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1.  Development of a highly sensitive method for detection of JAK2V617F 
Ph- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.
In the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.
We tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.
We have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.
PMCID: PMC3207960  PMID: 21985400
Tyrosine kinase; myeloproliferative neoplasms; JAK2; mutation; detection; diagnosis
2.  Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease 
PLoS ONE  2012;7(7):e38362.
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.
PMCID: PMC3388082  PMID: 22768290
3.  Differential Biological Activity of Disease-Associated JAK2 Mutants 
FEBS letters  2011;585(7):1007-1013.
The JAK2V617F mutation has been identified in most patients with myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia and primary myelofibrosis. Although JAK2V617F is the predominant allele associated with MPNs, other activating JAK2 alleles (such as K539L, T875N) also have been identified in distinct MPNs. The basis for the differences in the in vivo effects of different JAK2 alleles remains unclear. We have characterized three different classes of disease-associated JAK2 mutants (JAK2V617F, JAK2K539L and JAK2T875N) and found significant differences in biochemical, signaling and transforming properties among these different classes of JAK2 mutants.
PMCID: PMC3070755  PMID: 21362419
4.  Relevance of JAK2V617F positivity to hematological diseases - survey of samples from a clinical genetics laboratory 
JAK2V617F is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs) and has become a valuable marker for diagnosis of MPNs. However, it has also been found in many other hematological diseases, and some studies even detected the presence of JAK2V617F in normal blood samples. This casts doubt on the primary role of JAK2V617F in the pathogenesis of MPNs and its diagnostic value.
In the present study, we analyzed JAK2V617F positivity with 232 normal blood samples and 2663 patient blood, bone marrow, and amniotic fluid specimens obtained from a clinical genetics laboratory by using a simple DNA extraction method and a sensitive nested allele-specific PCR strategy.
We found JAK2V617F present in the majority (78%) of MPN patients and in a small fraction (1.8-8.7%) of patients with other specific hematological diseases but not at all in normal healthy donors or patients with non-hematological diseases. We also revealed associations of JAK2V617F with novel as well as known chromosomal abnormalities.
Our study suggests that JAK2V617F positivity is associated with specific hematological malignancies and is an excellent diagnostic marker for MPNs. The data also indicate that the nested allele-specific PCR method provides clinically relevant information and should be conducted for all cases suspected of having MPNs as well as for other related diseases.
PMCID: PMC3032761  PMID: 21235771
5.  JAK2 V617F-Dependent Upregulation of PU.1 Expression in the Peripheral Blood of Myeloproliferative Neoplasm Patients 
PLoS ONE  2011;6(7):e22148.
Myeloproliferative neoplasms (MPN) are multiple disease entities characterized by clonal expansion of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell). JAK2 mutations, such as the common V617F substitution and the less common exon 12 mutations, are frequently detected in such tumor cells and have been incorporated into the diagnostic criteria published by the World Health Organization since 2008. However, the mechanism by which these mutations contribute to MPN development is poorly understood. We examined gene expression profiles of MPN patients focusing on genes in the JAK–STAT signaling pathway using low-density real-time PCR arrays. We identified the following 2 upregulated genes in MPN patients: a known target of the JAK–STAT axis, SOCS3, and a potentially novel target, SPI1, encoding PU.1. Induction of PU.1 expression by JAK2 V617F in JAK2-wildtype K562 cells and its downregulation by JAK2 siRNA transfection in JAK2 V617F-positive HEL cells supported this possibility. We also found that the ABL1 kinase inhibitor imatinib was very effective in suppressing PU.1 expression in BCR-ABL1-positive K562 cells but not in HEL cells. This suggests that PU.1 expression is regulated by both JAK2 and ABL1. The contribution of the two kinases in driving PU.1 expression was dominant for JAK2 and ABL1 in HEL and K562 cells, respectively. Therefore, PU.1 may be a common transcription factor upregulated in MPN. PU.1 is a transcription factor required for myeloid differentiation and is implicated in erythroid leukemia. Therefore, expression of PU.1 downstream of activated JAK2 may explain why JAK2 mutations are frequently observed in MPN patients.
