JAK2信号通路与肿瘤血管新生

JAK2是 JAK 家族的成员之一,JAK2与 STAT 家族的多个成员共同构成多条信号转导通路,如JAK2／STAT3、JAK2／STAT5等。JAK2／STATs 信号通路通过配体和细胞表面的受体结合而诱导受体二聚化,并相互磷酸化,从而激活 JAK。激活了的 JAK2／STAT 信号通路参与了肿瘤的发生、发展、血管新生、侵袭和转移等多个环节。研究表明肿瘤细胞中活化的 JAK2／STATs 信号通路主要是通过上调多种血管生成相关因子如血管内皮生长因子（VEGF）、环氧化酶－2（COX －2）等表达来促进肿瘤血管生成,IFN －α、SHP －1、SOCS通过 JAK2／STATs 通路下调肿瘤细胞促血管生成因子表达,抑制肿瘤血管生成。本文就 JAK2信号通路与肿瘤血管新生作一综述。     

Myeloproliferative neoplasms: from JAK2 mutations discovery to JAK2 inhibitor therapies

Most BCR-ABL1-negative myeloproliferative neoplasms (MPN) carry an activating JAK2 mutation. Approximately 96% of patients with polycythemia vera (PV) harbors the V617F mutation in JAK2 exon 14, whereas the minority of JAK2 (V617F)-negative subjects shows several mutations in exon 12. Other mutation events as MPL, TET2, LNK, EZH2 have been described in chronic phase, while NF1, IDH1, IDH2, ASX1, CBL and Ikaros in blast phase of MPN. The specific pathogenic implication of these mutations is under investigation, but they may have a role in refinement of diagnostic criteria and in development of new prognostic models. Several trials with targeted therapy (JAK inhibitors) are ongoing mostly involving patients with PMF, post-PV MF and post-essential thrombocythemia (ET) MF. Treatment with ruxolitinib and TG101348 has shown clinically significant benefits, particularly in improvement of splenomegaly and constitutional symptoms in MF patients. On the other hand, JAK inhibitors have not thus far shown disease-modifying activity therefore any other deduction on these new drugs seems premature.     

JAK2抑制剂在肿瘤治疗中的应用价值

JAK家族是JAK-STAT信号传导通路中的非受体型酪氨酸蛋白激酶,JAK2-STAT3作为JAK-STAT通路中的一个重要信号轴,它在肿瘤中的持续性激活可以通过影响细胞的生长、凋亡、周期等起到促进肿瘤发生发展的作用.JAK2突变,尤其是JAK2V617F突变的发现引发了JAK2抑制剂的研究热潮,为肿瘤的治疗提供了新的方向.JAK2抑制剂能削弱肿瘤细胞的恶性生物学行为,在有JAK2V617F突变的血液系统肿瘤以及JAK2-STAT3信号异常的实体肿瘤中都具有一定的治疗价值.     

Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera.

After accounting for misdiagnosis and treatment effect, allele-specific (AS)-PCR detects the JAK2V617F mutation in >95% of polycythemia vera (PV) patients. Using database inquiry, we identified 6 of a total 220 cases with PV that were JAK2V617F-negative (prevalence=3%). Of these, five cases ( approximately 80%) were found to harbor one of the two JAK2 exon 12 mutations (F537-K539delinsL or N542-E543del) in bone marrow (BM) and/or peripheral blood cells. Similar screening of six additional cases - three each with idiopathic erythrocytosis (IE) or otherwise unexplained erythrocytosis (UE) - did not reveal either JAK2V617F or JAK2 exon 12 mutations. We found JAK2 exon 12 mutations in PV cases to be readily detected by both DNA sequencing and AS-PCR, regardless of whether BM or peripheral blood cells were used as the source for DNA. Although erythroid hyperplasia was the predominant histologic feature on BM examination, megakaryocyte abnormalities and reticulin fibrosis were noted in most PV patients harboring exon 12 mutations. However, similar BM morphologic changes can also be seen in some JAK2V617F-positive PV cases; therefore, distinct genotype-phenotype association cannot be established.     

