliferative neoplasms (MPN). However, its prognostic relevance to clinical outcome is not completely understood. We investigated the association of JAK2 V617F with vascular events in Korean patients with myeloproliferative neoplasms (MPN). We studied 283 patients from 15 centers, who were diagnosed with MPN. The JAK2 V617F status was evaluated by allele-specific polymerase chain reaction (PCR) and sequencing. The patients' diagnoses were essential thrombocythemia (ET n = 146), polycythemia vera (PV n = 120), primary myelofibrosis (n = 12), and unclassifiable MPN (MPNu n = 5). JAK2 V617F was detected in 89 (61%) patients with ET, 103 (86%) with PV, four (33%) with myelofibrosis, and four (80%) with MPNu. A higher number of leukocytes, haemoglobin levels and BM cellularity as well as an older age, lower platelet counts, and diagnosis of PV were significantly correlated with JAK2 V617F. Eighty-three and 43 episodes of thrombosis and bleeding occurred in 100 patients each before and after the diagnosis. Vascular events more frequently occurred in 37% of patients with JAK2 V617F than in 29% of those without the mutation (p = 0.045). Among 175 patients whose samples were available for sequencing, 28 patients with homozygous JAK2 V617F had vascular events more frequently (57%) than those who were heterozygotes (39%) or had the wild type (27%) (p = 0.03). The multivariate analysis showed that a JAK2 homozygous mutation, hypercholesterolemia and older age were independent risk factors for a vascular event. The results of this study showed that Korean patients with MPN had a similar JAK2 mutation rate and frequency of vascular events when compared to Western patients. The presence of V617F was significantly related to vascular events. Therefore, initial evaluation for the JAK2 mutation and careful monitoring for vascular events should be performed in MPN patients.

Results of a phase 2 study of pacritinib (SB1518), a JAK2/JAK2(V617F) inhibitor, in patients with myelofibrosis

PubMed Central

Seymour, John F.; Roberts, Andrew W.; Wadleigh, Martha; To, L. Bik; Scherber, Robyn; Turba, Elyce; Dorr, Andrew; Zhu, Joy; Wang, Lixia; Granston, Tanya; Campbell, Mary S.; Mesa, Ruben A.

2015-01-01

Pacritinib (SB1518) is a Janus kinase 2 (JAK2), JAK2(V617F), and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1. It demonstrated a favorable safety profile with promising efficacy in phase 1 studies in patients with primary and secondary myelofibrosis (MF). This multicenter phase 2 study further characterized the safety and efficacy of pacritinib in the treatment of patients with MF. Eligible patients had clinical splenomegaly poorly controlled with standard therapies or were newly diagnosed with intermediate- or high-risk Lille score. Patients with any degree of cytopenia were eligible. Thirty-five patients were enrolled. At entry, 40% had hemoglobin <10 g/dL and 43% had platelets <100 000× 109/L. Up to week 24, 8 of 26 evaluable patients (31%) achieved a ≥35% decrease in spleen volume determined by magnetic resonance imaging and 14 of 33 (42%) attained a ≥50% reduction in spleen size by physical examination. Median MF symptom improvement was ≥50% for all symptoms except fatigue. Grade 1 or 2 diarrhea (69%) and nausea (49%) were the most common treatment-emergent adverse events. The study drug was discontinued in 9 patients (26%) due to adverse events (4 severe). Pacritinib is an active agent in patients with MF, offering a potential treatment option for patients with preexisting anemia and thrombocytopenia. This trial was registered at www.clinicaltrials.gov as #NCT00745550. PMID:25762180

JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms

PubMed Central

Jones, Amy V; Chase, Andrew; Silver, Richard T; Oscier, David; Zoi, Katerina; Wang, Y Lynn; Cario, Holger; Pahl, Heike L; Collins, Andrew; Reiter, Andreas; Grand, Francis; Cross, Nicholas C P

2014-01-01

Chronic myeloproliferative neoplasms (MPNs) are a group of related conditions characterized by the overproduction of cells from one or more myeloid lineages. More than 95% of cases of polycythemia vera, and roughly half of essential thrombocythemia and primary myelofibrosis acquire a unique somatic 1849G>T JAK2 mutation (encoding V617F) that is believed to be a critical driver of excess proliferation1–4. We report here that JAK2V617F-associated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (polycythemia vera, n = 192, P = 2.9 × 10−16; essential thrombocythemia, n = 78, P = 8.2 × 10−9 and myelofibrosis, n = 41, P = 8.0 × 10−5). Furthermore, JAK2V617F specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of JAK2V617F-associated MPNs (OR = 3.7; 95% CI = 3.1–4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation. PMID:19287382

The Small Molecule Inhibitor G6 Significantly Reduces Bone Marrow Fibrosis and the Mutant Burden in a Mouse Model of Jak2-Mediated Myelofibrosis

PubMed Central

Kirabo, Annet; Park, Sung O.; Wamsley, Heather L.; Gali, Meghanath; Baskin, Rebekah; Reinhard, Mary K.; Zhao, Zhizhuang J.; Bisht, Kirpal S.; Keserű, György M.; Cogle, Christopher R.; Sayeski, Peter P.

2013-01-01

Philadelphia chromosome–negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F–mediated hyperplasia and a transgenic mouse model of Jak2-V617F–mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F–mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F–mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease. PMID:22796437

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms

PubMed Central

Tapper, William; Jones, Amy V.; Kralovics, Robert; Harutyunyan, Ashot S.; Zoi, Katerina; Leung, William; Godfrey, Anna L.; Guglielmelli, Paola; Callaway, Alison; Ward, Daniel; Aranaz, Paula; White, Helen E.; Waghorn, Katherine; Lin, Feng; Chase, Andrew; Joanna Baxter, E.; Maclean, Cathy; Nangalia, Jyoti; Chen, Edwin; Evans, Paul; Short, Michael; Jack, Andrew; Wallis, Louise; Oscier, David; Duncombe, Andrew S.; Schuh, Anna; Mead, Adam J.; Griffiths, Michael; Ewing, Joanne; Gale, Rosemary E.; Schnittger, Susanne; Haferlach, Torsten; Stegelmann, Frank; Döhner, Konstanze; Grallert, Harald; Strauch, Konstantin; Tanaka, Toshiko; Bandinelli, Stefania; Giannopoulos, Andreas; Pieri, Lisa; Mannarelli, Carmela; Gisslinger, Heinz; Barosi, Giovanni; Cazzola, Mario; Reiter, Andreas; Harrison, Claire; Campbell, Peter; Green, Anthony R.; Vannucchi, Alessandro; Cross, Nicholas C.P.

2015-01-01

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2V617F-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10−10) and rs2201862 (MECOM; meta-analysis P=1.96 × 10−9). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2V617F-positive cases. rs9376092 has a stronger effect in JAK2V617F-negative cases with CALR and/or MPL mutations (Breslow–Day P=4.5 × 10−7), whereas in JAK2V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ2 P=7.3 × 10−7). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype. PMID:25849990

JAK and MPL mutations in myeloid malignancies.

PubMed

Tefferi, Ayalew

2008-03-01

The Janus family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3 and tyrosine kinase 2) transduces signals downstream of type I and II cytokine receptors via signal transducers and activators of transcription (STATs). JAK3 is important in lymphoid and JAK2 in myeloid cell proliferation and differentiation. The thrombopoietin receptor MPL is one of several JAK2 cognate receptors and is essential for myelopoiesis in general and megakaryopoiesis in particular. Germline loss-of-function (LOF) JAK3 and MPL mutations cause severe combined immunodeficiency and congenital amegakaryocytic thrombocytopenia, respectively. Germline gain-of-function (GOF) MPL mutation (MPLS505N) causes familial thrombocytosis. Somatic JAK3 (e.g. JAK3A572V, JAK3V722I, JAK3P132T) and fusion JAK2 (e.g. ETV6-JAK2, PCM1-JAK2, BCR-JAK2) mutations have respectively been described in acute megakaryocytic leukemia and acute leukemia/chronic myeloid malignancies. However, current attention is focused on JAK2 (e.g. JAK2V617F, JAK2 exon 12 mutations) and MPL (e.g. MPLW515L/K/S, MPLS505N) mutations associated with myeloproliferative neoplasms (MPNs). A JAK2 mutation, primarily JAK2V617F, is invariably associated with polycythemia vera (PV). The latter mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). MPL mutational frequency in MPNs is substantially less (<10%). In general, despite a certain degree of genotype - phenotype correlations, the prognostic relevance of harbouring one of these mutations, or their allele burden when present, remains dubious. Regardless, based on the logical assumption that amplified JAK-STAT signalling is central to the pathogenesis of PV, ET and PMF, several anti-JAK2 tyrosine kinase inhibitors have been developed and are currently being tested in humans with these disorders.

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis

PubMed Central

Scott, Linda M.; Tong, Wei; Levine, Ross L.; Scott, Mike A.; Beer, Philip A.; Stratton, Michael R.; Futreal, P. Andrew; Erber, Wendy N.; McMullin, Mary Frances; Harrison, Claire N.; Warren, Alan J.; Gilliland, D. Gary; Lodish, Harvey F.; Green, Anthony R.

2010-01-01

BACKGROUND The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis

Roles of germline JAK2 activation mutation JAK2 V625F in the pathology of myeloproliferative neoplasms.

PubMed

Wu, Qing-Yun; Ma, Meng-Meng; Fu, Lin; Zhu, Yuan-Yuan; Liu, Yang; Cao, Jiang; Zhou, Ping; Li, Zhen-Yu; Zeng, Ling-Yu; Li, Feng; Wang, Xiao-Yun; Xu, Kai-Lin

2018-05-18

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

JAK2 inhibitor therapy in myeloproliferative disorders: rationale, preclinical studies and ongoing clinical trials.

PubMed

Pardanani, A

2008-01-01

The recent identification of somatic mutations such as JAK2V617F that deregulate Janus kinase (JAK)-signal transducer and activator of transcription signaling has spurred development of orally bioavailable small-molecule inhibitors that selectively target JAK2 kinase as an approach to pathogenesis-directed therapy of myeloproliferative disorders (MPD). In pre-clinical studies, these compounds inhibit JAK2V617F-mediated cell growth at nanomolar concentrations, and in vivo therapeutic efficacy has been demonstrated in mouse models of JAK2V617F-induced disease. In addition, ex vivo growth of progenitor cells from MPD patients harboring JAK2V617F or MPLW515L/K mutations is also potently inhibited. JAK2 inhibitors currently in clinical trials can be grouped into those designed to primarily target JAK2 kinase (JAK2-selective) and those originally developed for non-MPD indications, but that nevertheless have significant JAK2-inhibitory activity (non-JAK2 selective). This article discusses the rationale for using JAK2 inhibitors for the treatment of MPD, as well as relevant aspects of clinical trial development for these patients. For instance, which group of MPD patients is appropriate for initial Phase I studies? Should JAK2V617F-negative MPD patients be included in the initial studies? What are the likely consequences of 'off-target' JAK3 and wild-type JAK2 inhibition? How should treatment responses be monitored?

