Roles And Significances Of JAK2V617F Mutation In Myeloproliferative Disorders

In early 2005,several independent groups published virtually simultaneously on the presence of a specific mutation in the JAK2 associated with BCR/ABL-negative MPD.The mutation of JAK2 was the same base change(G to T)at nucleotide 1849,resulting in a substitution of valine 617 for a phenylalanine in the JH2 domain of JAK2(JAK2V617F).The frequency of JAK2V617F mutation was 65%～97%among patients with PV,23%～57%among patients with ET and 35%～57%among patients with IMF,which indicates JAK2V617F mutation plays an important role in the pathogenesis of MPD. JAK2V617F mutation caused a widespread concern,and the period afterwards was named as "JAK2 era of myeloproliferative disorders" However,JAK2V617F mutation among Chinese people has been scarcely reported;The diagnostic value of JAK2V617F mutation should be assessed further by considerable clinical data;The pathogenesis role of JAK2V617F in myeloproliferative disorders is still unclear;The signal pathway and downstream target gene of JAK2V617F mutation remains to be studied further.In this investigation,JAK2V617F mutation in 110 patients with malignant hematologic disease was detected,and a cell model was used to characterize the signal pathway and identify the target gene of this mutation.The research provides new targets as well as a theoretical basis for research and development of JAK2V617F-mutation-targeted drug,and will be of great significances in elucidating the mechanisms underling the mutation.OBJECTIVE①To analyze the specificity,incidence of JAK2V617F mutation in patients with BCR/ABL-negative myeloproliferative disorders(MPD)and explore the clinical significances, and develop a sensitive and specific clinical diagnosis test system for patients with MPD.②A cell model was used to characterize the signal pathway associated with the JAK2V617F mutation;③To further elucidate the mechanism underling the polycythaemia vera(PV),the target genes of JAK2V617F(JAK/STAT)signal pathway was identified and the relevance between this mutation and apoptosis and proliferation was studied.METHODS①Genomic DNA was isolated from granulocytes originating from peripheral-blood or bone marrow.Allele-specific polymerase chain reactions(AS-PCR),restriction enzyme digestion were performed to detect the mutation in genomic DNA.110 patients and ten normal controls were detected.The PCR product originating from 41 patients with BCR/ABL-negative MPDs and two normal controls was subject to DNA sequencing.②HEL cell line,which has a genetic background of erythrocyte and carries a homozygous JAK2V617F mutation confirmed by the three methods mentioned above,was selected as a cell model.We also investigated the morphology,cytochemical staining and chromosome karyotype properties of this model.③Western blot was performed to determine the effect of AG490 on the JAK2/STAT5 signal pathway by the antibodies against the JAK2,STATS,phospho-JAK2 and phospho-STATS.④CeilTiter-Glo(?) Luminescent cell viability assay and FITC-Annexin V/PI double staining and then Flow cytometry(FCM)were performed to determine the proliferation ability and apoptosis rate of the HEL cell,respectively.⑤RT-PCR was performed to screen the candidate gene(GATA1,GATA2,PTTG1,and bcl-x)to identify the downstream target of the JAK2/STAT5 signal pathway.Western blot and Flow Cytometry(FCM) were performed to analyze the effect of AG490 on the expression of PTTG1 protein further.⑥Dual Luciferase Reporter Assay was performed to detect the effect of AG490 on the promoter activity of PTTG1.RESULTS①The positive rate of JAK2V617F detected by AS-PCR and restriction enzyme was shown as follows:11(91.7%)of 12 with PV,8(53.3%)of 15 with ET,4(57.1%)of 7 with IMF,while in other patients including 7 patients with HES,25 patients with CML,44 patients with AL(including 24 patients with AML,18 patients with ALL,1 patients with CML-blast phase,1 patients with mixed-lineage acute leukemia (MAL)),JAK2V617F could not be detected.The PCR product prepared from 41 patients with BCR/ABL negative MPD patients and 2 normal controls were further subjected to sequencing analysis,and the results demonstrated that the 23 patients carrying JAK2V617F mutation detected by AS-PCR and restriction enzyme digestion were JAK2V617F positive and the others were wild-type.②The presence of the JAk2V617F in the HEL cell was confirmed by AS-PCR,restriction enzyme digestion and DNA sequencing,and morphological experiments and cytochemical staining suggested that the HEL cell has a genetic background of erythrocyte.③Western blot demonstrated that under the condition that the total protein loaded in each well were much the same,as the time extended(0h,12h,24h,48h,72h),the JAK2 total protein changed little, while the pospho-JAK2 protein decreased gradually,with the greatest decreasing rate of 95.3%;Similarly the STAT5 total protein changed little, while the phospho-STAT5 protein decreased obviously,with the greatest decreasing rate of 72.4%;In K562 cells,the protein expression of JAK2, phospho-JAK2,STAT5,phospho-STAT5 has little change when treated by AG490 for 72h.④When treated by 50μM AG490(0h,24h,48h,72h, 96h),the proliferation rate of HEL cells decreased dramatically compared with the control(P<0.01).⑤FITC-AnnexinV/PI double staining and FCM demonstrated that the apoptosis rate of HEL cell increased obviously in a time and dose dependent manner after treated by AG490.⑥RT-PCR demonstrated that when treated by AG490,the mRNA of PTTG1 was down-regulated obviously,and the down-regulation was strongly time and dose dependent;but the expression of transcriptional factor GATA1 and GATA2 changed little.⑦Western blot demonstrated that when treated by 100μM AG490(0h,12h,24h,48h,72h),PTTG1 protein decreased obviously as time extended;Flow cytometry(FCM)showed that the PTTG1 positive rate of the HEL cells was 55.9%under normal condition,and as the AG490 treating time extended,the positive rate decreased,which was only 9.0%at 72h;⑧Dual-luciferase assay showed that the PTTG1 promoter activity decreased by 2.5 fold when treated by AG490.CONCLUSION①The frequency of JAK2V617F mutation was more than 90%among patients with PV,more than 50%among patients with ET and IMF.AS-PCR and restriction enzyme digestion assay were sensitive,specific,convenient and shortcut tests for the JAK2V617F mutation;This mutation might be a molecular marker as well as a treatment target of MPD in the future;②In HEL cells,the muted JAK2V617F kinase and JAK2V617F/STAT5 signal pathway can be inhibited by the specific inhibitor of JAK2 kinase,resulting in the significantly decreasing of the cell proliferation activity and increasing of the apoptosis rate;AG490 caused an obviously decrease of Bcl-x(Bcl-x_L mRNA was the main species of Bcl-x found),which suggests that JAK2V617F might inhibit cell apoptosis through upregulation of Bcl-x_L.③On the inhibition of the JAK2V617F/STAT5 signal pathway by AG490, PTTG1 mRNA and protein were decreased significantly,which suggested that PTTG1 might be a downstream target of JAK2V617F signal pathway. Upregulation of PTTG1 on the transcriptional level might promote cell proliferation abnormally.