Effect Of Histone Deacetylase Inhibitor In NOTCH1 High-expressed Acute T Cell Leukemia And Its Underlying Mechanism

Objective T-cell acute lymphoblastic leukemia(T-ALL)is a hematological malignancy with high malignant degree and clinical progress rapidly.It is easy to relapse after remission of conventional chemotherapy.NOTCH1 activating mutations are found in 60%～70% of patients in T-ALL.At present,gamma-secretase inhibitor(GSI)targeting Notch1 signaling pathway is not effective.Histone deacetylases(HDACs)are involved in the regulation of downstream genes of Notch1 signaling pathway and its inhibitors have been successfully applied in other types of tumors,thus HDAC inhibitors(HDACis)are expected to be a new therapeutic option for T-ALL.Our study aims to analyze the expression of HDAC subtypes in T-ALL by searching Oncomine,explore the effects and its underlying mechanism of pan-and class I selective HDACi on NOTCH1 high expressed T-ALL cell lines,hoping for providing preliminary research data for T-ALL epigenetic treatment and combining HDACis with chemotherapy in future.Methods 1.The m RNA expression of HDAC1～11 in T-ALL were analyzed by searching in Oncomine;2.The expression of NOTCH1 and HDAC1～11 in common T-ALL cell lines were screened by PCR and/or RTq PCR.3.CCK-8 assay was used to determine the effects of pan-HDACi panobinostat and class I selective HDACi chidamide on the proliferation of T-ALL cell lines with different NOTCH1 expression levels;4.Flow cytometry was used to detect cell apoptosis and cell cycle;5.Western Blot was used to detect the proteins expression levels of histone H3K9 acetylation,apoptosis,Notch1 signaling pathway,C-MYC and so on.Result 1.The data of Oncomine showed that T-ALL patients overexpressed class I and class II HDACs;2.Class I and Class II HDAC were expressed in Molt-4 and THP-1 cell lines,while Class I and Class IIa HDAC were expressed in A3.The expression levels of HDAC subtypes were different in each cell line;3.Both panobinostat and chidamide inhibited the proliferation of A3 and Molt-4 cells in a time-and dosedependent manner,and induced apoptosis and G1-phase cell cycle arrest.The concentration of panobinostat induced apoptosis and cell cycle arrest in THP-1 was about 10 times higher than that in A3 and Molt-4 cells;4.Both panobinostat and chidamide can up-regulate the level of histone H3K9 acetylation in A3,Molt-4,THP-1.Panobinostat can down-regulate ICN1 and C-MYC proteins at a low concentration.Chidamide inhibited Notch1 pathway slightly,but regulated the levels of apoptosis proteins,down-regulated p-ERK1/2 protein levels.Conclusion 1.Pan-HDACi panobinostat can effectively inhibit the cell proliferation of NOTCH1-dependent T-ALL cell line A3 and Molt-4 in a time-and dose-dependent manner,and induce apoptosis and G1 phase cell cycle arrest.It may be related to the simultaneous role of histone H3K9 acetylation and C-MYC which is the non-histone substrate of HDACs in inhibiting the activation of Notch1 signaling pathway;2.Class I selective HDACi chidamide can effectively inhibit the cell proliferation of A3 and Molt-4,increase the level of histone H3K9 acetylation,but a weak effect on inhibiting Notch1 signaling pathway;The underlying mechanism of mitochondria apoptosis induced by chidamide in A3 and Molt-4 cells may be related to the inhibition of ERK/MAPK signaling pathway.