JAK2 V617F positive essential thrombocythemia developing in a patient with CD5⁻ chronic lymphocytic leukemia.

PubMed

Wei, Ju; Wang, Chun; Qin, You-Wen; Zhu, Jun; Gao, Yang-Rong; Cai, Qi; Yan, Shi-Ke

2012-06-01

Coexistence of chronic lymphocytic leukemia (CLL) and essential thrombocythemia (ET) in a patient is extremely rare, with only 10 cases reported thus far in literature. This paper describes a 94-year-old male having atypical B-CLL with CD5⁻ (CD5⁻) phenotype and ET. In this patient, we performed interphase fluorescence in situ hybridization (FISH) analysis which revealed 13q14.3 deletion in 31% of B-lymphocyte nuclei and RB1 deletion in 27% of B-lymphocyte nuclei, but not in neutrophils and T-lymphocytes. Furthermore, we identified JAK2 V617F mutation in the peripheral blood nucleated cells and neutrophils, but not in the B- and T-lymphocyte populations. Therefore, it was concluded that the occurrence of CD5− B-CLL and ET in this patient was pathogenically independent.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

PubMed

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

PubMed Central

Majumder, Anurima; Govindasamy, Lakshmanan; Magis, Andrew; Kiss, Róbert; Polgár, Tímea; Baskin, Rebekah; Allan, Robert W.; Agbandje-McKenna, Mavis; Reuther, Gary W.; Keserű, György M.; Bisht, Kirpal S.; Sayeski, Peter P.

2010-01-01

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. PMID:20667821

Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

PubMed

Park, Joonhong; Song, Minsik; Jang, Woori; Chae, Hyojin; Lee, Gun Dong; Kim, KyungTak; Park, Heekyung; Kim, Myungshin; Kim, Yonggoo

2017-02-01

We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Stimulation of the amino acid transporter SLC6A19 by JAK2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhavsar, Shefalee K.; Hosseinzadeh, Zohreh; Merches, Katja

Highlights: Black-Right-Pointing-Pointer The amino acid transporter SLC6A19 is upregulated by Janus kinase-2 JAK2. Black-Right-Pointing-Pointer The {sup V617F}JAK2 mutant, causing myeloproliferative disease, is more effective. Black-Right-Pointing-Pointer JAK2 inhibitor AG490 reverses stimulation of SLC6A19 by {sup V617F}JAK2. Black-Right-Pointing-Pointer JAK2 enhances SLC6A19 protein insertion into the cell membrane. Black-Right-Pointing-Pointer SLC6A19 may contribute to amino acid uptake into {sup V617F}JAK2 expressing tumor cells. -- Abstract: JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation {sup V617F}JAK2 mutant is found in the majority of myeloproliferativemore » diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na{sup +} coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, {sup V617F}JAK2 or inactive {sup K882E}JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I{sub le}), which was significantly increased following coexpression of JAK2 or {sup V617F}JAK2, but not by coexpression of {sup K882E}JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 {mu}M) resulted in a gradual decline of I{sub le}. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I{sub le} following inhibition of carrier insertion by brefeldin A (5 {mu}M) was

JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

PubMed Central

Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

2010-01-01

Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for

The JAK2 46/1 haplotype predisposes to MPL-mutated myeloproliferative neoplasms

PubMed Central

Jones, Amy V.; Campbell, Peter J.; Beer, Philip A.; Schnittger, Susanne; Vannucchi, Alessandro M.; Zoi, Katerina; Percy, Melanie J.; McMullin, Mary Frances; Scott, Linda M.; Tapper, William; Silver, Richard T.; Oscier, David; Harrison, Claire N.; Grallert, Harald; Kisialiou, Aliaksei; Strike, Paul; Chase, Andrew J.; Green, Anthony R.

2010-01-01

The 46/1 JAK2 haplotype predisposes to V617F-positive myeloproliferative neoplasms, but the underlying mechanism is obscure. We analyzed essential thrombocythemia patients entered into the PT-1 studies and, as expected, found that 46/1 was overrepresented in V617F-positive cases (n = 404) versus controls (n = 1492, P = 3.9 × 10−11). The 46/1 haplotype was also overrepresented in cases without V617F (n = 347, P = .009), with an excess seen for both MPL exon 10 mutated and V617F, MPL exon 10 nonmutated cases. Analysis of further MPL-positive, V617F-negative cases confirmed an excess of 46/1 (n = 176, P = .002), but no association between MPL mutations and MPL haplotype was seen. An excess of 46/1 was also seen in JAK2 exon 12 mutated cases (n = 69, P = .002), and these mutations preferentially arose on the 46/1 chromosome (P = .029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. The excess of 46/1 in JAK2 exon 12 cases is compatible with both the “hypermutability” and “fertile ground” hypotheses, but the excess in MPL-mutated cases argues against the former. No difference in sequence, splicing, or expression of JAK2 was found on 46/1 compared with other haplotypes, suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle. PMID:20304805

An accurate, simple prognostic model consisting of age, JAK2, CALR, and MPL mutation status for patients with primary myelofibrosis.

PubMed

Rozovski, Uri; Verstovsek, Srdan; Manshouri, Taghi; Dembitz, Vilma; Bozinovic, Ksenija; Newberry, Kate; Zhang, Ying; Bove, Joseph E; Pierce, Sherry; Kantarjian, Hagop; Estrov, Zeev

2017-01-01

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2 V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%); 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2 V617F allele burden and favorable survival and those with low Janus kinase 2 V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2 V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2 V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2 V617F allele burden or triple-negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis. Copyright© Ferrata Storti Foundation.

Jak2 FERM Domain Interaction with the Erythropoietin Receptor Regulates Jak2 Kinase Activity▿

PubMed Central

Funakoshi-Tago, Megumi; Pelletier, Stéphane; Moritake, Hiroshi; Parganas, Evan; Ihle, James N.

2008-01-01

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement. PMID:18160720

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

PubMed Central

Quintás-Cardama, Alfonso; Vaddi, Kris; Liu, Phillip; Manshouri, Taghi; Li, Jun; Scherle, Peggy A.; Caulder, Eian; Wen, Xiaoming; Li, Yanlong; Waeltz, Paul; Rupar, Mark; Burn, Timothy; Lo, Yvonne; Kelley, Jennifer; Covington, Maryanne; Shepard, Stacey; Rodgers, James D.; Haley, Patrick; Kantarjian, Hagop

2010-01-01

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC50] = 281nM), and proliferation of JAK2V617F+ Ba/F3 cells (IC50 = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F+ polycythemia vera patients (IC50 = 67nM) versus healthy donors (IC50 > 400nM). In a mouse model of JAK2V617F+ MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs. PMID:20130243

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

PubMed Central

dos Santos, Leonardo Caires; Ribeiro, Juliana Corrêa da Costa; Silva, Neusa Pereira; Cerutti, Janete; da Silva, Maria Regina Regis; Chauffaille, Maria de Lourdes Lopes Ferrari

2011-01-01

Background The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. Objectives The aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. Methods Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. Results Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia.

PubMed

Dos Santos, Leonardo Caires; Ribeiro, Juliana Corrêa da Costa; Silva, Neusa Pereira; Cerutti, Janete; da Silva, Maria Regina Regis; Chauffaille, Maria de Lourdes Lopes Ferrari

2011-01-01

The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. THE AIM OF THIS STUDY WAS TO DETECT THE FOLLOWING MUTATIONS: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L

SOCS3 tyrosine phosphorylation as a potential bio-marker for myeloproliferative neoplasms associated with mutant JAK2 kinases

PubMed Central

Elliott, Joanne; Suessmuth, Yvonne; Scott, Linda M.; Nahlik, Krystyna; McMullin, Mary Frances; Constantinescu, Stefan N.; Green, Anthony R.; Johnston, James A.

2009-01-01

JAK2 V617F, identified in the majority of patients with myeloproliferative neoplasms, tyrosine phosphorylates SOCS3 and escapes its inhibition. Here, we demonstrate that the JAK2 exon 12 mutants described in a subset of V617F-negative MPN cases, also stabilize tyrosine phosphorylated SOCS3. SOCS3 tyrosine phosphorylation was also observed in peripheral blood mononuclear cells and granulocytes isolated from patients with JAK2 H538QK539L or JAK2 F537-K539delinsL mutations. JAK kinase inhibitors, which effectively inhibited the proliferation of cells expressing V617F or K539L, also caused a dose-dependent reduction in both mutant JAK2 and SOCS3 tyrosine phosphorylation. We propose, therefore, that SOCS3 tyrosine phosphorylation may be a novel bio-marker of myeloproliferative neoplasms resulting from a JAK2 mutation and a potential reporter of effective JAK2 inhibitor therapy currently in clinical development. PMID:19229050

JAK2 mutations and clinical practice in myeloproliferative neoplasms.

PubMed

Tefferi, Ayalew

2007-01-01

With the discovery in the last 3 years of novel Janus kinase 2 (JAK2) and thrombopoietin receptor (MPL) mutations, the pathogenetic understanding of and clinical practice for myeloproliferative neoplasms (MPNs) have entered a new era. Each one of these newly discovered mutations, including JAK2V617F, MPLW515L, and a JAK2 exon 12 mutation, has been shown to result in constitutive activation of JAK-STAT signaling and also induce a MPN phenotype in mice. Thus, JAK2 is now considered to be a legitimate target for drug development in MPNs, and small molecule JAK2 inhibitors have already gone through successful preclinical testing, and early-phase human trials in primary myelofibrosis have already begun. Furthermore, JAK2 mutation screening has now become a front-line diagnostic test in the evaluation of both "erythrocytosis" and thrombocytosis and the 2001 World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis have now been revised to incorporate JAK2V617F mutation screening.

The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1-Negative Myeloproliferative Neoplasms.

PubMed

Lin, Yani; Liu, Enbin; Sun, Qi; Ma, Jiao; Li, QingHua; Cao, Zeng; Wang, Jun; Jia, Yujiao; Zhang, Hongju; Song, Zhen; Ai, Xiaofei; Shi, Lihui; Feng, Xiaofang; Li, Chenwei; Wang, Jianxiang; Ru, Kun

2015-07-01

To evaluate the mutation frequency of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 and the value of the combined tests in the diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). In the current study, mutations of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 were analyzed in 929 Chinese patients with BCR-ABL1-negative MPN, including 234 cases of polycythemia vera (PV), 428 ETs, 187 PMFs, and 80 unclassifiable MPNs (MPN-Us). Our result showed that the positive rate of any of four mutations in patients with PV, ET, PMF, and MPN-U was 89.3%, 83.4%, 87.2%, and 77.5%, respectively, which significantly improved the diagnostic rate, especially in ET and PMF. Meanwhile, we also found that the patients without any of four mutations were younger than those with one or more mutations. Unexpectedly, the coexistence of JAK2 V617F and CALR exon 9 was identified in six (0.6%) patients, and JAK2 V617F and MPL exon 10 were present simultaneously in two (0.2%) patients. In addition, we also identified several novel mutation types in CALR exon 9. The combined genetic tests of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 help improve the diagnostic rate for BCR-ABL1-negative MPN. Copyright© by the American Society for Clinical Pathology.