PMCID: PMC3138766  PMID: 21789226
6.  Germline and somatic JAK2 mutations and susceptibility to chronic myeloproliferative neoplasms 
Genome Medicine  2009;1(5):55.
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.
PMCID: PMC2689447  PMID: 19490586
7.  The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population 
BMC Medical Genetics  2012;13:6.
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.
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.
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.
Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive and JAK2 V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing JAK2 SNPs and quantifying JAK2 V617F mutations will provide further insights into the molecular pathogenesis of MPN.
PMCID: PMC3277458  PMID: 22251709
JAK2 V617F; SNP; myeloproliferative neoplasms
8.  The implication of identifying JAK2V617F in myeloproliferative neoplasms and myelodysplastic syndromes with bone marrow fibrosis 
Journal of Hematopathology  2008;1(2):111-117.
The myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) occasionally demonstrate overlapping morphological features including hypercellularity, mild/nonspecific dysplastic changes and variable bone marrow fibrosis. Thus, when the associated bone marrow fibrosis results in a suboptimal specimen for morphological evaluation, the descriptive diagnosis “fibrotic marrow with features indeterminate for MDS versus MPN” is often applied. The JAK2V617F mutation was recently shown to be frequently identified in MPN, but it is rarely present in other myeloid disorders. However, the diagnostic utility of JAK2V617F screening in hypercellular bone marrow specimens with fibrosis has not been previously investigated. Using a real-time polymerase chain reaction melting-curve assay capable of detecting JAK2V617F in archived fixed materials, we retrospectively studied JAK2V617F in 45 cases with fibrotic hypercellular bone marrow at initial presentation, including 19 cases initially described as “with features indeterminate for MDS versus MPN”. These 19 cases were reclassified into more specific categories of MDS (n = 14) or MPN (n = 5) based on the availability of subsequent clinical data and/or bone marrow examinations. The JAK2V617F allele was identified in 17 out of 18 BCR/ABL gene-negative MPN cases with marrow fibrosis, whereas only wild-type alleles were identified in the remaining non-MPN cases. Importantly, JAK2V617F alleles were seen in all five cases of “with features indeterminate for MDS versus MPN” at initial presentation that were later determined to be MPN, but they were absent in the 14 cases later determined to be MDS. Our results suggest that JAK2V617F allele evaluation can be a useful ancillary test for discriminating MDS from MPN in specimens with bone marrow fibrosis.
PMCID: PMC2713481  PMID: 19669209
Myeloproliferative neoplasm; Myelodysplastic syndrome; Bone marrow fibrosis; JAK2V617F
9.  Mouse models of myeloproliferative neoplasms: JAK of all grades 
Disease Models & Mechanisms  2011;4(3):311-317.
In 2005, several groups identified a single gain-of-function point mutation in the JAK2 kinase that was present in the majority of patients with myeloproliferative neoplasms (MPNs). Since this discovery, much effort has been dedicated to understanding the molecular consequences of the JAK2V617F mutation in the haematopoietic system. Three waves of mouse models have been produced recently (bone marrow transplantation, transgenic and targeted knock-in), which have facilitated the understanding of the molecular pathogenesis of JAK2V617F-positive MPNs, providing potential platforms for designing and validating novel therapies in humans. This Commentary briefly summarises the first two types of mouse models and then focuses on the more recently generated knock-in models.
