Molecular Features And Leukemogenesis Mechanisms Of Acute Myeloid Leukemia With FUS-ERG Fusion Gene

Part 1 The clinical features of acute myeloid leukemia patients with FUS-ERG fusion geneObjective:To systematically examine the clinical and biological characteristics of 7173 acute myeloid leukemia patients diagnosed and treated in the first affiliated hospital of Soochow University from 1998 to 2018.Given the differences of cytogenetics and fusion gene,to depict the clinical characteristics,cytogenetic features and prognosis of patients with different fusion gene.Methods:1.We completed the basic information collection of AML patients in the Hematology Department of the First Affiliated Hospital of Soochow University and then analyzed their clinical and cytogenetic characteristics.2.According to the results of fusion genes and cytogenetics test,the clinical characteristics of AML patients with different fusion gene were retrospectively analyzed.3.Patients with FUS-ERG fusion gene were selected as the research objects,and their clinical and prognostic characteristics were analyzed retrospectively.Results:1.Of all AML patients from the Department of Hematology of the First Affiliated Hospital of Soochow University,42.1%(3022/7173)present normal karyotype,6.5%(463/7173)of the patients don't have karyotype results,2.4%(172/7173)are out of mitotic phase,and 49%(3156/7173)possess clonal chromosome abnormalities:t(15;17)12.3%(884),t(8;21)11.2%(809),inv(16)1.8%(129),and t(9;22)1.4%(103).2.Hematology Department of the First Affiliated Hospital of Soochow University has being conducted genetic tests since 2006 and 3984 newly diagnosed patients has been detected,which showed that 62.6%(2492/3984)of the patients without fusion gene,of which AML1-ETO positive patients occupied 12.6%(500),PML-RARa positive patients occupied 13.2%(527),CBFB-MYH11 positive patients occupied 5.0%(198),BCR-ABL positive patients occupied 1.8%(71)and MLL rearrangement patients occupied 3.8%(152).The rare phenotypes were DEK-NUP214 fusion gene(17),FUS-ERG fusion gene(10),NPM-MLF1 fusion gene(3)and AML1-MTG16 fusion gene(2).We analyzed the clinical characteristics of these patients and found that the age of FUS-ERG positive patients was younger than others,as their median age of diagnosis was 24.5 years old(8-52 years old),Which is statistically different compared with other fusion gene groups(p<0.001).Given only 10 FUS-ERG positive AML patients were detected,these results may be biased.3.We collected 38 patients with FUS-ERG fusion gene,including 34 AML patients,3 ALL patients and a MPAL patient.The statistical results showed that 17.6%(6/34)of the AML patients with FUS-ERG fusion gene died in early stage and one course remission rate was 64.7%(22/34).50%(17/34)of the AML patients with FUS-ERG fusion gene had the chance to receive hematopoietic stem cell transplantation,overall survival rate and event free survival rate of the transplantation group were different from that of the chemotherapy group(P=0.0075,P=0.02 1),with means the transplantation group was relatively better.However,the recurrence rate after transplantation was 41.2%(7/1 7),and the prognosis of these relapse patients was quite poor.Conclusion:1.In our study,the cytogenetic characteristics of AML patients in the Department of Hematology from the First Affiliated Hospital of Soochow University are similar to those reported in other country.42.1%AML patients have normal karyotypes,while 49%have abnormal karyotypes,of which t(15;17),t(8;21),inv(16),t(9;22)account for 12.3%,11.2%,1.8%,1.4%.2.In the Hematology Department of the First Affiliated Hospital of Soochow University,the examination of fusion gene has been conducted since 2006.There were 3984 patients were detected,of which 60%patients were fusion gene negative,12.6%were AML 1-ETO positive,13.2%were PML-RARa positive,5.0%were CBFB-MYH11 positive,1.8%were BCR-ABL positive,3.8%were MLL arrangement.The rare fusion genes were DEK-NUP214(17),FUS-ERG(10),NPM-MLF1(3)and AML1-MTG16(2).We analyzed the clinical characteristics of these patients,we found that FUS-ERG positive AML patients age were younger,as the median age of onset was 24.5 years old(8-52 years old),meanwhile the age of other fusion genomes was statistically different(P<0.001).3.38 FUS-ERG fusion gene positive patients were studied including 34 AML patients indicating that 0.47%AML patients had FUS-ERG fusion gene,3 ALL patients and 1 MPAL patients.Moreover,of all FUS-ERG positive AML patients(FUS-ERG+AML patients),6 patients died in early stage;17 patients received transplantation and 7 patients relapse after transplantation.Part 2 Molecular features of acute myeloid leukemia patients with FUS-ERG fusion geneObjective:To investigate molecular features and pathogenic mechanism of FUS-ERG+AML patients diagnosed in the Hematology Department of the First Affiliated Hospital of Soochow University.Methods1.Cytogenetics and molecular biology of AML patients diagnosed by Hematology Department of the First Affiliated Hospital of Soochow University were analyzed.FUS-ERG+AML patients were selected as the research objects,and the DNA or RNA samples of FUS-ERG+AML patients were collected.2.The mutation features of FUS-ERG+AML patients were depicted by the whole exon sequencing technology,and the molecular abnormalities were examined to induce the possible characteristics of concomitant mutations.3.The gene expression and fusion gene subtype of ten FUS-ERG+ AML patients