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms.

PubMed

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-07-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC(50)) of <1 n, and had 30-50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL-JAK2 fusion gene; IC(50)=11-120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.

CALR, JAK2 and MPL mutation status in Argentinean patients with BCR-ABL1- negative myeloproliferative neoplasms.

PubMed

Ojeda, Mara Jorgelina; Bragós, Irma Margarita; Calvo, Karina Lucrecia; Williams, Gladis Marcela; Carbonell, María Magdalena; Pratti, Arianna Flavia

2018-05-01

To establish the frequency of JAK2, MPL and CALR mutations in Argentinean patients with BCR-ABL1-negative  myeloproliferative neoplasms (MPN) and to compare their clinical and haematological features. Mutations of JAK2V617F, JAK2 exon 12, MPL W515L/K and CALR were analysed in 439 Argentinean patients with BCR-ABL1-negative MPN, including 176 polycythemia vera (PV), 214 essential thrombocythemia (ET) and 49 primary myelofibrosis (PMF). In 94.9% of PV, 85.5% ET and 85.2% PMF, we found mutations in JAK2, MPL or CALR. 74.9% carried JAK2V617F, 12.3% CALR mutations, 2.1% MPL mutations and 10.7% were triple negative. In ET, nine types of CALR mutations were identified, four of which were novel. PMF patients were limited to types 1 and 2, type 2 being more frequent. In ET, patients with CALR mutation were younger and had higher platelet counts than those with JAK2V617F and triple negative. In addition, JAK2V617F patients had high leucocyte and haemoglobin values compared with CALR-mutated and triple-negative patients. In PMF, patients with mutant CALR were associated with higher platelet counts. Our study underscores the importance of JAK2, MPL and CALR genotyping for accurate diagnosis of patients with BCR-ABL1-negative MPN.

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

PubMed Central

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-01-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC50) of <1 n, and had 30–50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL–JAK2 fusion gene; IC50=11–120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs. PMID:22829185

Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

PubMed Central

Bartalucci, Niccolò; Tozzi, Lorenzo; Bogani, Costanza; Martinelli, Serena; Rotunno, Giada; Villeval, Jean-Luc; Vannucchi, Alessandro M

2013-01-01

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN. PMID:24237791

Germline and somatic JAK2 mutations and susceptibility to chronic myeloproliferative neoplasms

PubMed Central

2009-01-01

Myeloproliferative neoplasms (MPNs) are a group of closely related stem-cell-derived clonal proliferative diseases. Most cases are sporadic but first-degree relatives of MPN patients have a five- to seven-fold increased risk for developing an MPN. The tumors of most patients carry a mutation in the Janus kinase 2 gene (JAK2V617F). Recently, three groups have described a strong association of JAK2 germline polymorphisms with MPN in patients positive for JAK2V617F. The somatic mutation occurs primarily on one particular germline JAK2 haplotype, which may account for as much as 50% of the risk to first-degree relatives. This finding provides new directions for unraveling the pathogenesis of MPN. PMID:19490586

Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease

PubMed Central

Huijsmans, Cornelis J. J.; Poodt, Jeroen; Damen, Jan; van der Linden, Johannes C.; Savelkoul, Paul H. M.; Pruijt, Johannes F. M.; Hilbink, Mirrian; Hermans, Mirjam H. A.

2012-01-01

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci. PMID:22768290

Janus kinase 2 inhibitors in myeloproliferative disorders.

PubMed

Lucia, Eugenio; Recchia, Anna Grazia; Gentile, Massimo; Bossio, Sabrina; Vigna, Ernesto; Mazzone, Carla; Madeo, Antonio; Morabito, Lucio; Gigliotti, Vincenzo; De Stefano, Laura; Caruso, Nadia; Servillo, Pasquale; Franzese, Stefania; Bisconte, Maria Grazia; Gentile, Carlo; Morabito, Fortunato

2011-01-01

JAK2 is an obligatory kinase for the proliferation and differentiation of erythroid cells and megakaryocytes thus representing a relevant therapeutic target for agents that specifically inhibit its activity particularly in myeloproliferative disorders (MPD) harboring JAK2(V617F) mutations. We discuss the physiopathology of the JAK2 signaling pathway and review clinical trials of JAK2 inhibitors for the treatment of MPD using papers and meeting abstracts published up to September 2010. This review helps in understanding the potential role of JAK2 inhibitors in MPD clinical trials and provides a comprehensive review regarding their efficacy and safety in these disorders. JAK2 inhibitors may prove to be useful only for suppressing disease manifestations. However, unlike drugs such as IFN which are capable of eliminating the malignant clone, JAK2 inhibitors are unable to eradicate the disease. In fact, results to date indicate that although these inhibitors reduce splenomegaly and alleviate constitutional symptoms irrespective of JAK2 mutational status, most have only a modest impact on the JAK2(V617F) allele burden. Considering the relevant risk of serious complications in patients undergoing splenectomy, these drugs could find a suitable indication in patients with myelofibrosis awaiting bone marrow transplantation.

Aberrant let7a/HMGA2 signaling activity with unique clinical phenotype in JAK2-mutated myeloproliferative neoplasms

PubMed Central

Chen, Chih-Cheng; You, Jie-Yu; Lung, Jrhau; Huang, Cih-En; Chen, Yi-Yang; Leu, Yu-Wei; Ho, Hsing-Ying; Li, Chian-Pei; Lu, Chang-Hsien; Lee, Kuan-Der; Hsu, Chia-Chen; Gau, Jyh-Pyng

2017-01-01

High mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that is negatively regulated by let-7 microRNA through binding to it’s 3′-untranslated region. Transgenic mice expressing Hmga2 with a truncation of its 3′-untranslated region has been shown to exhibit a myeloproliferative phenotype. To decipher the let-7-HMGA2 axis in myeloproliferative neoplasms, we employed an in vitro model supplemented with clinical correlation. Ba/F3 cells with inducible JAK2V617F expression (Ton.JAK2.V617F cells) showed upregulation of HMGA2 with concurrent let-7a repression. Ton.JAK2.V617F cells treated with a let-7a inhibitor exhibited further escalation of Hmga2 expression, while a let-7a mimic diminished the Hmga2 transcript level. Hmga2 overexpression conferred JAK2-mutated cells with a survival advantage through inhibited apoptosis. A pan-JAK inhibitor, INC424, increased the expression of let-7a, downregulated the level of Hmga2, and led to increased apoptosis in Ton.JAK2.V617F cells in a dose-dependent manner. In samples from 151 patients with myeloproliferative neoplasms, there was a modest inverse correlation between the expression levels of let-7a and HMGA2. Overexpression of HMGA2 was detected in 29 (19.2%) of the cases, and it was more commonly seen in patients with essential thrombocythemia than in those with polycythemia vera (26.9% vs. 12.7%, P=0.044). Patients with upregulated HMGA2 showed an increased propensity for developing major thrombotic events, and they were more likely to harbor one of the 3 driver myeloproliferative neoplasm mutations in JAK2, MPL and CALR. Our findings suggest that, in a subset of myeloproliferative neoplasm patients, the let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. PMID:28057739

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population

PubMed Central

Silva, Sarah Pagliarini- e-; Santos, Bruna Cunha; de Figueiredo Pereira, Elizangela Mendes; Ferreira, Mari Ellen; Baraldi, Elaine Cristina; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2013-01-01

OBJECTIVE: The JAK2 46/1 haplotype has recently been described as a major contributing factor to the development of myeloproliferative neoplasm, whether positive or negative for the JAK2 V617F mutation. The G allele, identified by a single-nucleotide polymorphism known as JAK2 rs10974944, is part of the JAK2 46/1 haplotype. The aim of this study was to verify the association between the presence of the G allele and the development of BCR-ABL-negative chronic myeloproliferative neoplasms in our population. METHODS: Blood and oral mucosa swab samples were obtained from 56 patients of two local Brazilian hospitals who had previously been diagnosed with BCR-ABL-negative chronic myeloproliferative neoplasms. Blood samples from 90 local blood donors were used as controls. The presence of the G allele was assessed using a PCR-RFLP assay after extracting DNA from the samples. RESULTS: The presence of the G allele was strongly associated with the presence of BCR-ABL-negative chronic myeloproliferative neoplasms (p = 0.0001; OR = 2.674; 95% CI = 1.630−4.385) in the studied population. CONCLUSION: In agreement with previous reports, the JAK2 46/1 haplotype, represented in this study by the presence of the G allele, is an important predisposing factor in the oncogenetic development of these neoplasms in our population. PMID:23420150

Myeloproliferative neoplasms: JAK2 signaling pathway as a central target for therapy.

PubMed

Pasquier, Florence; Cabagnols, Xenia; Secardin, Lise; Plo, Isabelle; Vainchenker, William

2014-09-01

The discovery of the JAK2V617F mutation followed by the discovery of other genetic abnormalities allowed important progress in the understanding of the pathogenesis and management of myeloproliferative neoplasms (MPN)s. Classical Breakpoint cluster region-Abelson (BCR-ABL)-negative neoplasms include 3 main disorders: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Genomic studies have shown that these disorders are more heterogeneous than previously thought with 3 main entities corresponding to different gene mutations: the JAK2 disorder, essentially due to JAK2V617F mutation, which includes nearly all PVs and a majority of ETs and PMFs with a continuum between these diseases and the myeloproliferative leukemia (MPL) and calreticulin (CALR) disorders, which include a fraction of ET and PMF. All of these mutations lead to a JAK2 constitutive activation. Murine models either with JAK2V617F or MPLW515L, but also with JAK2 or MPL germ line mutations found in hereditary thrombocytosis, have demonstrated that they are drivers of myeloproliferation. However, the myeloproliferative driver mutation is still unknown in approximately 15% of ET and PMF, but appears to also target the JAK/Signal Transducer and Activator of Transcription (STAT) pathway. However, other mutations in genes involved in epigenetics or splicing also can be present and can predate or follow mutations in signaling. They are involved either in clonal dominance or in phenotypic changes, more particularly in PMF. They can be associated with leukemic progression and might have an important prognostic value such as additional sex comb-like 1 mutations. Despite this heterogeneity, it is tempting to target JAK2 and its signaling for therapy. However in PMF, Adenosine Tri-Phosphate (ATP)-competitive JAK2 inhibitors have shown their interest, but also their important limitations. Thus, other approaches are required, which are discussed in this review. Copyright © 2014

JAK2, MPL, and CALR mutations in Chinese Han patients with essential thrombocythemia.

