| Objective: To explore the molecular genetic characteristics of adult T cell acute lymphoblastic leukemia(T-ALL),and explore mechanism of chidamide on T-ALL.Methods: 1.The clinical features and prognosis of 97 adult T-ALL cases were retrospectively analyzed.Targeted next-generation sequencing(NGS)was performed on the newly diagnosed adult 45 T-ALL patients to analyze the gene mutation profile and the difference between ETP-ALL and non-ETP-ALL,and analyze associated factors and prognosis of mutations.2.The cell proliferation was detected by CCK-8,cell cycle and apoptosis by flow cytometry.The effect of chidamide on the NOTCH1 signal pathway was measured by Western blot(WB)and real-time quantitative PCR(q PCR),the mechanism of apoptosis pathway by WB,q PCR and co-immunoprecipitation.Results:A total of 97 adult T-ALL patients were enrolled in this study,of which 46 were ETP-ALL(including 6 near-ETP-ALL)and 51 were non-ETP-ALL,with the median follow-up time was 29(0.3-57)months.Compared with non-ETP-ALL,ETP-ALL was associated with older age,lower white blood cell count(WBC)and lower level of lactate dehydrogenase(LDH),and with lower incidence of mediastinal masses and central nervous system leukemia(CNSL).The complete remission(CR)rate of T-ALL was 53.24%,and the CR rate of ETP-ALL was significantly lower than that of non-ETP-ALL(37.14% vs.66.67%,p=0.0123).The 4-year event-free survival(EFS),relapse-free survival(RFS),and overall survival(OS)of 97 T-ALL patients were19.84±4.86%,39.22±7.39% and 42.38±8.84%,respectively,although ETP-ALL had a trend of lower survival,there was no significant statistical difference.Allogeneic hematopoietic stem cell transplantation(allo-HSCT)could significantly improve the survival of T-ALL,compared with patients who did not receive allo-HSCT,the hazard ratios(HRs)of EFS,RFS and OS were 0.3224(95% CI 0.1858-0.5592,p<0.0001),0.2064(95% CI 0.09306-0.4579,p=0.0001)and 0.1768(95% CI 0.08334-0.375,p<0.0001).We explored the gene mutation profile of the Chinese adult T-ALL patients.NGS was performed on the specimens of 45 T-ALL patients,of which 25 were ETP-ALL(including 3 near-ETP-ALL)and 20 were non-ETP-ALL,and at least 1 mutated gene was detected in all patients.The genes with higher mutation incidences were NOTCH1(57.78%),ETV6(24.44%),NRAS(22.22%),PHF6(20%),JAK3(17.78%),DNMT3A(17.78%),EZH2(15.56%),FBXW7(15.56%),IDH2(13.33%)and RUNX1(13.33%).The mutation incidences of NOTCH1 pathway(NOTCH1,FBXW7),signal transduction(JAK1,JAK2,JAK3,IL7 R,SH2B3,STATB5,BRAF,NRAS,KRAS,FLT3,PTEN),DNA methylation(DNMT3A,IDH1,IDH2),modification of histones(EZH2,EP300,CREBBP)and hematopoietic development(RUNX1,ETV6,GATA3,IKZF1,BCL11 B,EBF1)in T-ALL cases were 62.22%,64.44%,24.44%,26.66% and 51.11%,respectively.The proportion of ETP-ALL with NOTCH1 pathway mutations was 48%,which was significantly lower than 85% of non-ETP-ALL(p=0.0133).In T-ALL,NOTCH1 pathway mutations were associated with higher WBC(p=0.0328),DNA methylation and hematopoietic development mutations were associated with older age(p<0.0001 and 0.0043).In T-ALL patients,mutated SH2B3 was accompanied with mutated IL7R(p=0.0289),NRAS and IDH2(p=0.0011),EZH2 and ETV6(p=0.0061),NRAS and DNA methylation(p=0.0006),DNMT3 A and hematopoietic development(p=0.047).Mutations of modification of histones were mutually exclusive with mutated NRAS(p=0.0423),and signal transduction and hematopoietic development were mutually exclusive(p=0.0287).Adult T-ALL with NOTCH1/FBXW7,JAK3 and EZH2 mutations had lower OS than WT,HRs were 2.921(95% CI 1.135-7.514,p=0.0262),3.601(95% CI1.02-12.71),P=0.0464)and 13.75(95% CI 2.514-75.18,p=0.0025).The RFS of T-ALL with hematopoietic