The Phosphorylation Of TOPK By JAK2 And The Hot Spots Gene Mutation In The Chinese Patients With Burkitt's Lymphoma

Background:Burkitt's lymphoma(BL) is a highly aggressive B-cell non-Hodgkin's lymphoma,occurring mainly in children between 4 to 7 years of age,with a male to female ratio of approximately 2:1.The tumor cells often showed characteristic translocation of MYC gene and infection of Epstein Barr virus(EBV).After the first-line therapy,most of the patients have good treatment effects,but some patients still get poor outcomes,including the patients with significantly increased Lactate dehydrogenase(LDH),multiple complications and with high stage disease.There are no satisfactory treatment protocols for the patients with refractory BL,so the studies on the molecular mechanism of the high proliferation of BL are needed urgently to seek for new therapeutic targets and protocols.In 1994,some researchers have detected high expression of T-LAK cells originated protein kinase(TOPK)in BL,which is closely related to the cell cycle,proliferation,metastasis and apoptosis.However,the current reports of TOPK kinase are confined to the description of its high expression in BL,the mechanism have not yet been reported.In recent years,with the rapid development of genomic sequencing,some hot spots of gene mutation were explored in the patients of BL,but these studies are confined to the western populations,there are still not any reports on the gene mutation status in Chinese BL patients.Objectives:1.To explore the clinical features,serum lactic dehydrogenase level,lymphocyte to monocyte ratio(LMR),Ann Arbor clinical staging,international prognostic index(IPI),treatment and prognosis in Chinese patients with BL.2.To investigate the expression of TOPK in BL and the phosphorylation of TOPK by Janus Kinase 2(JAK2)in BL.3.To investigate the ID3,TCF3 and MYC gene hot spot mutation in Chinese patients with BL.Methods:1.The data of the general condition,clinical features and laboratory examination of the 35 BL patients were collected.All the patients were followed up and the prognostic information was recorded.The results of the collection were analyzed and summarized to get the information of the correlation between the clinical characteristics and prognosis of the patients with BL.2.(1)Immunohistochemical staining with the antibodies of TOPK,p-JAK2,p-STAT3,p-ERK1/2 were performed by immunohistochemistry technology.The survival analysis was performed with the immunohistochemical results and the corresponding prognosis of the patients.(2)Peptides of TOPK with different possible phosphorylation tyrosine site were designed and synthesized by PEPTIDE 2.0(Houston,TX,USA),and the in vitro kinase assay using the activated JAK2 kinase were perform to observe the possible phosphorylation site of TOPK by JAK2.(3)The expression of TOPK and JAK2 was detected by using Western Blot in 3 BL cell lines Daudi,P3HR1 and Raji.(4)In vitro binding assay was employed to determine the interactive binding of JAK2 and TOPK.(5) The effect of JAK2 agonist(IL-6)and JAK2 antagonist(AG490)on BL cell lines was observed.3.The integrated DNAs were extracted from the paraffin-embedded tissues of 32 patients with BL using the QIAGEN kit.PCR technology was used to amplify ID3 gene exon 1,TCF3 gene exon 17 and all 3 exons of MYC gene.The products were used to perform the electrophoresis,purification and sequencing.Results:1.In 35 cases of BL,the ratio of male to female was about 3:1,and there were 13cases(37.1%)were below the age of 18 years.The median age of onset was 22(1-74)years,20 patients with follow-up informations showed that the 5-year survival rate was51%.Patients with high clinical stage,high level of serum LDH,low LMR(LMR<3.57)or high IPI index and patients underwent surgery showed poor prognosis.2.In 35 patients with BL,TOPK expressions were diffuse strong positive in all cases,27 cases were p-JAK2 positive,9 cases were p-STAT3 positive,and p-ERK1/2 were negative in all the 35 cases.In vitro kinase assay showed that the TOPK can be phosphorylated by JAK2 at the Y74 site;the expressions of TOPK and JAK2 in Daudi cells were higher than P3HR1 and Raji;in vitro binding assay confirmed the interactive reaction of the JAK2 and TOPK either in vitro or in Daudi cells;the JAK2 agonist(IL-6)and antagonist(AG490)can up-regulated and down regulated the Y74 site phosphorylation level of TOPK respectively.3.The 32 Chinese patients with BL showed similar ID3,TCF3 and MYC genes mutation rate with Western populations reported in the literature,the rates were 35.5%,18.8% and 50%,respectively.For the first time,we found two novel recurrent mutation sites in the TCF3 gene and MYC gene: TCF3,C.2202 G > C p.L569V;MYC,C.1070 A > G p.G182 D.Conclusion:1.In this serials,the patients with BL is mainly adult male patients,high stage disease were seen in the majority of patients,more complications and low 5-year survival rate(51%)were found in these patients.High clinical stage,high serum LDH,low LMR(LMR<3.57)and high IPI,patients underwent surgery suggest poor prognosis.2.JAK2 can phosphorylated the TOPK at Y74 site in the BL cell line,suggesting that JAK2 may affect the proliferation of the BL cells through the serine / threonine kinase TOPK which plays an important role in the mitosis of tumor cells.3.The mutation rates of the ID3 gene,TCF3 gene and MYC gene in Chinese is similar with that of the western populations.For the first time,we found the TCF3 gene mutation site: C.2202 G > C p.L569 V and MYC gene mutation site: C.1070 A > G p.G182 D.