PubMed

Wang, Jing; Zhang, Biao; Chen, Bing; Zhou, Rong-Fu; Zhang, Qi-Guo; Li, Juan; Yang, Yong-Gong; Zhou, Min; Shao, Xiao-Yan; Xu, Yong; Xu, Xi-Hui; Ouyang, Jian; Xu, Jingyan; Ye, Qing

2017-04-01

Mutations in Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL), and CALR are highly relevant to Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms. Assessing the prevalence of molecular mutations in Chinese Han patients with essential thrombocythemia (ET), and correlating their mutational profile with disease characteristics/phenotype. Of the 110 subjects studied, 62 carried the JAK2 V617F mutation, 21 had CALR mutations, one carried an MPL (W515) mutation, and 28 had non-mutated JAK2, CALR, and MPL (so-called triple-negative ET). Mutations in JAK2 exon 12 were not detected in any patient. Two ET patients had both CALR and JAK2 V617F mutations. Comparing the hematological parameters of the patients with JAK2 mutations with those of the patients with CALR mutations showed that the ET patients with CALR mutations were younger (p = 0.045) and had higher platelet counts (p = 0.043). Genotyping for CALR could be a useful diagnostic tool for JAK2/MPL-negative ET, since the data suggest that CALR is much more prevalent than MPL, therefore testing for CALR should be considered in patients who are JAK2 negative as its frequency is almost 20 times that of MPL mutation.

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

PubMed Central

Tefferi, Ayalew

2009-01-01

Abstract Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- (e.g. FIP1L1-PDGFRA) or inter-chromosomal (e.g.ETV6-PDGFRB) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera (JAK2V617F and other JAK2 mutations), essential thrombocythemia (JAK2V617F and MPL515 mutations), primary myelofibrosis (JAK2V617F and MPL515 mutations), systemic mastocytosis (KITD816V and other KIT mutations) and stem cell leukaemia/lymphoma (ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets. PMID:19175693

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.

PubMed

Rumi, Elisa; Pietra, Daniela; Pascutto, Cristiana; Guglielmelli, Paola; Martínez-Trillos, Alejandra; Casetti, Ilaria; Colomer, Dolors; Pieri, Lisa; Pratcorona, Marta; Rotunno, Giada; Sant'Antonio, Emanuela; Bellini, Marta; Cavalloni, Chiara; Mannarelli, Carmela; Milanesi, Chiara; Boveri, Emanuela; Ferretti, Virginia; Astori, Cesare; Rosti, Vittorio; Cervantes, Francisco; Barosi, Giovanni; Vannucchi, Alessandro M; Cazzola, Mario

2014-08-14

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials. © 2014 by The American Society of Hematology.

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis

PubMed Central

Rumi, Elisa; Pietra, Daniela; Pascutto, Cristiana; Guglielmelli, Paola; Martínez-Trillos, Alejandra; Casetti, Ilaria; Colomer, Dolors; Pieri, Lisa; Pratcorona, Marta; Rotunno, Giada; Sant’Antonio, Emanuela; Bellini, Marta; Cavalloni, Chiara; Mannarelli, Carmela; Milanesi, Chiara; Boveri, Emanuela; Ferretti, Virginia; Astori, Cesare; Rosti, Vittorio; Cervantes, Francisco; Barosi, Giovanni; Vannucchi, Alessandro M.

2014-01-01

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials. PMID:24986690

The mutation profile of JAK2, MPL and CALR in Mexican patients with Philadelphia chromosome-negative myeloproliferative neoplasms.

PubMed

Labastida-Mercado, Nancy; Galindo-Becerra, Samantha; Garcés-Eisele, Javier; Colunga-Pedraza, Perla; Guzman-Olvera, Valeria; Reyes-Nuñez, Virginia; Ruiz-Delgado, Guillermo J; Ruiz-Argüelles, Guillermo J

2015-03-01

By using molecular markers, it is possible to gain information on both the classification and etiopathogenesis of chronic myeloproliferative neoplasias (MPN). In a group of 27 Mexican mestizo patients with MPNs, we studied seven molecular markers: the BCR/ABL1 fusion gene, the JAK2 V617F mutation, the JAK2 exon 12 mutations, the MPL W515L mutation, the MPL W515K mutation, and the calreticulin (CALR) exon 9 deletion or insertion. Patients with the BCR/ABL1 fusion gene were excluded. We studied 14 patients with essential thrombocythemia (ET), eight with polycythemia vera (PV), four with primary myelofibrosis (MF), and one with undifferentiated MPN. We found twelve individuals with the JAK2 V617F mutation; five of them had been clinically classified as PV, five as ET, and one as MF. One patient with the MPL W515L was identified with a clinical picture of ET. Five patients with the CALR mutation were identified, four ET and one MF. No individuals with either the MPL W515K mutation or the JAK2 exon 12 mutations were identified. The most consistent relationship was that between PV and the JAK2 V617F mutation (p=.01). Despite its small size, the study shows much less prevalence of JAK2 mutation in PV, ET and MF, which does not match international data. Copyright © 2015 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM and the risk of myeloproliferative neoplasms in Taiwanese population

PubMed Central

Chiang, Yi-Hao; Chang, Yu-Cheng; Lin, Huan-Chau; Huang, Ling; Cheng, Chun-Chia; Wang, Wei-Ting; Cheng, Hung-I; Su, Nai-Wen; Chen, Caleb Gon-Shen; Lin, Johnson; Chang, Yi-Fang; Chang, Ming-Chih; Hsieh, Ruey-Kuen; Chou, Wen-Chien; Lim, Ken-Hong; Kuo, Yuan-Yeh

2017-01-01

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM have been found to associate with myeloproliferative neoplasms (MPNs) in European populations. Whether these germline variations are associated with MPNs in Taiwanese population is obscure. Here we aimed to evaluate the association of five germline variations (JAK2 46/1 haplotype tagged by rs12343867, JAK2 intron 8 rs12339666, TERT rs2736100, HBS1L-MYB rs9376092 and MECOM rs2201862) and the risk of MPNs in Taiwanese population. A total of 178 MPN patients (109 essential thrombocythemia, 54 polycythemia vera and 15 primary myelofibrosis) were enrolled into this study. The information of 17033 control subjects was obtained from Taiwan Biobank database. The JAK2 46/1 haplotype, JAK2 rs12339666 and TERT rs2736100 were significantly associated with Taiwanese MPNs (P = 3.6×10-19, 1.9×10-19 and 3.1×10-6, respectively), and JAK2V617F-positive MPNs (n=121) (P = 5.6×10-21, 4.4×10-21 and 8.6×10-7, respectively). In JAK2V617F-negative cases (n=55), only the JAK2 46/1 haplotype and JAK2 rs12339666 remained statistically significant (P= 0.009 and 0.007, respectively). When stratified by disease subtypes, the JAK2 46/1 haplotype and JAK2 rs12339666 were significantly associated with all three MPN subtypes, but TERT rs2736100 was only associated with essential thrombocythemia and polycythemia vera. We did not find any association of these five SNPs with CALR mutations in our cohort. Furthermore, the risk alleles of MECOM rs2201862 and HBS1L-MYB rs9376092 were demonstrated to be negatively associated with the risk of developing polycythemia vera. In conclusion, germline variations at JAK2 (both the 46/1 haplotype and rs12339666) and TERT rs2736100 were associated with MPNs in Taiwanese population. PMID:29100304

The Amelioration of Myelofibrosis with Thrombocytopenia by a JAK1/2 Inhibitor, Ruxolitinib, in a Post-polycythemia Vera Myelofibrosis Patient with a JAK2 Exon 12 Mutation.

PubMed

Ikeda, Kazuhiko; Ueda, Koki; Sano, Takahiro; Ogawa, Kazuei; Ikezoe, Takayuki; Hashimoto, Yuko; Morishita, Soji; Komatsu, Norio; Ohto, Hitoshi; Takeishi, Yasuchika

2017-01-01

Less than 5% of patients with polycythemia vera (PV) show JAK2 exon 12 mutations. Although PV patients with JAK2 exon 12 mutations are known to develop post-PV myelofibrosis (MF) as well as PV with JAK2V617F, the role of JAK inhibitors in post-PV MF patients with JAK2 exon 12 mutations remains unknown. We describe how treatment with a JAK1/2 inhibitor, ruxolitinib, led to the rapid amelioration of marrow fibrosis, erythrocytosis and thrombocytopenia in a 77-year-old man with post-PV MF who carried a JAK2 exon 12 mutation (JAK2H538QK539L). This case suggests that ruxolitinib is a treatment option for post-PV MF in patients with thrombocytopenia or JAK2 exon 12 mutations.

[Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center].

PubMed

Li, M Y; Chao, H Y; Sun, A N; Qiu, H Y; Jin, Z M; Tang, X W; Han, Y; Fu, C C; Chen, S N; Wu, D P

2017-04-14

Objective: To explore the prevalences of JAK2, CALR and MPL gene mutations and the mutation types in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) , and to compare their clinical characteristics of different mutation types with each other and mutation negative group. Methods: The mutations of JAK2 V617F, JAK2 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 1 648 Ph negative MPNs patients were detected by direct sequencing. Results: ① The JAK2V617F mutation was found in 471 (92.7%) of 508 PV patients, 819 (78.1%) of 1 049 ET patients and 74 (81.3%) of 91 PMF patients respectively, with the total mutation rate as 82.8% (1 364/1 648) . The JAK2 exon12 mutation was found in 9 (1.7%) of 508 PV patients, none was found in ET or PMF patients, with the total mutation rate as 0.5% (9/1 648) . The CALR mutation was found in 132 (12.6%) of 1 049 ET patients and 11 (12.1%) of 91 PMF patients respectively, with the total mutation rate as 8.7% (143/1 648) ; the MPL mutation was found in 9 (0.9%) of 1 049 ET patients and 1 (1.1%) of 91 PMF patients respectively, with the total mutation rate as 0.6% (10/1 648) . The co-occurrence of any two types of driver gene mutations was not detected by direct sequencing. ②The median onset age of patients with JAK2V617F[61 (15-95) y] was significant higher than of with JAK2 exon12 mutation[49 (33-62) y] or without mutations[42 (3-78) y] ( P <0.001) , but not for patients with CALR[57 (17-89) y] or MPL mutation[59 (22-71) y] ( P >0.05) . Patients with JAK2V617F had higher white blood cell count and hemoglobin level ( P <0.05) when compared with patients with CALR mutation or without mutations, or only significantly higher white blood cell count when compared with patients with MPL mutation ( P =0.013) . The platelet count of patients with CALR mutation was significantly higher than of with JAK2V617F[966 (400-2 069) ×10(9)/L vs 800 (198-3 730) ×10(9)/L, P <0.001]. ③Karyotype analysis

Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms

PubMed Central

Atallah, Ehab; Verstovsek, Srdan

2016-01-01

The discovery of the Janus kinase (JAK)2 V617F mutation in patients with myeloproliferative neoplasms was a major milestone in understanding the biology of those disorders. Several groups simultaneously reported on the high incidence of this mutation in patients with myeloproliferative neoplasms: almost all patients with polycythemia vera harbor the mutation and about 50% of patients with essential thrombocythemia and primary myelofibrosis have the mutation, making the development of JAK2 tyrosine kinase inhibitors an attractive therapeutic goal. In addition, inhibition of JAK2 kinase may have a therapeutic role in other hematologic malignancies, such as chronic myeloid leukemia or lymphoma. A number of molecules that inhibit JAK2 kinase have been described in the literature, and several are being evaluated in a clinical setting. Here, we summarize current clinical experience with JAK2 inhibitors. PMID:19445582

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

PubMed

Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R; Combs, Andrew P; Vaddi, Kris; Haley, Patrick J; Burn, Timothy C; Rupar, Mark; Bromberg, Jacqueline F; Heaney, Mark L; de Stanchina, Elisa; Fridman, Jordan S; Levine, Ross L

2010-04-08

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis

PubMed Central

Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R.; Combs, Andrew P.; Vaddi, Kris; Haley, Patrick J.; Burn, Timothy C.; Rupar, Mark; Bromberg, Jacqueline F.; Heaney, Mark L.; de Stanchina, Elisa; Fridman, Jordan S.