development mutations was significantly higher than that of WT,HR=0.2408(95% CI 0.07585-0.7646,p=0.0157).ETP-ALL patients with NOTCH1/FBXW7,JAK3 and EZH2 mutations had poor survival outcomes.ETP-ALL patients with NOTCH1/FBXW7 mutations had worse prognosis on EFS,RFS and OS,HRs were 2.838(95% CI 1.049-7.674,p=0.0399),6.342(95% CI1.385-29.03,p= 0.0173)and 5.193(95% CI 1.556-17.33,p = 0.0074),respectively.ETP-ALL patients with mutated JAK3 and EZH2,and the corresponding signal transduction and modification of histones mutations had lower OS,HRs were 4.38(95% CI 1.015-18.9,p=0.0478),14.69(95% CI 2.563-84.13,p=0.0026),and 3.409(95% CI 1.003-11.59,p=0.0495),12.22(95% CI 2.206-67.64,p=0.0042).In non-ETP-ALL,patients with hematopoietic development mutations had better RFS,HR=0.1253(95% CI 0.02108-0.745,p=0.0224).The adult T-ALL had a dismal survival outcome and the NGS data suggested some genetic mutations are associated with poor prognosis.It was urgent to explore and discover some new approaches for T-ALL.Therefore,the mechanism of chidamide on T-ALL was explored.The inhibitory rate of chidamide on the proliferation of T-ALL cell lines Jurkat and Molt4 was in dose-dependent manner,the IC50 at 48 hours was 2.22±0.27μM and 1.32±0.13μM,respectively.The cell cycle was arrested in G2/M.The activated NOCTH1 protein(ICN1)was found to be down-regulated by chidamide,the expression of c-MYC protein was inhibited.The phosphorylation levels of AKT,4E-BP1 and p70 S6 K were not down-regulated significantly.Chidamide induced apoptosis of Jurkat and Molt4 cells by up-regulating the proapoptotic protein Bim,down-regulating the antiapoptotic protein Bcl-XL,and increasing the binding between Bim protein and Bcl-2 protein,and the apoptosis rate was in dose-dependent manner.The combination index(CI)of chidamide and Bcl-2inhibitor venetoclax in inhibiting cell proliferation of Jurkat and Molt4 was less than 1,suggesting that the two drugs have a synergistic effect.A low dose of venetoclax(0.1μM)could significantly increase the apoptosis by chidamide on T-ALL cell lines.Conclusion:1.The incidence of adult ETP-ALL was higher,and the clinical characteristics were significantly different from those of non-ETP-ALL.Adult T-ALL had a low CR rate and poor prognosis.Allo-HSCT significantly improved the survival of adult T-ALL2.The gene mutation profile of Chinese adult T-ALL was reported in this study.The genes with higher mutation incidences were NOTCH1(57.78%),ETV6(24.44%),NRAS(22.22%),PHF6(20%),JAK3(17.78%),DNMT3A(17.78%),EZH2(15.56%),FBXW7(15.56%),IDH2(13.33%)and RUNX1(13.33%).Except for the NOCTH1 pathway,which had a higher mutation incidence in non-ETP-ALL,there was no significant difference between ETP-ALL and non-ETP-ALL in other pathways.3.Adult T-ALL patients with NOCTH1/FBXW7,JAK3 and EZH2 mutations had shorter OS.ETP-ALL patients with NOTCH1/FBXW7 mutations had worse prognosis on EFS,RFS and OS.The ETP-ALL patients with JAK3 and EZH2 mutations,and their corresponding signal transduction and modification of histones mutations had worse OS.4.We reported the mechanism of chidamide on T-ALL for the first time.Chidamide inhibited the proliferation of T-ALL cells,and the cell cycle was arrested in G2/M phase.Chidamide inhibited the NOTCH1-MYC pathway.And chidamide induced T-ALL cells apoptosis by up-regulating the proapoptotic protein Bim,down-regulating the antiapoptotic protein Bcl-XL,and increasing the binding between Bim protein and Bcl-2 protein.Chidamide and venetoclax had a synergistic effect on inhibiting cell proliferation,and induced increased cell apoptosis significantly. |
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