Precise Diagnosis Of Fgfr1 Rearrangement And Pathogenetic Mechanism Study Of Fusion Gene Involving The FGFR1

Objective:1.To explore the incidence of 8p11/FGFR1 rearrangement by fluorescent in situ hybridization(FISH),identify the partner gene and the precise site of breakage mutation,analyze its clinical and other cytogenetic features.To decipher disease-driving genes responsible for the pathogenesis of this entity,we performed targeted NGS using a custom-designed gene panel.2.To provide more considerable insight into the mechanisms of FGFR1-related fusion genes in leukemia through molecular cloning and functional study of the fusion gene by the chromosome translocation der(8)inv(8)(p11q22)in a acute myeloid leukemia patient.For comparison,ZNF198-FGFR1 fusion was also cloned and studied.3.To investigate the effects of voxtalisib and arsenic trioxide in the KG-1 cell line.Methods:1.Patients harboring chromosome 8p11 abnormalities were studied by FISH.Clinical and laboratory features were analyzed.Different FGFR1-partner genes and the precise site of breakage mutation were identified by array-based comparative genomic hybridization(Array-CGH),reverse transcription-polymerase chain reaction(RT-PCR)and whole-transcriptome sequencing of mononuclear cells.Novel genetic variants were identified by next-generation sequencing(NGS).2.RT-PCR was used to detect the RBPMS-FGFR1 fusion transcript and to clone the full-length coding sequence of the fusion transcript which was afterwards inserted to a transfer plasmid-LV5.Full length ZNFl98-FGFRl was cloned by overlap extension PCR method.The fusion genes were transfected into BaF3 cells by DNA-calucium phosphate.Western blot was used to determine the fusion protein.Growth rate of the Ba F3 cells was detected by the cell counting kit 8(CCK8)and growth curve was made according to it.Growth ability of BaF3 cells in the methylcellulose with or without IL-3 was investigated to evaluate the malignant transformation ability of the fusion gene.Signal pathways were assessed by western blot.3.An in vitro adenosine triphosphate based chemotherapy response assay(ATP-CRA)was performed to evaluate the effects of a set of agents in KG-1 cells.Cells were examined using a water-soluble tetrazolium salt assay via Cell Counting kit-8.Cell apoptosis was analyzed using a flowcytometer.Quantitative real-time PCR(qRT-PCR)was performed to detect the expression of apoptosis-related genes.We quantified the number of KG-1 cell colonies formed on methylcellulose plated after treatment.Rabbit anti-AKT,p-AKT,?-actin,Bcl2 antibodies were used to evaluate the activity of related proteins by western blot.Results:1.Clinical and cytogenetic features of patients with FGFR1 alterationsFGFR1 rearrangements were observed in 8 patients(32%8/25).6 patients were men and 2 were women,with male significantly predominant,and the median age was 40 years(range 9–65years).All patients had leukocytosis,the mean white blood cell(WBC)count at diagnosis was 42.16×10~9/L(range 21.69–76.0×10~9/L)and eosinophilia(>6%of leukocytes)was noted in 62.5%(5/8)of the patients.Five different FGFR1-partner genes were identified,including CEP110(9q33,n=3),ZMYM2(13q12,n=2),MYO18A(17q23,n=1),and BCR(22q11,n=1).A new chromosomal translocation,inv(8)(p11;q12),which results in a novel fusion gene formed between the 5'region of RNA-binding protein with multiple splicing(RBPMS)and the 3'region of FGFR1 was identified,genomic studies to show the precise site of breakage mutation for patient 5 indicated its gene had resulted in the FGFR1 frame shift.RUNX1 was identified as the sole mutated transcription factor(66.7%,4/6 patients)by NGS in this study.2.Molecular cloning and leukemogenesis studies of fusion genes involving the