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。     

Predicted tyk2 protein contains two tandem protein kinase domains

Tyk2 was recently described as an 1187 amino acid protein with a putative protein-tyrosine kinase domain near its C-terminus and no similarity to other proteins in the approximately 900 amino acids preceding the kinase domain. I report here, however, that tyk2 contains an additional protein kinase domain, and that the two tandem kinase domains are preceded by an SH2-like, putative regulatory domain.     

Specificity of alpha-cyano-beta-hydroxy-beta-methyl-n-[4-(trifluoromethoxy)phe nyl]-propenamide as an inhibitor of the epidermal growth factor receptor tyrosine kinase.

The epidermal growth factor receptor (EGFR) tyrosine kinase has an essential function for the survival of human breast cancer cells. In a systematic effort to design potent and specific inhibitors of this receptor family protein tyrosine kinase (PTK) as antibreast cancer agents, we recently reported the construction of a three-dimensional homology model of the EGFR kinase domain. In this model, the catalytic site is defined by two beta-sheets that form an interface at the cleft between the NH2-terminal and COOH-terminal lobes of the kinase domain. Our modeling studies revealed a distinct, remarkably planar triangular binding pocket within the kinase domain with approximate dimensions of 15 A x 12 A x 12 A, and the thickness of the binding pocket is approximately 7 A with an estimated volume of approximately 600 A3 available for inhibitor binding. Molecular docking studies had identified alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy)phenyl]-p ropenamide (LFM-A12) as our lead inhibitor, with an estimated binding constant of 13 microM, which subsequently inhibited EGFR kinase in vitro with an IC50 value of 1.7 microM. LFM-A12 was also discovered to be a highly specific inhibitor of the EGFR. Even at very high concentrations ranging from 175-350 microM, this inhibitor did not affect the enzymatic activity of other PTKs, including the Janus kinases JAK1 and JAK3, the Src family kinase HCK, the Tec family member Bruton's tyrosine kinase, SYK kinase, and the receptor family PTK insulin receptor kinase. This observation is in contrast to the activity of a quinazoline inhibitor tested as a control, 4-(3-bromo, 4-hydroxyanilino)-6,7-dimethoxyquinazoline, which was shown to inhibit EGFR and other tyrosine kinases such as HCK, JAK3, and SYK.     

Somatic mutations of JAK1 and JAK3 in acute leukemias and solid cancers.

PURPOSE: The aim of this study was to see whether JAK1, JAK3, and TYK2 genes are altered in human cancers. EXPERIMENTAL DESIGN: We analyzed 494 tissues from 186 acute adulthood leukemias, 30 multiple myelomas, and 278 common solid cancers, including 90 breast, 47 gastric, 47 colon, 47 lung, and 47 hepatocellular carcinomas by single-strand conformation polymorphism analysis. RESULTS: Overall, we found six JAK1 mutations (four in acute leukemias, one in a lung carcinoma, and one in a breast carcinoma) and three JAK3 mutations (two in breast carcinomas and one in a gastric carcinoma). Of note, three JAK1 mutations were an identical p.V658F mutation, which is homologous to JAK2 p.V617F mutation. We also found two other JAK1 mutations that occurred at very close sites (p.T782M and p.L783F). We found three of the four leukemias with JAK1 mutations expressed mutated JAK1 at the mRNA level. For JAK3 mutations, one of them was JAK3 p.V715I that is homologous to the JAK1 p.L783F. These recurrent mutations in identical and homologous sites suggest a possibility that alterations of these amino acids might be important for tumor pathogenesis. With respect to the cancer types, T-acute lymphoblastic leukemia (T-ALL) showed the highest incidence of the mutations (3 of 11; 27.3%). CONCLUSION: Our data indicate that both JAK1 and JAK3 mutations occur in common human cancers and that JAK1 mutation in T-ALL is a frequent event. The data suggest that some of the JAK1 and JAK3 mutations may to be functional and contributes to cancer development, especially to T-ALL development.     

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.     

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.     