JAK2, a third member of the JAK family of protein tyrosine kinases.

We have isolated cDNA clones encoding a third, widely expressed, member of the JAK family of protein tyrosine kinases (PTKs). The anticipated amino acid sequence of JAK2 predicts the presence of two kinase-related domains, a feature characteristic of this family of PTKs. The structural similarity of JAK2 to the other members of this family extends towards their N-termini, beyond the two kinase-related domains, and reveals five further domains of substantial amino acid similarity. The C-terminal portion of one of these domains, the JH4 domain, bears an intriguing, albeit tenuous, similarity to the core element of the SH2 domain, whereas the remaining JAK homology domains do not appear to be a feature of other known proteins.     

Influence of JAK2 46/1 haplotype in the natural evolution of JAK2V617F allele burden in patients with myeloproliferative neoplasms

JAK2V617F allele burden was prospectively measured in untreated patients with polycythaemia vera (PV, n=26) or essential thrombocythaemia (ET, n=36) and compared according to JAK2 46/1 haplotype status. The mean increase in JAK2V617F allele burden per year was 1%, 0.8% and 6% for PV patients with the JAK2 46/1 haplotype in negative, heterozygous and homozygous status, respectively (p<0.001). The JAK2 46/1 haplotype had no influence in JAK2V617 allele burden in ET. In conclusion, untreated PV patients homozygous for the JAK2 46/1 haplotype show a progressive increase in the JAK2V617F allele burden during the evolution of the disease.     

JAK2 mutations and clinical practice in myeloproliferative neoplasms

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.     

Preclinical studies of Flonoltinib Maleate,a novel JAK2/FLT3 inhibitor,in treatment of JAK2V617F-induced myeloproliferative neoplasms

Janus kinase 2 (JAK2) hyperactivation by JAK2^V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2^V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2^V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.Subject terms: Myeloproliferative disease, Targeted therapies     

真性红细胞增多症患者JAK2基因突变的临床意义

真性红细胞增多症（polycythemia vera,PV）为一种以红细胞增多为特征的骨髓增殖性肿瘤（myeloproliferative neoplasm,MPN）。PV的发病机制尚未阐明,可能和JAK2基因的功能获得性体细胞突变相关,JAK2基因突变为PV提供了分子学诊断标准。JAK2基因突变和等位基因突变负荷可推测PV患者的临床特征和发展趋势。JAK2基因突变的发现促进了分子靶向治疗的发展,JAK2抑制剂ruxolitinib已应用于临床,治疗效果和安全性均良好。JAK2等位基因突变负荷与白细胞增多、转化为骨髓纤维化（myelofibrosis,MF）密切相关,高JAK2等位基因突变负荷可能为PV患者预后不良的危险因素。本文对PV患者JAK2基因突变的临床意义进行综述。      

Hypothesis: How do JAK2-inhibitors work in myelofibrosis

JAK2-inhibitors rapidly reduce spleen enlargement and clinical symptoms in persons with myelofibrosis but have little, if any, effect on the WBC, anemia, decreased platelets or bone marrow fibrosis. Also, JAK2-inhibitors are active in persons with and without the JAK2-mutation. Based on these and other data we suggest that the predominant effect of JAK2-inhibitors in persons with myelofibrosis is on normal rather than the abnormal clones.     