2010-01-01

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model. PMID:20154217

The role of JAK1/2 inhibitors in the treatment of chronic myeloproliferative neoplasms.

PubMed

Keohane, Clodagh; Mesa, Ruben; Harrison, Claire

2013-01-01

In 2005, the description of the JAK2V617F mutation for the first time provided a molecular key to enable more rapid diagnosis and target for novel therapeutics in the myeloproliferative neoplasms. In 2007, the first-in-class agent INC18424, ruxolitinib, JAKafi, or JAKAVI was first tested in patients with intermediate-risk 2 or high-risk myelofibrosis regardless of whether they possessed the JAK2V617F mutation. Patients treated with this agent had major reduction in splenomegaly as well as impressive reduction, and in some cases resolution, of symptoms. This study was followed by the two Controlled Myelofibrosis Study with Oral JAK Inhibitor Therapy (COMFORT) trials (the first-ever phase III trials in myelofibrosis), which confirmed results in these aspects were superior to either placebo or standard care, and updated results show a survival advantage with this therapy. This paper discusses these results and data from other JAK inhibitors while speculating on the future of these therapies. It also reflects on the fact that the true targets and agents' mode of action are uncertain. Unlike targeted therapy for chronic myeloid leukemia (CML), these agents do not deliver molecular remission, and it is not clear whether their predominant benefit is mediated via JAK2, JAK1, or both. Nonetheless, the advent of the JAK inhibitor is a welcome advance and has made a dramatic improvement to the therapeutic landscape of these conditions.

Two Clinical Phenotypes in Polycythemia Vera

PubMed Central

Spivak, Jerry L.; Considine, Michael; Williams, Donna M.; Talbot, Conover C.; Rogers, Ophelia; Moliterno, Alison R.; Jie, Chunfa; Ochs, Michael F.

2014-01-01

BACKGROUND Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown. METHODS We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women. RESULTS Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy. CONCLUSIONS Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. PMID:25162887

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders.

PubMed

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, S H; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders

PubMed Central

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, SH; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Background Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Material and Methods Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Results Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. Conclusions The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients. PMID:25914801

Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms

PubMed Central

Bhagwat, Neha; Koppikar, Priya; Keller, Matthew; Marubayashi, Sachie; Shank, Kaitlyn; Rampal, Raajit; Qi, Jun; Kleppe, Maria; Patel, Hardik J.; Shah, Smit K.; Taldone, Tony; Bradner, James E.; Chiosis, Gabriela

2014-01-01

The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK–signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting. PMID:24470592

MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.

PubMed

Pardanani, Animesh D; Levine, Ross L; Lasho, Terra; Pikman, Yana; Mesa, Ruben A; Wadleigh, Martha; Steensma, David P; Elliott, Michelle A; Wolanskyj, Alexandra P; Hogan, William J; McClure, Rebecca F; Litzow, Mark R; Gilliland, D Gary; Tefferi, Ayalew

2006-11-15

Recently, a gain-of-function MPL mutation, MPLW515L, was described in patients with JAK2V617F-negative myelofibrosis with myeloid metaplasia (MMM). To gain more information on mutational frequency, disease specificity, and clinical correlates, genomic DNA from 1182 patients with myeloproliferative and other myeloid disorders and 64 healthy controls was screened for MPL515 mutations, regardless of JAK2V617F mutational status: 290 with MMM, 242 with polycythemia vera, 318 with essential thrombocythemia (ET), 88 with myelodysplastic syndrome, 118 with chronic myelomonocytic leukemia, and 126 with acute myeloid leukemia (AML). MPL515 mutations, either MPLW515L (n = 17) or a previously undescribed MPLW515K (n = 5), were detected in 20 patients. The diagnosis of patients with mutant MPL alleles at the time of molecular testing was de novo MMM in 12 patients, ET in 4, post-ET MMM in 1, and MMM in blast crisis in 3. Six patients carried the MPLW515L and JAK2V617F alleles concurrently. We conclude that MPLW515L or MPLW515K mutations are present in patients with MMM or ET at a frequency of approximately 5% and 1%, respectively, but are not observed in patients with polycythemia vera (PV) or other myeloid disorders. Furthermore, MPL mutations may occur concurrently with the JAK2V617F mutation, suggesting that these alleles may have functional complementation in myeloproliferative disease.

Polycythemia Vera: An Appraisal of the Biology and Management 10 Years After the Discovery of JAK2 V617F

PubMed Central

Stein, Brady L.; Oh, Stephen T.; Berenzon, Dmitriy; Hobbs, Gabriela S.; Kremyanskaya, Marina; Rampal, Raajit K.; Abboud, Camille N.; Adler, Kenneth; Heaney, Mark L.; Jabbour, Elias J.; Komrokji, Rami S.; Moliterno, Alison R.; Ritchie, Ellen K.; Rice, Lawrence; Mascarenhas, John; Hoffman, Ronald

2015-01-01

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm that is associated with a substantial symptom burden, thrombohemorrhagic complications, and impaired survival. A decade after the seminal discovery of an activating mutation in the tyrosine kinase JAK2 in nearly all patients with PV, new treatment options are finally beginning to emerge, necessitating a critical reappraisal of the underlying pathogenesis and therapeutic modalities available for PV. Herein, we comprehensively review clinical aspects of PV including diagnostic considerations, natural history, and risk factors for thrombosis. We summarize recent studies delineating the genetic basis of PV, including their implications for evolution to myelofibrosis and secondary acute myeloid leukemia. We assess the quality of evidence to support the use of currently available therapies, including aspirin, phlebotomy, hydroxyurea, and interferon. We analyze recent studies evaluating the safety and efficacy of JAK inhibitors, such as ruxolitinib, and evaluate their role in the context of other available therapies for PV. This review provides a framework for practicing hematologists and oncologists to make rational treatment decisions for patients with PV. PMID:26324368

[MPLW515L point mutation in patients with myeloproliferative disease].

PubMed

Xia, Jun; Xu, Wei; Zhang, Su-Jiang; Fan, Lei; Qiao, Chun; Li, Jian-Yong

2008-12-01

In order to investigate the frequency of MPLW515L and JAK2V617F point mutations of the patients with myeloproliferative disease (MPD) in Nanjing area, MPLW515L and JAK2V617F point mutations were simultaneously detected by alleles specific polymerase chain reaction (AS-PCR) and sequencing in 190 MPD patients. The results showed that MPLW515L point mutation was detected in 1 out of 102 essential thrombocythemia (ET) patients (1.0%) and was not detected in 32 polycythemia vera (PV) patients, 13 idiopathic myelofibrosis (IMF) patients, 43 chronic myelogenous leukemia (CML) patients. JAK2V617F point mutation was detected in 20 out of 32 PV patients (62.5%), 43 out of 102 ET patients (42.2%), 5 out of 13 IMF patients (38.5%), and was not detected in 43 CML patients. It is concluded that MPLW515L point mutation exists in ET patient, but is not found in PV, IMF and CML. JAK2V617F point mutation exists in PV, ET and IMF, but not in CML.

The oral HDAC inhibitor pracinostat (SB939) is efficacious and synergistic with the JAK2 inhibitor pacritinib (SB1518) in preclinical models of AML

PubMed Central

Novotny-Diermayr, V; Hart, S; Goh, K C; Cheong, A; Ong, L-C; Hentze, H; Pasha, M K; Jayaraman, R; Ethirajulu, K; Wood, J M

2012-01-01

Acute myeloid leukemia (AML) is currently treated with aggressive chemotherapy that is not well tolerated in many elderly patients, hence the unmet medical need for effective therapies with less toxicity and better tolerability. Inhibitors of FMS-like tyrosine kinase 3 (FLT3), JAK2 and histone deacetylase inhibitors (HDACi) have been tested in clinical studies, but showed only moderate single-agent activity. High efficacy of the HDACi pracinostat treating AML and synergy with the JAK2/FLT3 inhibitor pacritinib is demonstrated. Both compounds inhibit JAK-signal transducer and activator of transcription (STAT) signaling in AML cells with JAK2V617F mutations, but also diminish FLT3 signaling, particularly in FLT3-ITD (internal tandem duplication) cell lines. In vitro, this combination led to decreased cell proliferation and increased apoptosis. The synergy translated in vivo in two different AML models, the SET-2 megakaryoblastic AML mouse model carrying a JAK2V617F mutation, and the MOLM-13 model of FLT3-ITD-driven AML. Pracinostat and pacritinib in combination showed synergy on tumor growth, reduction of metastases and synergistically decreased JAK2 or FLT signaling, depending on the cellular context. In addition, several plasma cytokines/growth factors/chemokines triggered by the tumor growth were normalized, providing a rationale for combination therapy with an HDACi and a JAK2/FLT3 inhibitor for the treatment of AML patients, particularly those with FLT3 or JAK2 mutations. PMID:22829971

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders

PubMed Central

Vainchenker, William; Leroy, Emilie; Gilles, Laure; Marty, Caroline; Plo, Isabelle; Constantinescu, Stefan N.

2018-01-01

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non- BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In

Quantification of the Mutant CALR Allelic Burden by Digital PCR: Application to Minimal Residual Disease Evaluation after Bone Marrow Transplantation.

PubMed

Mansier, Olivier; Migeon, Marina; Saint-Lézer, Arnaud; James, Chloé; Verger, Emmanuelle; Robin, Marie; Socié, Gérard; Bidet, Audrey; Mahon, François-Xavier; Cassinat, Bruno; Lippert, Eric

2016-01-01

With the recent discovery of CALR mutations, >80% of patients with myeloproliferative neoplasms carry a phenotype-driving mutation. For JAK2 V617F, the most frequent mutation in myeloproliferative neoplasms, accurate determination of mutational loads is of interest at diagnosis, for phenotypic and prognostic purposes, and during follow-up for minimal residual disease assessment. We developed a digital PCR technique that allowed the accurate determination of CALR allelic burdens for the main mutations (types 1 and 2). Compared with the commonly used fluorescent PCR product analysis, digital PCR is more precise, reproducible, and accurate. Furthermore, this method reached a very high sensitivity. We detected at least 0.025% CALR mutants. It can thus be used for patient characterization at diagnosis and for minimal residual disease monitoring. When applied to patients with primary myelofibrosis who underwent hematopoietic stem cell transplant, the digital PCR detected low levels of minimal residual disease. After negativation of the mutational load in all patients, the disease reappeared at a low level in one patient, preceding hematologic relapse. In conclusion, digital PCR adapted to type 1 and 2 CALR mutations is an inexpensive, highly precise, and sensitive technique suitable for evaluation of myeloproliferative neoplasm patients during follow-up. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Presence of calreticulin mutations in JAK2-negative polycythemia vera.

