The Mechanism Involved In Multiple Anti-acute B Lymphocytic Leukemia Effects Of Toll-like Receptor 9 Agonist

Acute lymphoblastic leukemia(ALL)is a highly heterogeneous disease that contains different biological and prognostic features.Although most patients with ALL can achieve complete remission after conventional chemotherapy or combined molecular targeted therapy,the minimal residual disease persists.BCR-ABL works as an independent adverse prognostic factor in B-ALL.Although with the application of imatinib for Ph-chromosomes positive B-ALL has improved efficacy,the overall survival is still dramatically low due to the emergence of drug resistance.Therefore,removal of minimal residual disease and reversal of imatinib resistance are key to preventing relapse and prolonging survival.It is extremely important to find a novel target that can induce anticancer immune responses as well as exert direct effects against cancer cells.Toll-like receptor 9(TLR9),one of the Toll-like receptors,serves as a bridge between innate and adaptive immunity in the antitumor responses.It has been shown that TLR9 stimulation by Cp G oligodeoxynucleotides(Cp G ODNs)appears to induce a strong T helper(Th1)-type immune response that is therapeutically important for antitumor immunities.The efficacy and safety of TLR9 agonists also have been demonstrated in clinical trials.However,the response to Cp G ODNs on B-cell malignancies exhibits high heterogeneity,particularly elicits the opposite biological effects.Besides,Study shows that the responses to Cp G ODN stimulation on Ph-chromosomal positive B-ALL cell lines(Sup-B15,KOPN57 bi,OP-1)are detectable,but the specific molecular mechanism has not been reported yet.Therefore,the mechanism of the immune regulation by TLR9 and the TLR9 downstream signaling pathway in B-ALL activated by Cp G 685 needs to be further explored.Objective: 1.Screen the potential targets for B-ALL treatment.2.Clarify the reason for the heterogeneity of Cp G 685 treatment on B-ALL,screen the characteristics of TLR9 agonists in sensitive individuals and look for the possible combination therapy.Methods: 1.Real-time Quantitative PCR(RT-PCR)was used to determine the expression level of TLR9 m RNA in clinical samples of B-ALL patients.SPSS23.0 statistical software was used to analyze the correlation between TLR9 expression levels and clinical characteristics.2.The effect of Cp G 685 on the proliferation,apoptosis,cell cycle and costimulatory regulation of B-ALL was detected by WST-1 assay and flow cytometry.3.The effect of Cp G 685 on the release of ATP in B-ALL cell lines was examined by ELISA to determine whether there existsimmunogenic cell death.4.Western blot was used to detect the activation of TLR9 downstream molecules by Cp G 685 stimulations and explore the mechanism of inducing apoptosis in BLIN-1 cells.Key molecular inhibitors were used to further explore the key molecules and regulatory mechanisms of apoptosis-related TLR9-downstream signaling pathways.5.Use the immunoprecipitation(IP)method to detect BAX activation.Results: 1.The expression of TLR9 was found in more than 95% patients with B-ALL,and TLR9 was highly expressed in patients with positive Ph-chromosome.2.Cp G 685 induces costimulatory molecules and NK cells activating ligand upregulation in B-ALL cells,thereby enhancing the immunogenicity of B-ALL cells.However,the surface molecule activation is heterogeneous.3.Cp G 685 induces apoptosis and G1-phase arrest,but no immunogenic cell death.4.The pathway of Cp G 685-induced apoptosis on BLIN-1 cell lines was achieved through the P53-dependent and non-dependent apoptosis pathway,up-regulation of BAX expression,and promote the activation of BAX,which was dependent on the high expression of C-MYC and the activation of the P38/P53/BAX signaling pathway.At the same time,C-MYC inhibits the sustained activation of NF-?B in turn,leading to the occurrence of apoptosiseventually.5.Resistance to Cp G 685 in patients with positive Ph-chromosomes is related to the blockade of BAX activation by BCR-ABL.6.The combination therapy of imatinib and Cp G 685 can reverse the drug resistance on using imatinib and Cp G 685 alone.Conclusion: 1.TLR9 is a potential target for B-ALL therapy and may be a target for reversing imatinib resistance and combination therapy after imatinib resistance in patients with Ph-positive B-ALL.2.Cp G 685 has multiple anti-tumor effects through direct killing of B-ALL cells and indirect killing by the immune system.3.Screening for C-MYC high expression levels and Ph-chromosomenegative B-ALL patients is the key to the treatment with Cp G 685.4.The combination of imatinib and Cp G 685 can help to reverse the resistance of imatinib,increase the therapeutic response to Cp G 685,reduce toxic side effects,and kill tumors to a greater extent.