Polycythaemia Vera among Sudanese Patients with Special Emphasis on JAK2 Mutations

Background: In recent years, a somatic point mutation in the Janus Kinase 2 (JAK2) gene (1849 G→T, V617F)has been reported to occur in over 90% of patients with polycythemia vera (PV). Another JAK2 mutation in exon 12had been described and shown capable of activating erythropoietin signaling pathways. Objective: In this study, weaimed to determine the frequency of Jak2 mutations (JAK2V617F and JAK2 exon 12) as well as their relationshipswith hematological parameters in Sudanese patients with myeloproliferative disorders (MPD). A comparison withfindings of published studies from other geographic regions was included. Materials and Methods: From each ofa total of 83 polycythaemia patients, six milliliters (ml) of venous blood were collected and processed for molecularanalysis and measurement of serum erythropoietin level by enzyme-linked immunoassay (ELISA). The JAK2 V617Fmutation was determined using an allele-specific competitive blocker (ACB) -PCR assay and High Resolution Melting(HRM) analysis was applied for the JAK2 exon 12 mutation. Results: According to patients’ history and the resultsfor EPO levels, nine (10.7 %) out of 83 patients were found to have secondary polycythaemia and 74 (89.3%) PV. Theoverall frequency of the 2 JAK2 mutations was 94.6% in our Sudanese PV patients, JAK2V617F being found in 91%and JAK2 exon 12 mutations in 8.1%.Conclusion: In summary JAK2 V617F and JAK2 exon 12 mutations are verycommon in Sudanese PC cases.     

地西他滨对MPN患者JAK2基因表达及突变的影响及临床意义

目的：探讨地西他滨对骨髓增殖性肿瘤（myeloproliferative neoplasm,MPN）患者非受体型酪氨酸激酶JAK2（Janus kinase 2,JAK2）基因表达及突变的影响及临床意义。方法选取17例MPN初治患者作为研究对象,健康采血者15例作为对照组。治疗前、后采用PCR方法检测JAK2-V617F基因突变及JAK2 mRNA表达。结果JAK2-V617F基因突变检测结果显示,10例MPN 患者存在JAK2-V617F 基因突变,阳性率为58．8％（10／17）。其中,真性红细胞增多症（polycythemia vera,PV）患者（PV组）的JAK2-V617F基因突变阳性率为80．0％（8／10）,原发性血小板增多症（essential thrombocythemia,ET）患者（ET组）为28．6％（2／7）。治疗4个周期后,PV组总缓解率为60．0％（6／10）;ET组为57．1％（4／7）。与对照组比较,PV组和ET组治疗前外周血JAK2 mRNA表达水平均升高（均P＜0．05）。PV组和ET组在治疗4个周期后JAK2 mRNA表达均低于治疗前（均P＜0．05）,并且PV组降低程度高于ET组（P＜0．05）。治疗4个周期后,JAK2-V617F基因突变在PV组中的转阴率为37．5％（3／8）,在ET组中转阴1例。结论地西他滨治疗MPN能取得很好的疗效,并能促进JAK2-V617 F基因突变转阴和降低JAK2基因的表达。     

Selective inhibition of JAK2-driven erythroid differentiation of polycythemia vera progenitors

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD) that is commonly characterized by mutant JAK2 (JAK2V617F) signaling, erythrocyte overproduction, and a propensity for thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression by human hematopoietic progenitors promoted erythroid colony formation and erythroid engraftment in a bioluminescent xenogeneic immunocompromised mouse transplantation model. A selective JAK2 inhibitor, TG101348 (300 nM), significantly inhibited JAK2V617F+ progenitor-derived colony formation as well as engraftment (120 mg/kg) in xenogeneic transplantation studies. TG101348 treatment decreased GATA-1 expression, which is associated with erythroid-skewing of JAK2V617F+ progenitor differentiation, and inhibited STAT5 as well as GATA S310 phosphorylation. Thus, TG101348 may be an effective inhibitor of JAK2V617F+ MPDs in clinical trials.     