Identification of a constitutively active mutant of JAK3 by retroviral expression screening

To identify transforming genes in acute myeloid leukemia (AML) we here constructed a retroviral cDNA expression library from an AML patient, and then used this library to infect a mouse cell line 32Dcl3-mCAT. cDNA inserts of the cell clones which proliferated in the presence of granulocyte colony-stimulating factor were derived from JAK3 encoding a JAK3 mutant with a valine-to-alanine substitution at codon 674 and two additional amino acid substitutions. The transforming activity of JAK3(V674A) was confirmed by its introduction into 32Dcl3-mCAT. Sequencing of the original JAK3 cDNA derived from the patient, however, failed to detect the V674A mutation.     

Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent

Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited.     

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.     

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     

The JAK2 inhibitor AG490 predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome.

The Janus kinase (JAK) family is one of intracellular protein tyrosine kinases (PTKs) present in hematopoietic and lymphoid cells and has been shown to play a crucial role in a variety of biological responses. It was reported that a human B-precursor leukemic cell line was potently inhibited in its proliferation by one of synthetic PTK inhibitors (tyrphostins), AG490, via anti-JAK2 activity. However, no extensive studies about it have been performed. In the present study, we tested 16 human lymphoid leukemic cell lines (B-precursor, 12; T cell, four) for their sensitivity to AG490 using 3H-thymidine incorporation and colony formation assays, and found that B-precursor cell lines with 11q23 translocation or Philadelphia chromosome (Ph1) whose JAK2 proved to be constitutively phosphorylated were predominantly sensitive to AG490 at a concentration that has few inhibitory effect on normal hematopoiesis. We first revealed the association of JAK2 with BCR-ABL in Ph1-positive cell lines and with Bruton's tyrosine kinase (BTK) in cell lines with 11q23 translocation by coimmunoprecipitation experiments. Of interest, AG490 markedly down-regulated phosphorylation of JAK2, but rather transiently up-regulated phosphorylation of BCR-ABL and BTK, suggesting direct implication of AG490 in the process of the JAK2 dephosphorylation. These results indicate that AG490 exerts a potent inhibitory activity to B-precursor leukemia with specific chromosomal abnormalities, and a therapeutic approach using AG490 is expected.     

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.     

Study of CALR,MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms

Objective: The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. Methods: The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F–negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. Results: 28.3% of JAK2 V617F–negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. Conclusion: Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.     

Evolution of alpha-fetoprotein: sequence comparisons among AFP species and with albumin species

Alpha-fetoprotein (AFP) and albumin are related proteins, which contain about 585 amino acid residues that are organized in a characteristic structure of 3 homologous domains of about 195 amino acid residues. We have compared the domains of mouse, rat, and human AFP and rat, bovine, and human albumin using computer programs designed to quantify relationships between proteins. The comparisons of corresponding domains of the AFPs (e.g. domain I of rat and human AFP) reveal that each domain is well conserved. Similar results were found for the comparisons of corresponding domains in the albumins. In contrast, there was much less similarity between corresponding domains of albumin and AFP. These comparisons between AFP and albumin revealed that: (1) the amino acid sequences in domain III, which is at their carboxy terminus, are most conserved, and (2) the amino acid sequences in domain I, which is at their amino terminus, are least conserved. This suggests that there are differences in the constraints on amino acid substitutions among the domains of AFP and albumin during the approximately 400 million years since they diverged from a common ancestor. Also, computer studies revealed that there are substantial differences between domains I, II, and III in each AFP and albumin species, which indicates that these domains are significantly different from their approximately 195 residue ancestral domain. Finally, we find that mouse and rat AFP are more dissimilar to the albumins than is human AFP. Overall, our computer analyses indicate that AFP and albumin can be considered to be composed of distinct, but related, approximately 195 residue proteins, each of which could differ in some of their properties; for example, the binding of fatty acids.     

JAK kinases overexpression promotes in vitro cell transformation

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokine-independent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway.     

Activating alleles of JAK3 in acute megakaryoblastic leukemia

Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.