JAK2V617F 点突变与骨髓增殖性疾病的临床相关性研究

目的:研究JAK2V617F点突变与骨髓增殖性疾病(myeloproliferative disease,MPD)的临床相关性,为MPD的基因学诊断及靶向治疗提供理论依据。方法:应用等位基因特异性聚合酶链反应(AS-PCR)检测JAK2V617F点突变。结果:102例的MPD患者中包括慢性粒细胞白血病(CML)患者9例、真性红细胞增多症(PV)患者21例、原发性血小板增多症(ET)患者37例、特发性骨髓纤维化(IMF)患者16例和分类不明的骨髓增殖性疾病(uMPD)患者19例,JAK2V617F突变阳性率依次为11%、71.4%、51.4%、75.0%、78.9%。结论:JAK2V617F点突变有助于不同类型MPD的诊断,在MPD疾病的诊断中起重要作用。     

Evaluation of the JAK2-V617F gene mutation in Turkish patients with essential thrombocythemia and polycythemia vera

An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80-90 % for patients with polycythemia vera (PV) and 40-70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Turkish patients with MPD and compared these findings with published studies from other geographic regions. A total of 148 patients were studied; of which, 70 were diagnosed with PV and 78 with ET. The mutation status of JAK2 was determined using a tetra-primer polymerase chain reaction. We found that 80 % of the PV group and 42 % of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed, the levels of white blood cells, hemoglobin and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). To our knowledge, this study is the first to evaluate the relationship between MPD and JAK2-V617F in Turkish patients. The JAK2-V617F mutation is frequently detected in the Turkish patients with MPD, and especially in patients with PV. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD.     

A tetra-primer polymerase chain reaction approach for the detection of JAK2 V617F mutation

Recently, an acquired somatic point mutation (p.V617F) in a highly conserved residue of the pseudokinase domain of the JAK2 tyrosine kinase was shown to be associated with myeloproliferative disorders. Because of the clinical importance of this mutation in diagnosing myeloproliferative disorders and its relevance for disease progression, we have developed a tetra-primer polymerase chain reaction (PCR) assay to detect JAK2 p.V617F. Titration studies showed that the assay could reliably detect one copy of the mutant allele in a mix of 50 wild-type alleles suggesting that the lower detection limit of this assay is estimated to be 2%. This study demonstrates that genotyping and quantifying of the JAK2 V617F mutation can be performed by tetra-primer PCR using both freshly isolated and formalin-fixed tissues. Our tetra-primer PCR assay is sensitive, low-cost, and easy to use method for the detection of JAK2 p.V617F, which could be used even in low-tech laboratories.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

Apoptotic index by Annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal hematologic malignancies characterized by pancytopenia, dysplastic hematopoiesis, and a propensity to leukemic transformation. Increased apoptosis has been noted in MDS as a possible explanation for ineffective hematopoiesis, with lower levels in progression to and in de novo acute leukemia. Apoptosis can be measured by binding of Annexin V to exposed membrane phosphatidylserine. We postulated that the apoptotic index would aid in the differential diagnosis of MDS versus other hematopoietic diseases. We examined 33 bone marrow aspirates suspected of hematopoietic malignancy for apoptotic index by Annexin V analysis using a Becton Dickinson FACStar+ flow cytometer. The apoptotic index was expressed as the percentage of Annexin V-positive cells divided by total mononuclear cells in the gate. By standard morphologic analysis, 16 cases were diagnosed as MDS (9 refractory anemia [RA], 2 refractory anemia with ringed sideroblasts [RARS], 1 refractory anemia with excess of blasts [RAEB], 3 chronic myelomonocytic leukemia [CMML], and 1 unclassified), 11 as acute leukemia (AL), 6 as myeloproliferative disorders (MPD). Eight cases (uninvolved marrow of five patients with lymphoproliferative disorders [LPD], one patient with multiple myeloma, and two patients with anemia of chronic disease) served as nonneoplastic controls. A higher degree of apoptosis was observed in MDS (mean = 44.7%; range = 29.5--60%) compared with MPD (mean = 8.2%; range = 2.3--15.4%), AL (mean = 16.1%; range = 5.1--29.4%), and control marrow samples (mean = 11.6%; range = 1.5--21%). Additionally, the apoptotic index was significantly higher in MDS compared with MPD (P < 0.0001). In conclusion, a high apoptotic index occurs in MDS, supporting previous reports and suggesting that Annexin V analysis can be used as an adjunct in the diagnosis of MDS versus MPD. This would be particularly useful for the often-difficult distinction between early MDS and early MPD cases with equivocal morphology.     

