Chidamide Synergistically Potentiated Imatinib Lethality In IM-resistant Chronic Myeloid Leukemia Cells

Background Chronic myeloid leukemia(CML)is a disorder of hematopoietic stem cells caused by constitutive activation of the Bcr-Abl tyrosine kinase.Imatinib mesylate(IM),as a first-generation ABL tyrosine kinase inhibitors,has dramatically improved the management of CML,but IM resistance is frequently observed,especially in patients with advanced-stage disease.Moreover,the first-and second-generation TKI are ineffective in CML cells harboring the T315I mutation.Therefore,it is urgent to find a promising therapeutic strategy to overcome the TKI resistant of CML.The selective histone deacetylase(HDAC)inhibitors have become promising anti-tumor agents in recent years,which had applied to several solid tumors and hematologic malignancies.Chidamide is a novel HDAC inhibitor of the benzamide class,which plays a potential role in restoring the sensitivity of drug-resistant cancer cells,and inhibiting tumor metastasis and recurrence by epigenetic regulation.The first indications of Chidamide is recurrent and refractory peripheral T cell lymphoma.Recent reports showed that chidamide also can kill acute myeloid leukemia cells,but the effect of chidamide on CML is still unknown.ObjectiveThis study was aim to explore the anti-leukimia effects of Chidamide and IM in TKI-resistant CML cells,further reveal the molecular mechanism of combination treatment drug,which provides a new therapy strategy to CML patients with TKI resistance.MethodWe chose human CML cell lines KBM5 and the KBM5-T315I harboring the T315I mutation as our research subject.1.To study the effect of Chidamide on the biological behavior of CML cells:After CML cell lines treated with incremental Chidamide,cell growth inhibition by CCK-8,cell cycle and apoptosis analysis by flow cytometry.2.To study the combined-effect of Chidamide and IM on CML cells:We used a low dose chidamide and/or IM treat with CML cells,and detected cell proliferation by CCK-8,apoptosis by flow cytometry,and observed the cells colony forming.The CalcuSyn software was used for analysing the combination effect of both drugs.3.Western bolt and RT-qPCR analyzed the protein and mRNA expression level of?-catenin in TKI-sensitive and-resistant primary cells and cell lines.4.The effect of combination therapy on ?-catenin signaling and Bcr-Abl activity and HDAC activity in CML cells:Western bolt were used to analyze the expression of?-catenin and its target gene C-myc and Surivin protein in CML cells after treating with the combination or drug alone.The inhibition of phosphorylation of Bcr-Abl,Stat5 and CrkL,the expression of histone H3 acetylation and Caspase-3/-8/-9 proteins were also detected by Western bolt.Result1.Chidamide can inhibit KBM5 and KBM5-T315I cells growth with IC50 4.5uM,8.4uM,respectively,and induce cell apoptosis and cell cycle arrested at G0/G1 phase.2.Low dose chidamide combined with IM greatly induced KBM5 and KBM5-T315I cell growth inhibition and cell death,compared to drugs alone.The combination index(CI)were both less than 1,which indicated a synergism against to CML cells by Chidamide and IM.3.?-catenin was higher expressed in the TKI-resistant CML primary cells and cell line KBM5-T315I,which had no correlation to the expression of BCR-ABL.It suggested that TKI-resistant cells existed ?-catenin signaling activation,probably related to the independent BCR-ABL induced TKI resistant mechanism.4.?-catenin,C-myc and Surivin protein were declined in CML cells after exposed to the combination of chidamide and IM,which indicated that the combination treatment inhibited the TKI-resistant CML cells through inhibiting the ?-catenin signaling Moreover,combination treatment can inhibited the phosphorylation of Bcr-Abl and Stat5,enhanced histone H3 acetylation and activated Caspase-9 and-3 pathway to induced CML cell apoptosis.ConclusionChidamide can inhibit CML cells growth,induce cell apoptosis and cell cycle arrested.Low dose chidamide combined with IM exhibited a synergistic anti-leukemia effect on TKI-resistant cells,through inhibiting the abnormal activation of ?-catenin signaling and Bcr-Abl activity,enhancing histone H3 acetylation.