Study On Effect Of Chidamide On T315I Mutant CML Cells And Exploration Of Effect Of HMGB1 On CML Cells

BackgroundChronic myeloid leukemia is characterized by t(9;22)(q34;q11.2).TKI significantly improves the survival of patients,but T315I mutation results in the resistance to TKI.Many researches were carried out on this issue.Reachers explored the effects on T315 mutant cells of other drugs alone or in combination with TKI.Studies reported that HDACi like LBH589 and LAQ824 could induced apoptosis of chronic myeloid leukemia stem cells,and they could enhance the sensitivity of chronic myeloid leukemia stem cells to TKI when combined with TKI.Therefore,it worth doing furture reseach that the effect on chronic myeloid leukemia cell harboring T3151 mutant.High mobility family protein B1(HMGB1)is a late inflammatory mediator,which plays an important role in tumorigenesis and development of tumor,but its role in hematological tumors has not been clarified.Besides cells exists which secrete HMGB1 actively in the bone marrow microenvironment,consisting of MSC,Macrophage and tumor cells.So a large amount of HMGB1 may be detected in bone marrow microenvironment.Previous studies have demonstrated that HMGB1’s receptor Tim-3 is highly expressed on the surface of chronic myeloid leukemia stem cells which overexpresses β-catenin.It is well known that β-catenin plays a vital role in balst crisis and resistance to TKI.Therefore It’s worth exploring that HMGB1 supports chronic myeloid leukemia cells.Objective(1)To explore the effect and mechanism of Chdamide on T3 15I mutant chronic myeloid leukemia cells.(2)To detect the content of HMGB1 in the bone marrow microenvironment of patients with chronic myeloid leukemia.(3)To explore the effect of HMGB1 on the expression of β-catenin in KBM5 cell line,providing proof that HMGB1 supports chronic myeloid leukemia cells through β-catenin.Method(1)The CCK8 method was used to analyze the effect of Chidamide on the proliferation of three group chronic myeloid leukemia cell lines.Flow cytometry was used to detect the pro-apoptotic effect of Chidamide alone and combination with IM on the T315I mutant cell lines;The effect of Chidamide on the colony-forming ability of T315I mutant cells was detected by colony-forming unit assay;A-TAC sequence was performed to find Chidamide-induced epigenetic changes on T315I mutant cells.Western Blot was to verify A-TAC sequence results and changes of transcription factor SITR1 downstream pro-apoptotic pathway FOXOs/BIM.Influence on localization of FOXOs in T315I mutant cells was analyzed by Immunofluorescence;SiRNAs of FOXOs were transferred into T315I mutant cell lines by electroporation,then we deteced changes in the pro-apoptotic effect of Chidamide on T315I mutant cells by flow cytometry.(2)We Collected bone marrow specimens from patients with different diseases,and detect the content of HMGB1 by ELISA.(3)Western Blot and flow cytometry were used to detect the effect of HMGB1 on β-catenin in KBM5 cell line;CCK8 method was used to detect whether HMGB1 weaken the effect of β-catenin specific inhibitor PRI-724 on KBM5 cell line.Results(1)T315I mutant cells were more sensitive to Chidamide;Chidamide and IM synergistically killed T3 1 5I mutant cells;Chidamide inhibited colony forming ability of T315I mutant cells;Chidamide down-regulated the transcriptional activity of SIRT1 in T315I mutant cells and activated its downstream apoptosis Pathway FOXOs/BIM;Chidamide promoted the nuclear expression of FOXOs;the pro-apoptotic effect of Chidamide is significantly reduced after down-regulating FOXOs expression.(2)The bone marrow supernatant of patients with chronic myeloid leukemia contained high levels of HMGB1,with an average value of 110 ug/ml.In addition,the content of HMGB1 in bone marrow supernatants of other hematological malignancies were also higher than that of healthy people,with an average value greater than 100 ug/ml..(3)HMGB1 promoted the expression of total β-catenin protein and nuclear protein;it partially reversed the inhibitory effect of PRI-724 on the KBM5 cell line.Conclusion(1)Chidamide could inhibit the proliferation and colony-forming ability.Chidamide and IM can synergistically killed T3 15I mutant cells;Chidamide induced apoptosis of T315I mutant cells by reducing SIRT1 transcriptional activity and activiating its downstrem FOXO3-meditating apoptotic pathway.This provides a new therapeutic drug for solving the resistance to TKI of T315I-mutated chronic myeloid leukemia cells.(2)Patients with chronic myeloid leukemia contained high levels of HMGB1 in the bone marrow microenvironment,and HMGB1 may become a biomarker for chronic myeloid leukemia.(3)HMGB1 promoted β-catenin expression in chronic myeloid leukemia cell lines,and may play an important role in the maintenance,blast crisis and TKI resistance of chronic myeloid leukemia cells.