Evaluation of clinical and laboratory findings with JAK2 V617F mutation as an independent variable in essential thrombocytosis

Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia and thrombocytes are increased with abnormal functions. Discovery of the protein tyrosine kinase JAK2 V617F allele contributed to better understanding of the pathogenetic mechanisms of MPNs. Acquired single point mutation in the JAK2 V617F was determined approximately 50–60 % of patients with ET. In this study we aimed to investigate the relationship between JAK2 V617F gene mutation, hematologic, biochemical markers and the complications in the ET patients. A total of 268 patients diagnosed with ET and 219 of those studied for JAK2 gene mutation were followed at the hematology clinics of three major hospitals between 2008 and 2013 were screened retrospectively. Laboratory, clinical and hematologic parameters were compared for JAK2 V617F positive and JAK2 V617F negative patients with ET. 102 (46 %) patients were positive with the JAK2 V617F mutation. The complications were observed in 61 (28 %) patients and 38 (62 %) of them had JAK2 V617F mutation. The levels of white blood cells, neutrophil, basophil, red blood cells, hemoglobin, hematocrit, mean platelet volume, thrombocytes, eosinophil; urea, creatinine were significantly different in patients with the JAK2 V617F mutation (P < 0.05). Presence of the JAK2 V617F mutation supports the diagnosis of ET. It would be useful to investigate the JAK2 V617F mutation and the hematologic and biochemical markers at diagnosis with respect to consider the risk of developing complications and to take the precautions against these complications.     

Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of affymetrix single-nucleotide-polymorphism genotyping microarrays

Loss of heterozygosity (LOH), either with or without accompanying copy-number loss, is a cardinal feature of cancer genomes that is tightly linked to cancer development. However, detection of LOH is frequently hampered by the presence of normal cell components within tumor specimens and the limitation in availability of constitutive DNA. Here, we describe a simple but highly sensitive method for genomewide detection of allelic composition, based on the Affymetrix single-nucleotide-polymorphism genotyping microarray platform, without dependence on the availability of constitutive DNA. By sensing subtle distortions in allele-specific signals caused by allelic imbalance with the use of anonymous controls, sensitive detection of LOH is enabled with accurate determination of allele-specific copy numbers, even in the presence of up to 70%-80% normal cell contamination. The performance of the new algorithm, called "AsCNAR" (allele-specific copy-number analysis using anonymous references), was demonstrated by detecting the copy-number neutral LOH, or uniparental disomy (UPD), in a large number of acute leukemia samples. We next applied this technique to detection of UPD involving the 9p arm in myeloproliferative disorders (MPDs), which is tightly associated with a homozygous JAK2 mutation. It revealed an unexpectedly high frequency of 9p UPD that otherwise would have been undetected and also disclosed the existence of multiple subpopulations having distinct 9p UPD within the same MPD specimen. In conclusion, AsCNAR should substantially improve our ability to dissect the complexity of cancer genomes and should contribute to our understanding of the genetic basis of human cancers.     

Fullerene derivative prevents cellular transformation induced by JAK2 V617F mutant through inhibiting c-Jun N-terminal kinase pathway