Bone marrow stroma-secreted cytokines protect JAK2(V617F)-mutated cells from the effects of a JAK2 inhibitor

Signals emanating from the bone marrow microenvironment, such as stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative neoplasms (MPN). To examine this hypothesis, we established a coculture platform [cells cocultured directly (cell-on-cell) or indirectly (separated by micropore membrane)] designed to interrogate the interplay between Janus activated kinase 2-V617F (JAK2(V617F))-positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET-2) and mouse (FDCP-EpoR) JAK2(V617F)-positive cells as well as primary blood or bone marrow mononuclear cells from patients with polycythemia vera; however, these effects were attenuated when any of these cell types were cocultured (cell-on-cell) with human marrow stromal cell lines (e.g., HS5, NK.tert, TM-R1). Coculture with stromal cells hampered the ability of atiprimod to inhibit phosphorylation of JAK2 and the downstream STAT3 and STAT5 pathways. This protective effect was maintained in noncontact coculture assays (JAK2(V617F)-positive cells separated by 0.4-μm-thick micropore membranes from stromal cells), indicating a paracrine effect. Cytokine profiling of supernatants from noncontact coculture assays detected distinctly high levels of interleukin 6 (IL-6), fibroblast growth factor (FGF), and chemokine C-X-C-motif ligand 10 (CXCL-10)/IFN-γ-inducible 10-kD protein (IP-10). Anti-IL-6, -FGF, or -CXCL-10/IP-10 neutralizing antibodies ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2(V617F)-positive cells. Therefore, our results indicate that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, thus underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes.     

JAK2 and Beyond: Mutational Study of JAK2V617 in Myeloproliferative Disorders and Haematological Malignancies in Kashmiri population

Background: Janus Tyrosine Kinase-2 (JAK2 V617F), a novel point mutation affecting the MPD’S is a somatic gain-of-function mutation. It alters a highly conserved amino acid valine in the negative regulatory JH2 domain to phenylalanine predicted to dysregulate kinase activity. Aim: To evaluate the prevalence and clinical significance of JAK2 V617F mutation in various MPD’s as well as in hematological malignancies. Subjects and Methods: JAK2 mutation was assessed in 90 patients with myeloproliferative disorders and 47 leukemic patients. In addition, peripheral blood samples from 90 healthy donors were also collected as control. We used a highly sensitive Allele-Specific polymerase chain reaction (AS-PCR) for the detection and confirmed the mutation further by direct sequencing. Results: Our results showed significant differences between various disorders with respect to either the proportion of positivity or that of mutant alleles. JAK2-V617F was detected in 67/90 MPD patients and 02/17 for AML,01/11 for ALL-L1,02/12 for ALL-L2 and 02/07 for CML and 90 healthy controls. Conclusion: From the above findings it is evident that the JAK2 V617F mutation is widespread not only in MPD's but also in hematological malignancies, which might as well lead to the new classification of MPD'S. Our data also suggest that different genetic events may lead to JAK-STAT pathway activation in different malignancies.     

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

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.     

The V617F JAK2 mutation and the myeloproliferative disorders

The discovery this year of a single mutation in the Janus Kinase (JAK)-2 gene in a high percentage of cases of polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis suggests that it maybe the underlying molecular mechanism for these disorders. Different approaches from the inhibition of the tyrosine kinase JAK2, widespread search for mutations in tyrosine kinases, and investigation of the short arm of chromosome 9 where JAK2 is located all led to the discovery of the V617F JAK2 mutation. Substitution of a valine for a phenylalanine destabilizes the JH2 domain of JAK2 causes loss of the auto-inhibitory activity of this domain and explains some of the biological phenomena observed in patients with myeloproliferative disorders (MPD). The V617F JAK2 mutation can be detected by PCR-direct sequencing using DNA from the granulocyte lineage or with increased sensitivity by the amplification refractory mutation system using DNA from unfractionated blood. Pyrosequencing assays can be used to quantitate allele ratios to accurately define homozygote and heterozygote status. This single mutation is widespread having been detected in related MPD and other haematological malignancies. This leads to a number of further questions about the role of this single mutation in the clinical pattern of disease.