Effects Of CpG 685 On Different Types Of B-Cell Acute Lymphoblastic Leukemia And Their Different Mechanism

Background:Immunotherapy for B-cell acute lymphoblastic leukemia(B-ALL)has advanced quickly in recent years,which has improved the response rate of relapsed refractory B-ALL from 30%to 90%.However,current treatments suffer from side effects and limited efficacy.Therefore,it is important to find new strategies to reduce side effects and prolong survival.Toll-like receptor 9(TLR9)agonists have shown good safety and efficiency as immune adjuvants.It not only has immunomodulatory effect,but also has a direct effect on tumor cells.Besides,it can enhance the anti-tumor immunity as well as affect the suppression immune microenvironment of the cancer,so as to promote the immune system to better recognize the tumor and turn“cold”tumors“hot”.However,previous studies mainly focused on the immune regulation of TLR9 agonists,while the direct effect of TLR9 agonists on tumors often been ignored.Especially,the direct effect of TLR9 agonists on B-ALL has not been reported.According to our previous study,TLR9 agonist can inhibit B-ALL proliferation,induce them apoptosis,and promote them cell cycle arrest.But its effect on different types of B-ALL are heterogeneous.Thus,clarifying the role and molecular mechanism of TLR9 agonist in B-ALL patients with different subtypes may be helpful to explore the biomarkers and patient characteristics of the effective treatment of TLR9 agonists and optimize the precise therapy of B-ALL.Objectives:1.Clarify the relationship between TLR9 expression and clinicopathological characteristics as well as prognosis of B-ALL patients.2.Explore the effect of CpG 685 on B-ALL.3.Find the apoptotic molecular mechanism mediated by CpG 685 in B-ALL cells and explore the biomarker for therapy.4.Identify the therapeutic strategies for different types of B-ALL patients and the characteristics of sensitive populations for CpG 685 application.Methods:1.Detect TLR9 expression by real-time quantitative RT-PCR and flow cytometry.Screen the relationship between TLR9 expression and clinicopathological features as well as prognosis by statistical analysis.2.Clarify the direct effect of CpG 685 in the role of different types on B-ALL by WST-1 proliferation experiment,Annexin?/7-AAD apoptosis detection,and flow cytometry.3.To determine the engraftment and tumorgenicity of BLIN-1 and Sup-B15cells in vivo,NCG mouse xenograft models have been made.And B-ALL cells from boom marrow,peritoneal cavity and spleen of each mouse were detected by flow cytometry.Besides,survival time of mice was observed,so as to clarify the difference of the effects of CpG 685 on B-ALL cells in vivo.4.After CpG 685 stimulation,western blot,co-immunoprecipitation,immunofluorescence co-localization,and chromatin immunoprecipitation were used to detect the expression of proteins related to TLR9 downstream signaling pathways and the action sites of genes through pretreating with or without related signaling pathways inhibitors.The expression of mi RNA involved in the pathway was determined by RT-PCR and real-time quantitative RT-PCR.By exploring the mechanism of CpG 685 on B-ALL,the predictive biomarkers of the efficacy of CpG685 in the treatment of B-ALL were explored.5.Ficol density gradient centrifugation was used to isolate peripheral blood mononuclear cells from clinical samples,and the feasibility of cell line-based predictive markers in clinical application was determined with or without CpG 685stimulation.6.Flow cytometry was used to detect the proportion of various immune cells in BALB/C mice transplanted tumor models to determine the regulation of anti-tumor immunity in mice by CpG ODN.Results:1.TLR9 expression in PBMCs was higher in B-ALL patients than in healthy controls.B-ALL patients with high TLR9 expression have a better prognosis.2.Heterogeneous effects on B-ALL cells.Proliferation suppression,apoptosis,cell cycle arrest,costimulatory molecules upregulation,tumor burden reduced,and survival time prolonged for BLIN-1 mediated by CpG 685.However,no significant effect on Sup-B15.3.CpG 685 induce apoptosis in BLIN-1 cells through P38/P53/BAX signaling and rely on C-MYC overexpression.C-MYC overexpression can promote P53stabilization as well as activation,enhance BAX transcription,and mediate conformational changes associated with BAX activation.4.In Ph(+)B-ALL cells,BCR-ABL kinase blocks BAX activation through multiple pathways,such as C-MYC and JAK/STAT5,leading to CpG 685 resistance.After imatinib pretreatment,inhibition of BAX activation by BCR-ABL kinase as well as by C-MYC was observed,and CpG 685 resistance was reversed.Besides,CpG 685 downregulated mi R-21,further upregulated PTEN,and ultimately reversed the drug resistance of imatinib caused by BCR-ABL1-independent AKT phosphorylation in Ph(+)B-ALL.5.CpG 685 can significantly promote the apoptosis of primary B-ALL cells with C-MYC overexpression.6.CpG ODN promote B-cell as well as pDC proliferation,and inhibit MDSC proliferation,which further increase the proportion of NK cells,T cells,and M1/M2.Conclusion:1.TLR9 expression was associated with prognosis in patients with B-ALL.2.TLR9 is a promising target for B-ALL therapy.CpG 685 induces apoptosis in B-ALL cells through P38/P53/BAX signaling and relys on C-MYC overexpression.3.C-MYC may be a biomarker to help predict the efficacy of CpG 685monotherapy in B-ALL patients.4.Combinational use of CpG 685 and imatinib can reverse resistance to imatinib or CpG 685 treatment alone.And this combination therapy can not only directly induce apoptosis of Ph(+)B-ALL cells,but also enhance the immunogenicity of B-ALL cells.5.CpG ODN can promote by CD8~+T cell differentiation and increase the rate of M1/M2,which further active the antitumor immunity.