PubMed

Broséus, Julien; Park, Ji-Hye; Carillo, Serge; Hermouet, Sylvie; Girodon, François

2014-12-18

Calreticulin (CALR) mutations have been reported in Janus kinase 2 (JAK2)- and myeloproliferative leukemia (MPL)-negative essential thrombocythemia and primary myelofibrosis. In contrast, no CALR mutations have ever been reported in the context of polycythemia vera (PV). Here, we describe 2 JAK2(V617F)-JAK2(exon12)-negative PV patients who presented with a CALR mutation in peripheral granulocytes at the time of diagnosis. In both cases, the CALR mutation was a 52-bp deletion. Single burst-forming units-erythroid (BFU-E) from 1 patient were grown in vitro and genotyped: the same CALR del 52-bp mutation was noted in 31 of the 37 colonies examined; 30 of 31 BFU-E were heterozygous for CALR del 52 bp, and 1 of 31 BFU-E was homozygous for CALR del 52 bp. In summary, although unknown mutations leading to PV cannot be ruled out, our results suggest that CALR mutations can be associated with JAK2-negative PV. © 2014 by The American Society of Hematology.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

PubMed Central

Milosevic Feenstra, Jelena D.; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N.; Cazzola, Mario

2016-01-01

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. PMID:26423830

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

PubMed

Milosevic Feenstra, Jelena D; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N; Cazzola, Mario; Kralovics, Robert

2016-01-21

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

PubMed Central

Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D. Gary; Levine, Ross L

2006-01-01

Background The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). Methods and Findings DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Conclusions Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative

MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

PubMed

Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D Gary; Levine, Ross L

2006-07-01

The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF

The mutation profile of JAK2 and CALR in Chinese Han patients with Philadelphia chromosome-negative myeloproliferative neoplasms

PubMed Central

2014-01-01

Mutations in JAK2, MPL and CALR are highly relevant to the Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs). We performed high resolution melting analysis and Sanger sequencing together with T-A cloning to elucidate the unique mutation profile of these genes, in Chinese patients with MPNs. Peripheral blood DNA samples were obtained from 80 patients with polycythemia vera (PV), 80 patients with essential thrombocytosis (ET) and 50 patients with primary myelofibrosis (PMF). Ten PV patients were identified with diverse JAK2 exon 12 mutations. Five novel JAK2 Exon 12 mutation patterns (M532V/E543G, N533D, M535I/H538Y/K549I, E543G and D544N) were described. JAK2 V617F was detected in 140 samples (66 PV, 45 ET and 29 PMF). JAK2 Exon 12 mutations were prevalent (13%) and variable in the Chinese patients. Compared with PV patients with JAK2 V617F mutations, PV patients with JAK2 exon 12 mutations had an earlier median onset of disease (P = 0.0013). MPL W515L/K mutations were discerned in 4 ET and 3 PMF patients. Two kinds of CALR mutation, c. 1179_1230del and c. 1234_1235insTTGTC were detected in 20 ET and 16 PMF patients. A novel CALR mutation pattern (c. 1173_1223del/c. 1179_1230del) was identified in 2 PMF samples. In addition, 17 scattered point mutations in CALR c.1153 to c.1255 were also detected in 13 cases with CALR frame-shifting variations and 2 cases without CALR frame-shifting variations. Female patients showed a predisposition to CALR mutations (P = 0.0035). Chinese Ph-negative MPN patients have a unique mutation landscape in the common molecular markers of MPN diagnosis. Validation of the molecular diagnostic pipeline should be emphasized since there is a considerable ethnical diversity in the molecular profiles of Ph-negative MPNs. PMID:25023898

Targeting glutamine metabolism in myeloproliferative neoplasms

PubMed Central

Zhan, Huichun; Ciano, Kristen; Dong, Katherine; Zucker, Stanley

2016-01-01

JAK2V617F mutation can be detected in the majority of myeloproliferative neoplasm (MPN) patients. The JAK2 inhibitor Ruxolitinib is the first FDA-approved treatment for MPNs. However, its use is limited by various dose related toxicities. Here, we studied the metabolic state and glutamine metabolism of BaF3-hEPOR-JAK2V617F and BaF3-hEPOR-JAK2WT cells. We found that the JAK2V617F-mutant cells were associated with increased oxygen consumption rate and extracellular acidification rate than the JAK2WT cells and there was an increased glutamine metabolism in JAK2V617F-mutant cells compared to wild-type cells. Glutaminase (GLS), the key enzyme in gluta-mine metabolism, was upregulated in the JAK2V617F-mutant BaF3 cells compared to the JAK2WT BaF3 cells. In MPN patient peripheral blood CD34+ cells, GLS expression was increased in JAK2V617F-mutant progenitor cells compared to JAK2 wild-type progenitor cells from the same patients and GLS levels were increased at the time of disease progression compared to at earlier time points. Moreover, GLS inhibitor increased the growth inhibitory effect of Ruxolitinib in both JAK2V617F-mutant cell lines and peripheral blood CD34+ cells from MPN patients. Therefore, GLS inhibitor should be further explored to enhance the therapeutic effectiveness of JAK2 inhibitor and allow the administration of lower doses of the drug to avoid its toxicity. PMID:26227854

CHZ868, a Type II JAK2 Inhibitor, Reverses Type I JAK Inhibitor Persistence and Demonstrates Efficacy in Myeloproliferative Neoplasms

PubMed Central

Meyer, Sara C.; Keller, Matthew D.; Chiu, Sophia; Koppikar, Priya; Guryanova, Olga A.; Rapaport, Franck; Xu, Ke; Manova, Katia; Pankov, Dmitry; O’Reilly, Richard J.; Kleppe, Maria; McKenney, Anna Sophia; Shih, Alan H.; Shank, Kaitlyn; Ahn, Jihae; Papalexi, Eftymia; Spitzer, Barbara; Socci, Nick; Viale, Agnes; Mandon, Emeline; Ebel, Nicolas; Andraos, Rita; Rubert, Joëlle; Dammassa, Ernesta; Romanet, Vincent; Dölemeyer, Arno; Zender, Michael; Heinlein, Melanie; Rampal, Rajit; Weinberg, Rona Singer; Hoffman, Ron; Sellers, William R.; Hofmann, Francesco; Murakami, Masato; Baffert, Fabienne; Gaul, Christoph; Radimerski, Thomas; Levine, Ross L.

2015-01-01

Summary Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasms (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated if CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2- and MPL-mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients. PMID:26175413

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

PubMed

Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

2011-04-01

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms

PubMed Central

Rice, K L; Lin, X; Wolniak, K; Ebert, B L; Berkofsky-Fessler, W; Buzzai, M; Sun, Y; Xi, C; Elkin, P; Levine, R; Golub, T; Gilliland, D G; Crispino, J D; Licht, J D; Zhang, W

2011-01-01

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3–23.3 (n=1), 9q33.1–34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31–36.33 (n=6), 17q21.2–q21.31 (n=5) and 17q25.1–25.3 (n=5) and deletions affecting 18p11.31–11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a ‘HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal. PMID:22829077

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2V617F positive polycythemia vera: a case report.

PubMed

Pang, Ying; Gupta, Garima; Yang, Chunzhang; Wang, Herui; Huynh, Thanh-Truc; Abdullaev, Ziedulla; Pack, Svetlana D; Percy, Melanie J; Lappin, Terence R J; Zhuang, Zhengping; Pacak, Karel

2018-03-13

The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome. A female presented with a history of JAK2 V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells. This is the first report which provides

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms.

PubMed

Kovačić, Marijana; Mitrović-Ajtić, Olivera; Beleslin-Čokić, Bojana; Djikić, Dragoslava; Subotički, Tijana; Diklić, Miloš; Leković, Danijela; Gotić, Mirjana; Mossuz, Pascal; Čokić, Vladan P

2018-06-26

Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34 + cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN

Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

PubMed

Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

2018-03-09

The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

Molecular analyses of 15,542 patients with suspected BCR-ABL1-negative myeloproliferative disorders allow to develop a stepwise diagnostic workflow

PubMed Central

Schnittger, Susanne; Bacher, Ulrike; Eder, Christiane; Dicker, Frank; Alpermann, Tamara; Grossmann, Vera; Kohlmann, Alexander; Kern, Wolfgang; Haferlach, Claudia; Haferlach, Torsten

2012-01-01

We investigated 15,542 patients with suspected BCR-ABL1- negative myeloproliferative or myelodysplastic/myeloproliferative neoplasm (including 359 chronic myelomonocytic leukemia) by a molecular marker set. JAK2V617F was detected in the suspected categories as follows: polycythemia vera 88.3%, primary myelofibrosis 53.8%, essential thrombocythemia 50.2%, and not further classifiable myeloproliferative neoplasms 38.0%. JAK2 exon 12 mutations were detected in 40.0% JAK2V617F-negative suspected polycythemia vera, MPLW515 mutations in 13.2%JAK2V617F-negative primary myelofibrosis and 7.1% JAK2V617F-negative essential thrombocythemia. TET2 mutations were distributed across all entities but were most frequent in suspected chronic myelomonocytic leukemia (77.8%). CBL mutations were identified in suspected chronic myelomonocytic leukemia (13.9%), primary myelofibrosis (8.0%), and not further classifiable myeloproliferative neoplasm (7.0%). This leads to a stepwise workflow for suspected myeloproliferative neoplasms starting with JAK2V617F and investigating JAK2V617F-negative patients for JAK2 exon 12 or MPL mutations, respectively. In cases in which a myeloproliferative neoplasm cannot be established, analysis for TET2, CBL and EZH2 mutations may be indicated. PMID:22511494

MECOM, HBS1L-MYB, THRB-RARB, JAK2, and TERT polymorphisms defining the genetic predisposition to myeloproliferative neoplasms: A study on 939 patients.

