Effects And Mechanisms Of Hydroxychloroauine On Tvrosine Kinase Inhibitors-Resistant Chronic Mveloid Leukemia Cell

Chapter 1 Targeting autophagy with Hydroxychloroquine potentiates TKIs induced cell death in the cells with BCR-ABL T315IIntroduction:Tyrosine kinase inhibitors(TKIs)resistance due to BCR-ABL mutation is a great challenge in treating chronic myeloid leukemia(CML),and T315I mutation is common.The mutation of the kinase gatekeeper residue from threonine to isoleucine results in the change of kinase conformation,which is important for multidrug resistence.The third generation TKIs-Ponatinib is the first-line medicine for CML with T315I mutation.In addition,it has been reported that vascular epidermal growth factor receptor inhibitor-Axitinib can effectively kill CML cell with T315I in vitro.Autophagy is a powerful weapon for cancer cell against drugs.Targeting BCR-ABL imatinib can induce autophagy in CML cell,then autophagy weakens the efficacy of imatinib,and autophagy inhibition can enhance the anti-CML efficiency of imatinib.Whether axitinib and ponatinib can induce autophagy by inhibiting BCR-ABLT315I,whether CML cell with T315I can utilize autophay to escape killing by the drugs,and whether autophagy inhibitor-hydroxychloroquine(HCQ)can potentiates axitinib and ponatinib induced cell death in CML with T315I have not been reported.Aim:To clarify whether HCQ can enhance the efficacy of axitinib and ponatinib in treating CML with T315I mutation,and further to reveal the mechanism.Methods:The 32D cells stably expressing BCR-ABLT315I(32Dp210-T315I cells)were used as main object.Western Blot and confocal immunofluorescence microscopy were used to test whether axitinib and ponatinib can induce autophagy in 32Dp210-T315I cells.Compare cell apoptosis,cell clonality,cell cycle and cell senesce after treating with axitinib/ponatinib combined with or without HCQ.To confirm whether HCQ play its role by inhibiting autophagy in this research,we inhibited autophagy by knocking down ATG7 in 32Dp210-T315I cells,and then tested whether ATG7 knockdown can enhance the toxicity of axitinib and ponatinb to 32Dp210-T315I cells.Verify the synergies effects of HCQ and axitinib/ponatinib in nude mouse with subcutaneous tumor.Results:Both axitinib and ponatinib can induce autophagy in 32Dp210-T315I cells.HCQ can inhibit autophagy induced by axitinib/ponatinib.And HCQ potentiate efficacy of axitinib/ponatinib on cell apoptosis and cell clonality,but not on cell cycle and cell senesce.Knocking down ATG7 can effectively inhibit autophagy induced by axitinib/ponatinib.Similar to HCQ,ATG7 knockdown can potentiate efficacy of axitinib/ponatinib on cell apoptosis,but not on cell cycle.Conclusion:HCQ can potentiate axitinib/ponatinib induced cell death in 32Dp210-T315I cell through autophagy inhibition.Chapter 2 HCQ promotes NKG2D-ULBP4 mediated elimination of TKIs-resistant CML cells by V?9V?2 T cellIntroduction:Through targeting autophagy HCQ can not only enhance the anti-tumor efficiency of chemotherapy but also promote cancer cell elimination by immunocyte.Autophagy inhibition in lung cancer and melanoma cells can sensitize the cells to NK and CTL cell mediated cytotoxicity.Immunotherapy represents the future tendency of leukemia treatment.V?9V?2 T cell is powerful in tumor elimination,it even can effectively kill TKIs-resistant CML cell,which imply V?9V?2 T cell is the"future star" in cell immunotherapy.This study was designed to investigate whether HCQ can promote the elimination of TKIs-resistant CML cells by V?9V?2 T cell,and to reveal its mechanisms.Aim:To investigate whether HCQ can promote the elimination of TKIs resistant CML cells by V?9V?2 T cell,and to reveal the mechanisms.Methods:K562/GO1 cells(imatinib-resistant CML cell line)and K562 cells(imatinib sensitive CML cell line)were used as research object.LC3?.P62 were tested by Western Blot,and autophagosome were detected with transmission electron microscopy to analyze whether HCQ can inhibit autophagy in K562/GO1 and K562 cells.HCQ or PBS pretreated K562/GO1 or K562 cells were used as target cells,and V?9V?2 T cell from healthy donors were used as effecter cells.CML cells and V?9V?2 T cells were co cultured at different effecter-target ratios to compare the sensitivity of target cell to effecter cell.Test the degranulation of V?9V?2 T cells co cultured with HCQ or PBS pretreated CML cell lines or CML primary cells by flow cytometry.Inhibit autophagy with shRNA targeting ATG7,then analyze whether autophagy inhibition can sensitize CML cell to V?9V?2 T cell mediated lysis;in addition we also tested whether HCQ can sensitize ATG7 defective CML cell to V?9V?2 T cell mediated lysis.The expression of NKG2DL(MICA/B ULBP1-6)on CML cell surface were tested with flow cytometry after HCQ or PBS treatment.NKG2D blocking antibody were used to block the interaction of NKG2D and NKG2DL to investigate whether HCQ sensitize CML cells to Vy9V82 T cells mediated lysis through inducing ULBP4 expression on CML cell membrane.The expression of ULBP4 in whole cell was detected by Western Blot,fluorescence quantitative PCR and flow cytometry,and the distribution of ULBP4 in cells were tested by confocal immunofluorescence microscopy to reveal the mechanism of the expression of ULBP4 regulated by HCQ.Results:HCQ can effectively block autophagy in K562/GO1 cells and K562 cells,and sensitize K562/GO1 and K562 cells to V?9V?2 T cytotoxicity.However,blocking autophagy by knocking down ATG7 cannot mimic the sensitization effects of HCQ,and even in ATG7 defective K562/GO1 and K562 cells HCQ still can improve the eliminaton of CML cell by V?9V?2 T.These results suggested that HCQ sensitize CML to V?9V?2 T cytotoxicity independent of autophagy.HCQ induced ULBP4 expression on K562/GO1 and K562 cell membranes.After blocking the interaction of NKG2D and NKG2DL the sensitization effects of HCQ almost disappeared,suggesting that HCQ induced ULBP4 expression on CML cell surface promotes the elimination of CML cell by V?9V?2 T cell.HCQ did not improve the transcription and synthesis of ULBP4,but induced ULBP4 transfer from intracellular to the cell membrane.Conclusion:HCQ induced the transfer of ULBP4 from K562/GO1 and K562 cytoplasm to cell membrane,and promoted the elimination of K562/GO1 and K562 cells by V?9V?2 T cells.