PMCID: PMC3097453  PMID: 21558064
10.  Myeloproliferative Neoplasm Animal Models 
Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow cells into irradiated syngeneic mice established the field of MPN animal modeling and the retroviral bone marrow transplantation (BMT) assay has been used extensively since. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN associated genetic abnormalities on the hematopoietic stem and progenitor cell (HSPC) compartment and xenograft models have allowed the study of primary human MPN-propagating cells in vivo. All models have facilitated the pre-clinical development of MPN therapies. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models. MPN animal models have also been used to investigate additional genetic lesions found in human MPN and to evaluate the bone marrow microenvironment in these diseases. Finally, several genetic lesions, although not common, somatically mutated drivers of MPN in humans induce a MPN phenotype in mice. Future uses for MPN animal models will include modeling compound genetic lesions in MPN and studying myelofibrotic transformation.
PMCID: PMC3459181  PMID: 23009938
Myeloproliferative neoplasms; preclinical murine models; BCR-ABL; JAK2V617F; hematopoietic stem cells; bone marrow microenvironment; myelofibrosis; oncogenes
11.  The Constitutive Activation of Jak2-V617F is Mediated by a π Stacking Mechanism Involving Phe 595 and Phe 617 
Biochemistry  2010;49(46):9972-9984.
Somatic mutations in the Jak2 allele that lead to constitutive kinase activation of the protein have been identified in human disease conditions such as the myeloproliferative neoplasms (MPNs). The most common mutation in these patients is a V617F substitution mutation, which is believed to play a causative role in the MPN pathogenesis. As such, identifying the molecular basis for the constitutive activation of Jak2-V617F is important for understanding its clinical implications and potential treatment. Here, we hypothesized that conversion of residue 617 from Val to Phe resulted in the formation of novel π stacking interactions with neighboring Phe residues. To test this, we first examined the Jak2 structure via molecular modeling and identified a potential π stacking interaction between F594, F595 and F617. Disruption of this interaction through site directed mutagenesis impaired Jak2 auto-phosphorylation, Jak2 dependent gene transcription and in vitro kinase activity of the Jak2-V617F protein. Further, substitution of F594 and F595 with Trp did not affect Jak2 function significantly, but replacement with charged residues did, showing the conservation of aromaticity and hydropathy index at these positions. Using molecular dynamics (MD) simulations, we found that the π stacking interaction between residues 595 and 617 in the Jak2-V617F protein was of much greater energy and conformed to the properties of π stacking, relative to the Jak2-WT or Jak2-V617F/F594A/F595A. In summary, we have identified a π stacking interaction between F595 and F617 that is specific to and is critical for the constitutive activation of Jak2-V617F.
PMCID: PMC2982877  PMID: 20958061
Janus kinase 2 (Jak2); Pi stacking; Constitutive Activation
12.  The allele burden of JAK2 V617F can aid in differential diagnosis of Philadelphia Chromosome-Negative Myeloproliferative Neoplasm 
Blood research  2013;48(2):128-132.
We aimed to evaluate the feasibility of using the allele burden of Janus kinase 2 (JAK2) V617F as a criterion for discriminating 3 subtypes of Philadelphia chromosome-negative myeloproliferative neoplasm (Ph-MPN): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).
We collected 70 peripheral blood (PB) and 81 bone marrow (BM) samples from patients diagnosed with Ph-MPN. Real-time quantitative PCR (RQ-PCR) and Amplification Refractory Mutation System (ARMS) assays were performed for each sample. We compared the allele burden of JAK2 V617F for each subtype of Ph-MPN and determined the concordance rates of the results between the 2 tests.
The JAK2 V617F allele burden differed significantly among the 3 disease categories in both PB (P=0.045) and BM (P=0.011) samples. Subsequent subgroup analysis revealed that the median allele burden of JAK2 V617F for ET (21.71% for PB and 24.95% for BM) was significantly lower than that for PV (56.88% for PB, P=0.047; 72.66% for BM, P=0.003) and PMF (56.16% for PB, P=0.050; 59.04% for BM, P=0.049). Concordance rate between the RQ-PCR and ARMS data was 90.7%. Of the 14 discrepant cases, 12 were RQ-PCR(+)/ARMS(-) and 2 were RQ-PCR(-)/ARMS(+).