PubMed

Trifa, Adrian P; Bănescu, Claudia; Bojan, Anca S; Voina, Cristian M; Popa, Ștefana; Vișan, Simona; Ciubean, Alina D; Tripon, Florin; Dima, Delia; Popov, Viola M; Vesa, Ștefan C; Andreescu, Mihaela; Török-Vistai, Tünde; Mihăilă, Romeo G; Berbec, Nicoleta; Macarie, Ioan; Coliţă, Andrei; Iordache, Maria; Cătană, Alina C; Farcaș, Marius F; Tomuleasa, Ciprian; Vasile, Kinga; Truică, Cristina; Todincă, Adriana; Pop-Muntean, Lavinia; Manolache, Raluca; Bumbea, Horia; Vlădăreanu, Ana-Maria; Gaman, Mihaela; Ciufu, Cristina M; Popp, Radu A

2018-01-01

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are classical myeloproliferative neoplasms (MPN), characterized by specific somatic mutations in JAK2, CALR or MPL genes. JAK2 46/1 and TERT rs2736100 polymorphisms are known to significantly predispose to MPN. This study aimed to establish the additional contribution of the recently described MECOM rs2201862, HBS1L-MYB rs9376092 and THRB-RARB rs4858647 polymorphisms to the occurrence of MPN. These three polymorphisms, along with JAK2 46/1 and TERT rs2736100 were genotyped in 939 MPN patients (454 with ET, 337 with PV and 148 with PMF) and 483 controls. MECOM rs2201862 associated significantly with each MPN entity, except for ET, and with all major molecular sub-types, especially those CALR-mutated (OR = 1.4; 95% CI = 1.1-1.8; P-value = .005). HBS1L-MYB rs9376092 associated only with JAK2 V617F-mutated ET (OR = 1.4; 95% CI = 1.1-1.7; P-value = .003). THRB-RARB rs4858647 had a weak association with PMF only (OR = 1.5; 95% CI = 1-2.1; P-value = .04). Surprisingly, JAK2 46/1 haplotype was associated significantly not only with JAK2 V617F-mutated MPN, but also with CALR-mutated MPN (OR = 1.4; 95% CI = 1.1-1.8; P-value = .01). TERT rs2736100 was associated equally strong with all MPN, regardless of phenotype or molecular sub-type. In conclusion, JAK2 46/1, TERT rs2736100 and MECOM rs2201862 are the chief predisposing polymorphisms to MPN. © 2017 Wiley Periodicals, Inc.

Stem and Progenitor Cell Subsets Are Affected by JAK2 Signaling and Can Be Monitored by Flow Cytometry

PubMed Central

Iida, Ryuji; Welner, Robert S.; Zhao, Wanke; Alberola-lla, José; Medina, Kay L.; Zhao, Zhizhuang Joe; Kincade, Paul W.

2014-01-01

Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation. PMID:24699465

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster

PubMed Central

Anderson, Abigail M.; Bailetti, Alessandro A.; Rodkin, Elizabeth; De, Atish; Bach, Erika A.

2017-01-01

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling. PMID:28620086

High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

PubMed Central

Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

2009-01-01

JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

PubMed

Yang, Tianfeng; Shi, Xianpeng; Kang, Yuan; Zhu, Man; Fan, Mengying; Zhang, Dongdong; Zhang, Yanmin

2018-07-01

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Involvement of mast cells by the malignant process in patients with Philadelphia chromosome negative myeloproliferative neoplasms.

PubMed

Wang, J; Ishii, T; Zhang, W; Sozer, S; Dai, Y; Mascarenhas, J; Najfeld, V; Zhao, Z J; Hoffman, R; Wisch, N; Xu, M

2009-09-01

The Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematologic malignancies frequently characterized by a mutation in JAK2 (JAK2V617F). Peripheral blood (PB) CD34(+) cells from patients with polycythemia vera (PV) and primary myelofibrosis (PMF) generated in vitro significantly fewer mast cells (MCs) than normal PB CD34(+) cells. The numbers of MC progenitors assayed from MPN CD34(+) cells were, however, similar to that assayed from normal CD34(+) cells. A higher percentage of the cultured MPN MCs expressed FcvarepsilonRIalpha, CD63 and CD69 than normal MCs, suggesting that cultured MPN MCs are associated with an increased state of MC activation. Further analysis showed that a higher proportion of cultured PV and PMF MCs underwent apoptosis in vitro. By using JAK2V617F, MplW515L and chromosomal abnormalities as clonality markers, we showed that the malignant process involved MPN MCs. JAK2V617F-positive MC colonies were assayable from the PB CD34(+) cells of each of the 17 JAK2V617F positive MPN patients studied. Furthermore, erlotinib, a JAK2 inhibitor, was able to inhibit JAK2V617F-positive PV MC progenitor cells, indicating that malignant MC progenitor cells are a potential cellular target for such JAK2 inhibitor-directed therapy.

FLT3 and JAK2 Mutations in Acute Myeloid Leukemia Promote Interchromosomal Homologous Recombination and the Potential for Copy Neutral Loss of Heterozygosity.

PubMed

Gaymes, Terry J; Mohamedali, Azim; Eiliazadeh, Anthony L; Darling, David; Mufti, Ghulam J

2017-04-01

Acquired copy neutral LOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G 0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N -acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. Cancer Res; 77(7); 1697-708. ©2017 AACR . ©2017 American Association for Cancer Research.

Characteristics and clinical correlates of MPL 515W>L/K mutation in essential thrombocythemia.

PubMed

Vannucchi, Alessandro M; Antonioli, Elisabetta; Guglielmelli, Paola; Pancrazzi, Alessandro; Guerini, Vittoria; Barosi, Giovanni; Ruggeri, Marco; Specchia, Giorgina; Lo-Coco, Francesco; Delaini, Federica; Villani, Laura; Finotto, Silvia; Ammatuna, Emanuele; Alterini, Renato; Carrai, Valentina; Capaccioli, Gloria; Di Lollo, Simonetta; Liso, Vincenzo; Rambaldi, Alessandro; Bosi, Alberto; Barbui, Tiziano

2008-08-01

Among 994 patients with essential thrombocythemia (ET) who were genotyped for the MPLW515L/K mutation, 30 patients carrying the mutation were identified (3.0%), 8 of whom also displayed the JAK2V671F mutation. MPLW515L/K patients presented lower hemoglobin levels and higher platelet counts than did wild type (wt) MPL; these differences were highly significant compared with MPLwt/JAK2V617F-positive patients. Reduced hemoglobin and increased platelet levels were preferentially associated with the W515L and W515K alleles, respectively. MPL mutation was a significant risk factor for microvessel disturbances, suggesting platelet hyperreactivity associated with constitutively active MPL; arterial thromboses were increased only in comparison to MPLwt/JAK2wt patients. MPLW515L/K patients presented reduced total and erythroid bone marrow cellularity, whereas the numbers of megakaryocytes, megakaryocytic clusters, and small-sized megakaryocytes were all significantly increased. These data indicate that MPLW515L/K mutations do not define a distinct phenotype in ET, although some differences depended on the JAK2V617F mutational status of the counterpart.

Impact of genotype on leukaemic transformation in polycythaemia vera and essential thrombocythaemia.

PubMed

Alvarez-Larrán, Alberto; Senín, Alicia; Fernández-Rodríguez, Concepción; Pereira, Arturo; Arellano-Rodrigo, Eduardo; Gómez, Montse; Ferrer-Marin, Francisca; Martínez-López, Joaquín; Camacho, Laura; Colomer, Dolors; Angona, Anna; Navarro, Blanca; Cervantes, Francisco; Besses, Carlos; Bellosillo, Beatriz; Hernández-Boluda, Juan Carlos

2017-09-01

The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow-up of 7·2 years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2-0·9, P = 0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9-4·6, P = 0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR-mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1-5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1-3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F-positive. JAK2 V617F-mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F-negative leukaemia. Survival was longer in JAK2 V617F-unmutated leukaemia (343 days vs. 95 days, P = 0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F-negative clone is TP53 independent and shows better survival. © 2017 John Wiley & Sons Ltd.

Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms.

PubMed

Wolach, Ofir; Sellar, Rob S; Martinod, Kimberly; Cherpokova, Deya; McConkey, Marie; Chappell, Ryan J; Silver, Alexander J; Adams, Dylan; Castellano, Cecilia A; Schneider, Rebekka K; Padera, Robert F; DeAngelo, Daniel J; Wadleigh, Martha; Steensma, David P; Galinsky, Ilene; Stone, Richard M; Genovese, Giulio; McCarroll, Steven A; Iliadou, Bozenna; Hultman, Christina; Neuberg, Donna; Mullally, Ann; Wagner, Denisa D; Ebert, Benjamin L

2018-04-11

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2 V617F , the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2 V617F -expressing neutrophils and that PAD4 is required for Jak2 V617F -driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2 V617F -positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2 V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Aggressive B-cell lymphomas in patients with myelofibrosis receiving JAK1/2 inhibitor therapy.

PubMed

Porpaczy, Edit; Tripolt, Sabrina; Hoelbl-Kovacic, Andrea; Gisslinger, Bettina; Bago-Horvath, Zsuzsanna; Casanova-Hevia, Emilio; Clappier, Emmanuelle; Decker, Thomas; Fajmann, Sabine; Fux, Daniela A; Greiner, Georg; Gueltekin, Sinan; Heller, Gerwin; Herkner, Harald; Hoermann, Gregor; Kiladjian, Jean-Jacques; Kolbe, Thomas; Kornauth, Christoph; Krauth, Maria-Theresa; Kralovics, Robert; Muellauer, Leonhard; Mueller, Mathias; Prchal-Murphy, Michaela; Putz, Eva Maria; Raffoux, Emmanuel; Schiefer, Ana-Iris; Schmetterer, Klaus; Schneckenleithner, Christine; Simonitsch-Klupp, Ingrid; Skrabs, Cathrin; Sperr, Wolfgang R; Staber, Philipp Bernhard; Strobl, Birgit; Valent, Peter; Jaeger, Ulrich; Gisslinger, Heinz; Sexl, Veronika

2018-06-14

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 MPN patients including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4/69 patients (5.8%) upon JAK1/2 inhibition compared to 2/557 (0.36%) with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 MPN patients. Considering primary myelofibrosis only (N=216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) versus 1/185 controls (0.54%). Lymphomas were of aggressive B-cell type, extra-nodal or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a pre-existing B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1 -/- mice: 16/24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B-cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a pre-existing B-cell clone may identify individuals at risk. Copyright © 2018 American Society of Hematology.

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

PubMed Central

Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina

2018-01-01

The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL) and calreticulin (CALR), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression. PMID:29641446

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

PubMed

Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina; Albano, Francesco

2018-04-11

The germline JAK2 haplotype known as "GGCC or 46/1 haplotype" (haplotype GGCC_46/1 ) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 ( INLS4 ) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a "GGCC" combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotype GGCC_46/1 and mutations in other genes, such as thrombopoietin receptor ( MPL ) and calreticulin ( CALR ), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotype GGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotype GGCC_46/1 and blood cell count, survival, or disease progression.

[Clinical Analysis of Driver Mutations in Patients with Ph Negative Myeloproliferative Neoplasms].