FGFR1 geneIn this study,we identified a patient with leukemia harboring der(8)inv(8)(p11q22),which results in an out-frame fusion of FGFR1 to RNA binding protein with multiple splicing(RBPMS).RT-PCR was used to detect the fusion transcript RBPMS-FGFR1 and ZNF198-FGFR1,and to clone the full-length coding sequence of the fusion transcript which was afterwards inserted to a transfer plasmid-LV5.Both fusion genes were transfected into BaF3 cells by DNA-calucium.RBPMS-FGFR1 has two different fusion transcripts.ZNF198-FGFR1 fusion protein could make Ba F3 cells grow well without IL3,but not RBPMS-FGFR1.The overexpression of ZNF198-FGFR1 and RBPMS-FGFR1 can promote the phosphorylation activity of Akt,STAT3,and STAT5,and inhibit the phosphorylation activity of AMPK,but have no effects on ERK1/2 protein.Arsenic trioxide(As2O3)induces remarkable apoptosis and shows superior effects to compared agents.3.The effects of Voxtalisib and Arsenic trioxide(As2O3)in KG-1 cellsATP-CRA data were successfully generated from 13 agents tested.Cell viabilitywas measured by CCK-8 method.Voxtalisib and As2O3 alone demonstrated concentration-dependent inhibition of the cell viability of the KG-1 cells.At high doses,Both Voxtalisib and As2O3 resulted in>70%inhibition.We quantified the number of KG-1 cell colonies formed on methylcellulose plates after treatment.Compared with control,voxtalisib and As2O3 alone significantly inhibit the clonogenic activity of KG-1cells(P<0.05),whereas the combination of voxtalisib and As2O3 significantly inhibited the clonogenic activity of KG-1 cells compared with voxtalisib and As2O3 alone or control(P<0.05).Apoptosis increased after treatment with voxtalisib and As2O3 on KG-1 cells.The expression of apoptosis-related genes,including caspase-3,Bcl-2 was detected by Q-PCR.Both voxtalisib and As2O3 inhibited the mRNA expression of caspase-3 and Bcl-2(P<0.05).Western blot showed the phosphorylation levels PI3K and Akt were also suppressed.Conclusion:1.All patients with EMS had leukocytosis,Bone marrow(BM)aspirate revealedhypercellular,and eosinophilia was noted in most cases.The frequent FGFR1-partner geneswereidentified,includingCEP110(9q33,n=3),ZMYM2(13q12,n=2),MYO18A(17q23,n=1),and BCR(22q11,n=1).RUNX1 was identified as the sole mutated transcription factor(66.7%,4/6 patients)by NGS,strong higher than other series such as AML and myelodysplastic syndrome(MDS).All CEP110-FGFR1-positive patients habored RUNX1 mutations.The high frequency of combined RUNX1 mutation and FGFR1 rearrangement suggests that these 2 aberrations may cooperate as drivers in leukemogenesis.2.We identified a novel chromosome 8p11 abnormalities,der(8)inv(8)(p11q22),which results in an out-frame fusion of FGFR1 to RNA binding protein with multiple splicing(RBPMS).RT-PCR was used to detect the RBPMS-FGFR1 fusion transcript and to clone the full-length coding sequence of the ZNF198-FGFR1 and RBPMS-FGFR1 fusion transcripts which was afterwards inserted to a transfer plasmid-LV5.The fusion gene was transfected into Ba F3 cells by DNA-calucium phosphate.Our data showed that ZNF198-FGFR1 made IL3-independent growth to BaF3 cells,but not RBPMS-FGFR1.The overexpression of ZNF198-FGFR1 and RBPMS-FGFR1 can promote the phosphorylation activity of Akt,STAT3,and STAT5,and inhibit the phosphorylation activity of AMPK,but both genes have no effects on ERK1/2 protein.Arsenic trioxide(As2O3)induces remarkble apoptosis and shows superior effects to compared agents.Further study was required to explore the mechanism.3.Both the dual PI3K/Akt inhibitors voxtalisib and(As2O3)showed superior effects in KG-1 cells.Regulating the expression of Bcl-2 and Caspase3 and inhibiting of PI3K/Akt signal pathway may contribute KG-1 cell apptosis.