Role And Mechanism Of FEV In Homing And Expansion Of Acute Myeloid Leukemia Cells

BackgroundAcute myeloid leukemia(AML)is a heterogeneous malignant disease caused by abnormal cloning of hematopoietic tissue,and is the most common acute hematologic malignant in adults.Leukemia stem cells(LSCs)are the main cause of drug resistance and relapse of leukemia.Fifth Ewing Variant(FEV)is a transcription factor,which was previously found to be expressed in LSCs but not in normal hematopoietic stem cells,and specifically regulate LSCs.However,the role and mechanism of FEV in AML have not been elucidated.ObjectiveTo elucidate the function and mechanism of FEV in AML cells,and explore whether the FEV-related signal pathways can be candidate targets for AML.MethodsReal-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of FEV in bone marrow mononuclear cells(MNCs)samples from 69 AML patients and 14 healthy donors,which was validated in vizome database.The expression of FEV in AML cell lines was also detected.In cell lines highly expressed FEV,the shRNA was used to knocked down the FEV expression.The clone formation,proliferation,cell cycle and apoptosis in vitro,the survival of mice and leukemia cell infiltration in vivo,and homing,migration and adhesion were detected.RNA sequencing was performed in iFEV/NSC MV4-11 cells to find the downstream target genes of FEV.Target gene was rescued in iFEV MV4-11 cells to detected the ability of homing and proliferation.The chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays were performed to investigate whether FEV directly regulates the transcription.Using natalizumab(NZM)to block the function of target protein,and using shRNA to knockdown target gene expression and then detected whether the expansion and homing of AML cells was affected.The MNCs and LSCs from AML patients were treated with NZM and then detected the clone formation,migration and adhesion assays.Results1.The expression of FEV in bone marrow MNCs from AML patients was significantly higher than that in normal bone marrow(AML vs.normal:0.2119±0.0601 vs.0.0000±0.0000,P<0.05),which was consistent with vizome database(AML vs.normal:0.4065±0.2626 vs.0.0016±0.0015,P<0.01).In AML samples,the mRNA level of FEV was significantly higher in the relapsed samples than that in the diagnostic samples(relapse vs.diagnosis:3.328±2.744 vs.0.212±0.061,P<0.001).In FEV highly expressed MV4-11,THP-1 and KG-1 cells,knockdown FEV markedly reduced the colony-forming cells(CFCs,iFEV vs.NSC:1.333±0.577 vs.30.67±3.055,P<0.001),and elevated the fraction of G1 phase(iFEV vs.NSC:58.73±0.97 vs.51.43±0.57,P<0:01).The mice receiving iFEV cells had extensively prolonged survival time compared with their NSC counterparts(iFEV vs.NSC:26 days vs.16 days,P<0.001).Homing(iFEV vs.NSC:0.010±0.003 vs.0.028±0.002,P<0.01),migration(iFEV vs.NSC:6.15±1.179 vs.66.95±10.93,P<0.001)and adhesion(iFEV vs.NSC:10.09±3.171 vs.69.38±13.72,P<0.001)were significantly inhibited.2.Gene transcriptome examination of iFEV MV4-11 cells indicated that integrin signaling pathway was significantly inhibited(P<0.01).A significant correlation between FEV expression and ITGA4(integrin α4)expression in AML samples was observed(r=0.397,P<0.01).Overexpression of ITGA4 in FEV knockdown cells resulted in increased CFCs(iFEV+ITGA4 vs.iFEV+vector:87.67±4.26 vs.29.33±3.18,P<0.001)and reduced proportion of G1 phase(iFEV+ITGA4 vs.iFEV+vector:54.93±0.48 vs.65.07±0.27,P<0.01).The mice who received ITGA4-expressed iFEV cells showed a remarkably reduced survival time(iFEV+ITGA4 vs.iFEV+vector:23 days vs.28 days,P<0.01)and increasing homing cells(iFEV+ITGA4 vs.iFEV+vector:0.023±0.001 vs.0.008±0.001,P<0.001).The ChIP assay with MV4-11 leukemic cells demonstrated that FEV could bind to both regions.Luciferase reporter assay suggested that FEV positively regulated wild-type ITGA4 expression in a dose-dependent manner.3.In knockdown or blocked ITGA4 MV4-11 cell,the CFCs(sh-ITGA4 vs.NSC:31.11±2.94 vs.82.67±5.29,P<0.05),migrated cells(sh-ITGA4 vs.NSC:28.18±4.90 vs.76.49±0.79,P<0.05)and adhesion cells(sh-ITGA4 vs.NSC:19.17±2.04 vs.59.11±5.79,P<0.01)was decreased,the mice who received sh-ITGA4 cells showed prolonged survival time(sh-ITGA4 vs.NSC:28 days vs.19 days,P<0.01),and homing cells was reduced(sh-ITGA4 vs.NSC:0.008±0.001 vs.0.016±0.001,P<0.01).In the bone marrow MNCs from AML patients,the ITGA4 antibody NZM(Natalizumab)significantly inhibited clone formation(NZM vs.IgG:28.08±18.85 vs.75.83±19.95,P<0.05),migration(NZM vs.IgG:20.50±9.41 vs.50.24±4.31,P<0.05)and adhesion(NZM vs.IgG:50.73±19.84 vs.100.80±26.17,P<0.01)of FEV+groups,but in FEV-groups the clone formation(NZM vs.IgG:41.37±13.32 vs.39.85±11.79,P≥0.05),migration(NZM vs.IgG:62.41±6.64 vs.64.45±6.78,P≥0.05)and adhesion(NZM vs.IgG:27.26±6.61 vs.28.40±7.49,P≥0.05)were not significantly affected.The clone formation,migration and adhesion were inhibited by NZM in sample from FEV+AML both at diagnosis and at relapse stage.CD34+ LSCs were sorted from AML cells,and the clone formation(NZM vs.IgG:32.33±17.40 vs.56.47±18.15,P<0.05),migration(NZM vs.IgG:31.11±2.82 vs.46.33±4.81,P<0.05)and adhesion(NZM vs.IgG:27.44±9.28 vs.82.33±25.02,P<0.05)were inhibited by NZM in FEV+group,which was in consistent with MNCs.ConclusionThe expression of FEV in AML was significantly higher than that in normal bone marrow MNCs,and was higher at relapsed stage.FEV could directly regulate the transcription of ITGA4 and modulate the homing and expansion of AML cells,which affecting the progression of AML.In FEV+AML MNCs and LSCs,the clone formation,migration and adhesion could be inhibited by NZM.ITGA4 could be a novel target for AML therapy.