The constitutively activated mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs). The development of a novel chemical compound to suppress JAK2 V617F mutant-induced onset of MPNs and clarification of the signaling cascade downstream of JAK2 V617F mutant will provide clues to treat MPNs. Here we found that a water-soluble pyrrolidinium fullerene derivative, C(60)-bis (N, N-dimethylpyrrolidinium iodide), markedly induced apoptosis of JAK2 V617F mutant-induced transformed cells through a novel mechanism, inhibiting c-Jun N-terminal kinase (JNK) activation pathway but not generation of reactive oxygen species (ROS). Pyrrolidinium fullerene derivative significantly reduced the protein expression level of apoptosis signal-regulating kinase 1 (ASK1), one of the mitogen-activated protein kinase kinase kinases (MAPKKK), resulting in the inhibition of upstream molecules of JNK, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7). Strikingly, the knockdown of ASK1 enhanced the sensitivity to pyrrolidinium fullerene derivative-induced apoptosis, and the treatment with a JNK inhibitor, SP600125, also induced apoptosis of the transformed cells by JAK2 V617F mutant. Furthermore, administration of both SP600125 and pyrrolidinium fullerene derivative markedly inhibited JAK2 V617F mutant-induced tumorigenesis in nude mice. Taking these findings together, JAK2 V617F mutant-induced JNK signaling pathway is an attractive target for MPN therapy, and pyrrolidinium fullerene derivative is now considered a candidate potent drug for MPNs.     

Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.     

Activated Jak2 with the V617F point mutation promotes G1/S phase transition

Hematopoietic stem cells in myeloproliferative diseases mostly retain the potential to differentiate but are characterized by hyper-responsiveness to growth factors, as well as partial factor-independent growth. The V617F activating point mutation in Jak2 has recently been associated with myeloproliferative disorders. Using various cell line models, mechanisms that contribute to Jak2V617-mediated signaling were investigated. Treatment of the Jak2V617F mutant-expressing erythroid leukemia cell line HEL with a small molecule Jak2 inhibitor was associated with a dose-dependent G(1) cell cycle arrest. This inhibition correlated with decreased expression of cyclin D2 and increased expression of the cell cycle inhibitor p27(Kip). Inhibition of Jak2V617F with a Jak2-targeted small interfering RNA approach resulted in a similar phenotype. Mechanisms leading to altered p27(Kip) and cyclin D2 likely involve inhibition of STAT5, a major target of Jak2 in hematopoietic cells, because a constitutively active form of STAT5 reduced p27(Kip) and increased cyclin D2 expression. Jak2V617F and constitutively active STAT5 also induced high levels of reactive oxygen species, which are sufficient to promote G(1)/S phase transition. In contrast, treatment of HEL cells with the antioxidant N-acetylcysteine decreased cell growth or expression of cyclin D2 and increased expression of p27(Kip). Similar results were obtained in BaF3 cells transfected with Jak2V617F, but these cells required coexpression of the erythropoietin receptor for optimal signaling. These results suggest that regulation of cyclin D2 and p27(Kip) in combination with redox-dependent processes promotes G(1)/S phase transition downstream of Jak2V617F/STAT5 and therefore hint at potential novel targets for drug development that may aid traditional therapy.     

Identification of oncostatin M as a JAK2 V617F-dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.     

Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.     

Differential biological activity of disease-associated JAK2 mutants

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 Janus kinase 2 (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.     

The Polymorphisms in LNK Gene Correlated to the Clinical Type of Myeloproliferative Neoplasms

ObjectiveLNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN).MethodsA total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing.ResultsMissense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01).ConclusionMutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.     

Recurrent DNMT3A R882 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.     

Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2

Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.     

Molecular mechanisms that produce secondary MDS/AML by RUNX1/AML1 point mutations

RUNX1/AML1 point mutations have been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. A heterozygous germline mutation of the RUNX1 gene causes a familial platelet disorder with a predisposition to AML. RUNX1 mutations have also been detected with high frequency in minimally differentiated AML M0 subtypes and myelodysplastic/myeloproliferative neoplasms. Here we propose a new disease category of myelodysplastic neoplasms (MDN) consisting of MDS refractory anemia with excess blasts and AML with myelodysplasia-related changes, including therapy-related cases. RUNX1 mutations have been detected in about 20% of patients with "MDN". Among the MDN cases, histories of radiation exposure, therapy-related myeloid neoplasms after successful treatment for acute promyelocytic leukemia, and leukemic transformation of myeloproliferative neoplasms have been reported to have a strong association with RUNX1 mutations. The mutations occur in a normal, a receptive, or a disease-committed hematopoietic stem cell. It is suspected that the "MDN" phenotypes are defined by the RUNX1 mutations in addition to some other abnormalities.     

Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.