The allele burden of JAK2 V617F was significantly lower for ET than that for PV or PMF in both PB and BM samples. The JAK2 V617F allele burden is a diagnostic tool for differentiating PV or PMF from ET.
PMCID: PMC3698398  PMID: 23826582
Allele; Discrimination; Janus Kinase 2; Mutation; Myeloproliferative disorders; Real-time polymerase chain reaction
13.  Heterozygous and Homozygous JAK2V617F States Modeled by Induced Pluripotent Stem Cells from Myeloproliferative Neoplasm Patients 
PLoS ONE  2013;8(9):e74257.
JAK2V617F is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2V617F, respectively. In the patient with homozygous JAK2V617F, additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA+CD41+ cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.
PMCID: PMC3774801  PMID: 24066127
14.  The involvement of Galectins in the modulation of the JAK/STAT pathway in myeloproliferative neoplasia 
In patients with myeloproliferative neoplasia (MPN) the development of fibrosis and increased vessel density correlate with poor prognosis. The JAK2V617F mutation constitutively activates JAK2, which phosphorylates signal transducer activator of transcription (STAT), up-regulating vascular endothelial growth factor (VEGF), which might be responsible for angiogenesis in MPN. Galectins are involved in the development of fibrosis and angiogenesis and might also be involved in activation of the JAK/STAT pathway in MPN.
106 MPN patients, 36 essential thrombocythemia (ET), 25 polycythemia vera (PV) and 45 primary myelofibrosis (PMF), were analyzed for the expression pattern of galectin-1, galectin-3, pSTAT3, pSTAT5 and MVD by immunostaining of bone marrow biopsy sections followed by automated image analysis. The JAK2 mutational status was analysed through real time PCR in blood samples.
The expression of galectin-1 was significantly higher in all MPN patients compared to normal controls. Galectin-3 was expressed more in PV patients. MVD was significantly higher in all MPN patients and correlated with galectin-1 and pSTAT5 expression. pSTAT5 expression showed a trend of higher expression in patients carrying the JAK2V617F mutation as well as in PV patients. PMF patients and all JAK2V617F positive patients showed a significantly higher pSTAT3 expression compared to control and ET patients.
The findings suggest the involvement of galectin-1 in MPN development, regardless of the subtype. Furthermore involvement of galectin-3 in PV development, pSTAT5 in that of PV and JAK2V617F positive patients and angiogenesis, as well as pSTAT3 is involved in the pathogenesis of PMF.
PMCID: PMC3384397  PMID: 22762031
MPN; myeloproliferative neoplasia; galectin; JAK; STAT; angiogenesis; MVD
15.  Allelic Expression Imbalance of JAK2 V617F Mutation in BCR-ABL Negative Myeloproliferative Neoplasms 
PLoS ONE  2013;8(1):e52518.
The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs.
PMCID: PMC3551963  PMID: 23349688
16.  A germline JAK2 SNP is associated with predisposition to the development of JAK2V617F-positive myeloproliferative neoplasms 
Nature genetics  2009;41(4):455-459.
Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis1–5. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2V617F) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis6–10, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci11. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2V617F-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2V617F is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition.
PMCID: PMC3676425  PMID: 19287384
17.  JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms 
PLoS ONE  2010;5(8):e12165.
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.
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 testing.
PMCID: PMC2921382  PMID: 20730051
18.  Advantages of the quenching probe method over other PCR-based methods for detection of the JAK2 V617F mutation 
Oncology Letters  2012;4(2):205-208.
The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.
PMCID: PMC3402723  PMID: 22844354
janus kinase 2; myeloproliferative neoplasms; allele-specific polymerase chain reaction; PCR-restriction fragment length polymorphism; high-resolution melting analysis; quenching probe
19.  Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells 
Cancer cell  2010;17(6):584-596.