were tested by the whole transcriptome sequencing technology.The gene expression characteristics of F FUS-ERG+ AML patients and their difference with other AML patients were studied,and the possible pathogenicity of FUS-ERG fusion gene was explored.4.The targeted next-generation sequencing technolgy were performed on 13 cases of FUS-ERG fusion gene positive AML patients to compare the single nucleotide variations,which contained 51 blood tumor related genes mutation.Results:1.DNA samples of bone marrow cells from 10 FUS-ERG+ AML patients were collected for whole exon sequencing.The results showed that mutation including RPL14,MUC4,LNP1,CAMKK2,FADS6 and NBPFL4 were the mutations with more than 6 times in these ten patients,and no common gene mutation AML was detected.2.The whole transcriptome sequencing technongy were performed on ten newly diagnosed AML patients' bone marrow cell samples.Moreover,pathways that significant in malignance including PI3K-Akt and Rap1 signal transduction pathway were extremely up-regulated in FUS-ERG+ AML patients.Results from the GSEA analysis showed that these pathways were mainly related to metabolism:anti-cyclic acid and aldehyde metabolism pathway,cysteine and methionine metabolism pathway and histidine metabolism pathway.3.The Ion torrent S5 sequencing technology were performed on bone marrow cells'DNA samples collected from 13 newly diagnosed FUS-ERG+ AML patients.There were 20 gene mutation detected:PTPN11 activated missene mutation was most common with a mutation frequency of 30.8%(4/13),followed by WT1 mutation.It was speculated that PTPN11 mutation may play an important role in the occurrence and progress of the disease.Conclusion:Resorting to the GSEA analysis technology,it is concluded that the main differences between FUS-ERG+ AML patients and other AML patients are the metabolism related pathways.20 gene mutation are detected,of which the most frequent mutation is PTPN11 activate mutation(4/13)and it is speculated to play an important role in the progress of the disease.Part 3 The pathogenesis of FUS-ERG fusion gene inducing leukemiaObjective:To elucidate the pathogenesis of FUS-ERG fusion gene in vitro and in vivo.Methods:1.Using PCR Cloning Technology,we constructed FUS-ERG fusion gene overexpression vector,then packaged lentivirus,and infected HL-60 and 32D cells respectively,afterwards,analyzed the effect of FUS-ERG on cell proliferation,apoptosis and differentiation.2.We constructed Retrovirus Expression Vector to transfect cord blood CD34 positive cells,then selected CD34+GFP+samples to carry out primary cell clone formation and liquid culture myeloid differentiation experiment to observe the difference of clone number and type,and the influence of primary cell differentiation ability.3.We took advantage of lentivirus overexpression vector to overexpress mutation including PTPN11-WT,PTPN11-D61V,PTPN11-E76K mutants in 32D cells to study the effect of PTPN11 wild type and mutant protein on cell proliferation and differentiation.4.After construction of Lentivirus Expression Vector,we transfected Babl/c mouse bone marrow CD117+ cells,then transplanted these cells into the same Babl/c mouse bone marrow and observed the incidence of mice.5.We constructed FUS-ERG fusion gene knock-in mice with VAV-1 as the promoter,propagated and purified,and observed the incidence of mice.Results:1.In FUS-ERG fusion gene knock-in human leukemia cell line experiments,the results showed that FUS-ERG fusion gene could increase proliferation and decrease apoptosis of HL-60 cells and the differentiation of 32D cells were blocked after transformation.2.The CD34+ cells were isolated from cord blood and infected with FUS-ERG and vector virus.The results showed that FUS-ERG fusion gene could increase the ability of cord blood cell clone formation,block its differentiation by hindering in the early stage of medullary progenitor and granulocyte formation.3.FUS-ERG fusion gene cooperated with PTPN11 mutation in vitro can significantly increase the proliferation ability of 3 2D cells and block the myeloid differentiation,activating of PI3K-Akt signal pathway,and promoted the downregulation of downstream HIF-la,IKKa and CASP9 genes.4.In constructing bone marrow transplantation model experiment,incidence of mice after transplantation was observed that all FUS-ERG fusion gene carried mice died while one mouse in vector group died and there was a significant statistical difference in the overall survival rate between the two groups(P=0.075).Results from flow cytometry and HE stain of pathological sections indicated that FUS-ERG fusion gene alone could lead to T lymphocytic and/or erythrocytic leukemia in mice,which was similar to the effect of FUS-ERG fusion gene in clinical patients.Conclusion:1.FUS-ERG fusion gene can increase proliferation and decrease apoptosis of HL-60 cells,meanwhile blocking the differentiation of 32D cells.2.FUS-ERG fusion gene can facilitate the ability of cord blood cells to proliferate but block the differentiation in the early stage of medullary progenitor cell and granulocyte.3.FUS-ERG fusion gene cooperates with PTPN11 mutation to accelerate the proliferation and differentiation of 32D cells,activates the PI3K-Akt signal pathway,and down-regulates the downstream HIF-1a,IKKa and CASP9 gene.4.FUS-ERG fusion gene alone can lead to the leukemia development in mice,but the occurrence of mouse diseases is diverse,which is similar to the clinical condition.