The Role And The Significance Of BTK Regulates Ikaros In Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia(ALL)is a clonal lymphocytic malignant disease.Due to the high incidence,low remission rates,high relapse rates and lack of specific targets therapy,ALL endangers human health.As a protein tyrosine kinase,expressed in hematopoietic cells(except T cells),Bruton Tyrosine Kinase(BTK),is a member of the Tec family kinase.It can promote the differentiation and maturation of B lymphocytes.Previous research found that BTK may involve in the occurrence,development and prognosis of many types of hematological malignancies,including ALL,chronic lymphoblastic leukemia(CLL)and malignant lymphoma.However,the exact effects of BTK in ALL is not clear.Ikaros family zinc finger 1(IKZF1)gene and its coding protein Ikaros play important roles in lymphocytes differentiation and lymphocytic malignancy suppression.IKZF1 is associated with the relapse,prognosis and overall survival of ALL.Recently,pediatric ALL studies found,BTK,as a partner and posttranslational regulator of Ikaros,phosphorylates Ikaros,thereby augmenting its nuclear localization and sequence-specific DNA binding activity.It is currently not clear whether similar regulation also exist in adult ALL.Therefore,it is necessary to study the relationship between BTK and Ikaros,and their roles in adult ALL,which may be useful in ALL target therapy.Objective: 1.Based on public database,to explore the interactions between BTK and Ikaros and the biological process,cell component,molecular function and the pathways involved.2.To investigate the role and significance of BTK regulation on Ikaros in ALL cell lines,sup-b15 and nalm6 cells,and to explore the possible mechanisms.Methods: 1.The interactions between BTK and Ikaros: To verify of the relationship betweenBTK and Ikaros through bioinformatics analysis,and to explore the molecular mechanism.2.The role and the significance of BTK regulates Ikaros in ALL: The B cell malignant cell lines: sup-b15 and nalm6 cells were treated with different concentration of Ibrutinib.The cell proliferation was detected by CCK-8 methods and BTK and Ikaros expression are detected by Western blot.Coimmunoprecipitation(co-ip)was used to investigate the interaction between BTK and Ikaros.Results: 1.According to the database analysis,there is a correlation between BTK and Ikaros(R=0.66)in diffuse large B cell lymphoma(DLBCL)cells;the current study predicts that there exists interaction and regulation between BTK and Ikaros.The enrichment analysis indicates that BTK and Ikaros are enriched in biological process(BP),including mesoderm development and lymphocyte differentiation,while in cell component(CC),they are enriched in nucleus and cytoplasm.For molecular function(MF),they are enriched in protein binding and metal ion binding.The most significantly enriched KEGG pathways of BTK are NF-kappa B signaling pathway,osteoclast differentiation,platelet activation,B cell receptor signaling pathway,Fc epsilon RI signaling pathway and pathways involved in Primary immunodeficiency.2.ALL cell lines sup-b15 and nalm6 cells were treated with different concentrations of BTK inhibitor(Ibrutinib)(0.5,1,2,4,6,8 μmol/L)for 24 h and 48 h.The results show that Ibrutinib can inhibit the proliferation of sup-b15 and nalm6 cells by decreasing the expression of BTK and increasing the expression of Ikaros.As the concentration increases,the level of BTK expression decreases,and the opposite of Ikaros.The effects were time and drug density dependent.The co-ip results revealed that there was a direct interaction between BTK and Ikaros,suggesting that BTK regulates Ikaros through binding it directly.Conclusion: 1.The bioinformatic analysis results indicated that there may exist a correlationbetween BTK and Ikaros in B cell malignancy(R=0.66).The enrichment analysis indicates that BTK and Ikaros are enriched in biological process(BP),including mesoderm development and lymphocyte differentiation,while in cell component(CC),they are enriched in nucleus and cytoplasm.For molecular function(MF),they are enriched in protein binding and metal ion binding.The most significantly enriched KEGG pathways of BTK are NF-kappa B signaling pathway,osteoclast differentiation,platelet activation,B cell receptor signaling pathway,Fc epsilon RI signaling pathway and pathways involved in Primary immunodeficiency.2.Ibrutinib inhibits the proliferation of ALL cell lines(sup-b15 and nalm6)in drug-concentration and time dependent manner.These inhibition effects were done by decreasing the expression of BTK and increasing the expression of Ikaros in two cell lines.BTK regulates the expression and function of Ikaros through binding it directly.