We report a Jak2V617F knock-in 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 towards 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.
The JAK2V617F mutation is a promising candidate for molecularly targeted therapy in MPN. Early data from JAK2 inhibitor clinical trials have called into question the capacity of these compounds to alter the natural history of JAK2V617F mediated MPN. Determining the effect of JAK2 inhibitors on the disease-initiating population requires a model in which the JAK2V617F allele is expressed at physiological levels in hematopoietic stem and progenitor cells, as it is in humans. Our model demonstrates that JAK2V617F causes expansion of erythroid progenitors but that only the HSC compartment can initiate disease in a transplanted mouse. We further demonstrate that the HSC compartment, the definitive target for curative therapy of JAK2V617F mediated MPN, is resistant to treatment with a JAK2 inhibitor.
PMCID: PMC2909585  PMID: 20541703
20.  Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms 
Leukemia  2011;26(4):708-715.
The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified 5 different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib compared to native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the ‘gatekeeper’ mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.
PMCID: PMC3974504  PMID: 21926964
Myeloid neoplasia; JAK2; drug resistance
21.  Intrinsic resistance to JAK2 inhibition in myelofibrosis 
Recent results have demonstrated that myeloproliferative neoplasms (MPN) are strongly associated with constitutive activation of the JAK2 tyrosine kinase. However, JAK2 inhibitors currently approved or under development for treating myeloproliferative neoplasms do not selectively deplete the malignant clone, and the inhibition of activity of the drug target (JAK2) has not been rigorously evaluated in clinical studies. Therefore in this study we developed an in vitro assay to gain insight into how effectively JAK2 activity is inhibited in patient samples.
Experimental Design
We treated primary cells from normal donors and patients with MPN with JAK2 inhibitors and measured phosphorylation of downstream targets STAT5 and STAT3 by flow cytometry. Obtained results were next correlated with JAK2 V617F allele burden and plasma cytokines level.
We observed a dose-dependent decrease in pSTAT5 and pSTAT3 in ex vivo treated granulocytes. However, phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis was significantly less inhibited when compared to cells from patients with polycythemia vera, essential thrombocytosis, and normal donors. Sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. Mixing studies using plasma from patients with myelofibrosis did not transfer resistance to sensitive cells. Likewise, no single cytokine measured appeared to account for the observed pattern of resistance. Taken together these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5.
PMCID: PMC3618591  PMID: 23386690
JAK2 inhibition; myeloproliferative neoplasms; biomarkers; phospho-specific flow cytometry
22.  Effect of NS-018, a selective JAK2V617F inhibitor, in a murine model of myelofibrosis 
Blood Cancer Journal  2014;4(1):e174-.
A single somatic mutation, V617F, in Janus kinase 2 (JAK2) is one of the causes of myeloproliferative neoplasms (MPNs), including primary myelofibrosis, and the JAK2V617F mutant kinase is a therapeutic target in MPN. However, inhibition of wild-type (WT) JAK2 can decrease the erythrocyte or platelet (PLT) count. Our selective JAK2 inhibitor, NS-018, suppressed the growth of Ba/F3 cells harboring JAK2V617F more strongly than that of cells harboring WT JAK2. The 4.3-fold JAK2V617F selectivity of NS-018 is higher than the 1.0- to 2.9-fold selectivity of seven existing JAK2 inhibitors. NS-018 also inhibited erythroid colony formation in JAK2V617F transgenic mice at significantly lower concentrations than in WT mice. In keeping with the above results, in a JAK2V617F bone marrow transplantation mouse model with a myelofibrosis-like disease, NS-018 reduced leukocytosis and splenomegaly, improved bone marrow fibrosis and prolonged survival without decreasing the erythrocyte or PLT count in the peripheral blood. By exploring the X-ray co-crystal structure of NS-018 bound to JAK2, we identified unique hydrogen-bonding interactions between NS-018 and Gly993 as a plausible explanation for its JAK2V617F selectivity. These results suggest that NS-018 will have therapeutic benefit for MPN patients through both its efficacy and its reduced hematologic adverse effects.