PubMed

He, Zhi-Peng; Tian, Hui-Yun; Tan, Ming; Wu, Yong

2018-06-01

To explore the relationship between driver mutations and clinical characteristics in patients with Philadelphia chromosome (Ph) negative myeloproliferative neoplasms (MPN), so as to provide evidence for diagno-sis and treatment of the disease. The clinical data of 410 patients with classic Ph negative MPN including 150 cases of polycythemia vera (PV), 188 cases of essential thrombocythemia (ET) and 72 cases of primary myelofibrosis (PMF) from January 2013 to December 2016 in Fujian Medical University Union Hospital were retrospectively analyzed. The PCR or DNA sequencing were used for JAK2 V617F, JAK2 exon12, CALR and MPL W515L/K mutation analyses, and follow-up information on patients was updated by direct phone call or follow-up in outpatient. Among the 410 patients with Ph negative MPN, 136 (33.2%) cases were asymptomatic at diagnosis. 389 cases were sequenced and JAK2 V617F was detected in 87.1% (122/140) of PV, 64.1% (118/184) of ET, 64.6% (42/65) of PMF; JAK2 exon 12 mutation in 1 case of PV; MPL W515L/K mutation in 1 case of ET and PMF, respectively; CALR mutation in 18(9.8%) cases of ET and 5 (7.7%) cases of PMF. JAK2 V617F mutated PV patients ocourred in older age: the white blood cell count, platelet count and incidence of splenomegaly were higher than JAK2-negative PV cases(P<0.05). Compared with JAK2 V617F mutated ET patients, CALR mutated ET cases displayed younger age, lower leukocyte count, higher platelet count and lower incidence of thrombosis; JAK2-negative ET cases had younger age, lower leukocyte count, lower hemoglobin level, higher platelet count and lower incidence of thrombosis(P<0.05). The incidence of splenomegaly in JAK2 V617F or CALR mutated PMF patients was both higher than that in JAK2-negative PMF cases, but the incidence of leukemia transformation in JAK2-negative PMF patients was higher than that in JAK2 V617F mutated cases (P<0.05). The types of driver mutations are closely related with the clinical features and prognosis in Ph

AKT is a therapeutic target in myeloproliferative neoplasms

PubMed Central

Khan, Irum; Huang, Zan; Wen, Qiang; Stankiewicz, Monika J.; Gilles, Laure; Goldenson, Benjamin; Schultz, Rachael; Diebold, Lauren; Gurbuxani, Sandeep; Finke, Christy M.; Lasho, Terra L.; Koppikar, Priya; Pardanani, Animesh; Stein, Brady; Altman, Jessica K.; Levine, Ross L.; Tefferi, Ayalew; Crispino, John D.

2014-01-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K, and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with Ruxolitinib in suppressing the growth of JAK2V617F mutant SET2 cells. Importantly MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis (PMF) and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs. PMID:23748344

AKT is a therapeutic target in myeloproliferative neoplasms.

PubMed

Khan, I; Huang, Z; Wen, Q; Stankiewicz, M J; Gilles, L; Goldenson, B; Schultz, R; Diebold, L; Gurbuxani, S; Finke, C M; Lasho, T L; Koppikar, P; Pardanani, A; Stein, B; Altman, J K; Levine, R L; Tefferi, A; Crispino, J D

2013-09-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

MPL W515L/K Mutations in Chronic Myeloproliferative Neoplasms.

PubMed

Akpınar, Timur Selçuk; Hançer, Veysel Sabri; Nalçacı, Meliha; Diz-Küçükkaya, Reyhan

2013-03-01

The MPL gene encodes the thrombopoietin receptor. Recently MPL mutations (MPL W515L or MPL W515K) were described in patients with essential thrombocythemia (ET) and primary (idiopathic) myelofibrosis (PMF). The prevalence and the clinical importance of these mutations are not clear. In the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. A total of 77 patients (66 were diagnosed with ET and 11 with PMF) and 42 healthy controls were included in the study. Using peripheral blood samples, the presence of MPL W515L/K mutations and JAK-2 V617F mutation were analyzed by real-time polymerase chain reaction. In our study, MPL W515L/K or JAK-2 V617F mutations were not observed in healthy controls. JAK-2 V617F mutation was present in 35 patients, of whom 29 had ET (43.9%, 29/66) and 6 had PMF (54.5%, 6/11). In the patient group, MPL W515L/K mutations were found in only 2 PMF cases, and these cases were negative for JAK-2 V617F mutation. The prevalence of MPL W515L/K mutations in the patient group was 2.6%, and the prevalence of MPL W515L/K mutations among the cases negative for the JAK-2 V617F mutation was found to be 4.8%. The 2 cases with MPL W515L/K mutations had long follow-up times (124 months and 71 months, respectively), had no thrombotic or hemorrhagic complications, and had no additional cytogenetic anomalies. MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation. None declared.

HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans

PubMed Central

Marubayashi, Sachie; Koppikar, Priya; Taldone, Tony; Abdel-Wahab, Omar; West, Nathan; Bhagwat, Neha; Caldas-Lopes, Eloisi; Ross, Kenneth N.; Gönen, Mithat; Gozman, Alex; Ahn, James H.; Rodina, Anna; Ouerfelli, Ouathek; Yang, Guangbin; Hedvat, Cyrus; Bradner, James E.; Chiosis, Gabriela; Levine, Ross L.

2010-01-01

JAK2 kinase inhibitors were developed for the treatment of myeloproliferative neoplasms (MPNs), following the discovery of activating JAK2 mutations in the majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown limited efficacy and apparent toxicities in clinical trials. We report here that an HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2 protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of JAK-STAT signaling, normalization of peripheral blood counts, and improved survival in MPN models at doses that did not degrade JAK2 in normal tissues or cause substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele burden in mice. These data establish what we believe to be a novel therapeutic rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN. PMID:20852385

HDAC8 overexpression in mesenchymal stromal cells from JAK2+ myeloproliferative neoplasms: a new therapeutic target?

PubMed Central

Ramos, Teresa L.; Sánchez-Abarca, Luis Ignacio; Redondo, Alba; Hernández-Hernández, Ángel; Almeida, Antonio M.; Puig, Noemí; Rodríguez, Concepción; Ortega, Rebeca; Preciado, Silvia; Rico, Ana; Muntión, Sandra; González Porras, José Ramón; Cañizo, Consuelo Del; Sánchez-Guijo, Fermín

2017-01-01

Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment. PMID:28390197

Erythrocytes from patients with myeloproliferative neoplasms and splanchnic venous thrombosis show greater expression of Lu/BCAM.

PubMed

Novitzky-Basso, I; Spring, F; Anstee, D; Tripathi, D; Chen, F

2018-05-13

Lutheran/BCAM protein (Lu) on the surface of erythrocytes is key for their adhesion to the endothelium, and erythrocytes from individuals with JAK2V617F-mutated myeloproliferative neoplasms (MPN) have increased endothelial adhesion. Splanchnic vein thrombosis (SVT) is a devastating thrombotic complication of MPN, and frequently, the only diagnostic feature is the JAK2V617F mutation. We sought to examine whether erythrocytes from patients with JAK2V617F mutated SVT (MPN-SVT) exhibited increased Lu expression, thereby supporting a mechanistic contribution to the development of thrombosis. We report the validation of a novel flow cytometry assay for Lu expression on erythrocytes. We examined the expression of Lu on erythrocytes from a cohort of MPN patients with and without SVT, and healthy controls. Samples were obtained from 20 normal individuals, 22 with MPN (both JAK2V617F-mutated and wild-type) and 8 with JAK2V617F-mutated MPN-SVT. Lu expression by erythrocytes from patients with MPN and MPN-SVT is significantly increased compared to erythrocytes from healthy individuals (P < .05), but there was no significant difference between patients with MPN-SVT and MPN. Patients with MPN have increased expression of the red cell Lu/BCAM adhesion molecule. Further work is required to determine the role of the increased Lu/BCAM adhesion to the endothelium in the development of thrombosis in MPN of all genotypes. © 2018 John Wiley & Sons Ltd.

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

PubMed Central

Oh, Stephen T.; Simonds, Erin F.; Jones, Carol; Hale, Matthew B.; Goltsev, Yury; Gibbs, Kenneth D.; Merker, Jason D.; Zehnder, James L.; Nolan, Garry P.

2010-01-01

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis. PMID:20404132

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations.

PubMed

Szpurka, Hadrian; Jankowska, Anna M; Makishima, Hideki; Bodo, Juraj; Bejanyan, Nelli; Hsi, Eric D; Sekeres, Mikkael A; Maciejewski, Jaroslaw P

2010-08-01

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases.

PubMed

Roskoski, Robert

2016-09-01

The Janus kinase (JAK) family of non-receptor protein-tyrosine kinases consists of JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase-2). Each of these proteins contains a JAK homology pseudokinase (JH2) domain that regulates the adjacent protein kinase domain (JH1). JAK1/2 and TYK2 are ubiquitously expressed whereas JAK3 is found predominantly in hematopoietic cells. The Janus kinase family is regulated by numerous cytokines including interleukins, interferons, and hormones such as erythropoietin, thrombopoietin, and growth hormone. Ligand binding to cytokine and hormone receptors leads to the activation of associated Janus kinases, which then mediate the phosphorylation of the receptors. The SH2 domain of STATs (signal transducers and activators of transcription) binds to the receptor phosphotyrosines thereby promoting STAT phosphorylation by the Janus kinases and consequent activation. STAT dimers are translocated to the nucleus where they participate in the regulation of the expression of thousands of proteins. JAK-STAT dysregulation results in autoimmune disorders such as rheumatoid arthritis, ulcerative colitis, and Crohn disease. JAK-STAT dysregulation also plays a role in the pathogenesis of myelofibrosis, polycythemia vera, and other myeloproliferative illnesses. An activating JAK2 V617F mutation occurs in 95% of people with polycythemia vera and in a lower percentage of people with other neoplasms. JAK1/3 signaling participates in the pathogenesis of inflammatory afflictions while JAK1/2 signaling participates in the development of several malignancies including leukemias and lymphomas as well as myeloproliferative neoplasms. Tofacitinib is a pan-JAK inhibitor that is approved by the FDA for the treatment of rheumatoid arthritis and ruxolitinib is a JAK1/2 inhibitor that is approved for the treatment of polycythemia vera and myelofibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Is it time to revisit contraindications to organ donation from donors with a JAK-2 mutation? Safe use of a liver allograft from a donor with essential thrombocythaemia.

PubMed

Haldar, Debashis; Chen, Frederick; Byron, Jane; Elsharkawy, Ahmed Mohamed; Perera, M Thamara Pr

2015-07-01

Transplantation can cure end-stage liver disease and hepatocellular carcinoma. However, the balance of organ demand and provision is heavily tipped to the detriment of patients. Patients awaiting transplantation rely on the greater use of marginal donors that may carry a risk to the recipient. UK authorities have decreed donor haematological malignancy an absolute contraindication. The authors describe the first report of a patient being safely transplanted with a liver from a donor who suffered from JAK2 V617F mutation-driven essential thrombocythaemia to a patient with a critical burden of hepatocellular carcinoma. A year after transplantation, the patient has neither evidence of acquisition of the donor's pathology, nor evidence of carcinoma recurrence. The case highlights the responsibility of the recipient team to maximize the use of organs by expert risk assessment. Dissemination of experience should inform future decisions, benefit patients and bolster utility in an era of growing waiting-list mortality. © 2015 Steunstichting ESOT.

Different expression patterns of LGALS1 and LGALS3 in polycythemia vera, essential thrombocythemia and primary myelofibrosis.

PubMed

Moura, L G; Tognon, R; Nunes, N S; Rodrigues, L Cataldi; Ferreira, A F; Kashima, S; Covas, D T; Santana, M; Souto, E X; Perobelli, L; Simões, B P; Dias-Baruffi, M; Castro, F A

2016-10-01

Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/