PMCID: PMC3913942  PMID: 24413068
JAK2; V617F; myelofibrosis; kinase inhibitor; NS-018
23.  Comprehensive review of JAK inhibitors in myeloproliferative neoplasms 
Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem-cell disorders, characterized phenotypically by the abnormal accumulation of mature-appearing myeloid cells. Polycythemia vera, essential thrombocythemia, primary myelofibrosis (also known as ‘BCR-ABL1-negative’ MPNs), and chronic myeloid leukemia (CML) are the primary types of MPNs. After the discovery of the BCR-ABL1 fusion protein in CML, several oncogenic tyrosine kinases have been identified in ‘BCR-ABL1-negative’ MPNs, most importantly, JAK2V617F mutation. The similarity in the clinical characteristics of the BCR-ABL1-negative MPN patients along with the prevalence of the Janus kinase mutation in this patient population provided a strong rationale for the development of a new class of pharmacologic inhibitors that target this pathway. The first of its class, ruxolitinib, has now been approved by the food and drug administration (FDA) for the management of patients with intermediate- to high-risk myelofibrosis. Ruxolitinib provides significant and sustained improvements in spleen related and constitutional symptoms secondary to the disease. Although noncurative, ruxolitinib represents a milestone in the treatment of myelofibrosis patients. Other types of JAK2 inhibitors are being tested in various clinical trials at this point and may provide better efficacy data and safety profile than its predecessor. In this article, we comprehensively reviewed and summarized the available preclinical and clinical trials pertaining to JAK inhibitors.
PMCID: PMC3629759  PMID: 23610611
primary myelofibrosis; polycythemia vera; essential thrombocythemia; Janus kinase 2
24.  A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms 
BioMed Research International  2014;2014:458457.
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.
PMCID: PMC3960525
25.  Correlations between Janus Kinase 2 V617F Allele Burdens and Clinicohematologic Parameters in Myeloproliferative Neoplasms 
Annals of Laboratory Medicine  2012;32(6):385-391.
This study evaluated potential correlations between the allele burden of the Janus kinase 2 (JAK2) V617F mutation and clinicohematologic characteristics in patients with myeloproliferative neoplasms (MPN).
Clinical and hematologic features were reviewed for 103 MPN patients, including patients with polycythemia vera (PV, 22 patients), essential thrombocythemia (ET, 64 patients), and primary myelofibrosis (PMF, 17 patients). JAK2 V617F allele status and allele burdens were measured by allele-specific PCR and pyrosequencing, respectively.
The JAK2 V617F mutation was detected in 95.5%, 68.8%, and 52.9% of PV, ET, and PMF patients, respectively. JAK2 V617F-positive ET patients were significantly older and exhibited higher neutrophil fractions, a higher frequency of thrombotic events, and a higher myelofibrosis rate than JAK2 V617F-negative patients (P <0.05). PV patients carried the highest mean T allele burden (66.0%±24.9%) compared with ET (40.5%±25.2%) and PMF patients (31.5%±37.0%) (P =0.00). No significant correlations were detected between V617F allele burden and patient age, white blood cell count, Hb, Hct, or the platelet count for PV, ET, or PMF patients. ET patients with organomegaly had a higher JAK2 V617F allele burden (53.4%±23.7%) than patients without organomegaly (35.6%±24.3%) (P =0.03).
The JAK2 V617F mutational status and its allele burden correlate with the clinicohematologic phenotypes of ET patients, including older age, higher neutrophil count, and greater rates of organomegaly, thrombotic events, and myelofibrosis. For PV and PMF patients, larger-scale studies involving more MPN patients are needed.
PMCID: PMC3486931  PMID: 23130336
Janus kinase 2; Allele; Myeloproliferative neoplasm

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