Expression And Role Of CD146 In Adult Philadelphia Chromosome-positive Leukemia And Its Mechanism

Objective1.To analyze the expression,clinical features and prognosis of CD146 in hematological malignancies.2.To study the role of CD 146 in normal hematopoietic process.3.To study the role and mechanism of CD 146 in Philadelphia chromosome(Ph)positive leukemia.Methods1.Multiple methods(flow cytometry,Immunohistochemistry,real-time-PCR)were used to detect the expression of CD 146 in hematologic malignancies and healthy donors in our center.2.We tried to analyze the expression of CD 146 in hematologic malignancies from the RNA-seq database in our center and the online GEO database(Genes Expression Omnibus,GEO).3.Among these 87 de novo Ph-positive acute B-lymphoblastic leukemia(Ph+ B-ALL)patients in our center from January 2009 to June 2019.We analyzed the clinical features of these(Ph+ B-ALL)patients.In this study,we set the cut-off value for CD 146 expression by the software X-Title.We analyzed the effects of CD 146 expression on relapse-free survival(RFS),event-free survival(EFS)and overall survival(OS).4.Detection of CD 146 expression on the surface of hematopoietic cells from healthy donors by flow cytometry.5.To construct conditional knockout CD 146 genotype mice by Crispr/cas9 and cross them with Vav-Cre genotype mice to generate hematopoietic system-specific knockout CD 146 genotype mice.Flow cytometry was used to detect the difference of hematopoietic cells between the CD 146 knockout mice and the control mice.6.We respectively transduced hematopoietic specific knockout CD 146 genotype mice and control mice with BCR/ABL1 to explore the function of CD 146 in Ph positive leukemia progress.7.To construct four cell line-derived xenograft mice models(CDX)used BaF3 cells(BaF3-Venus,BaF3-CD146,BaF3-BA,BaF3-BA-CD146)to explore the role of CD146 overexpressing.8.To investigate the phenotypes of K562 and SUP-B15 cells after RNA silencing of CD 146 and MEG01 cells after RNA overexpression of CD 146.Cell proliferation was detected by cells counting assay and cell colony forming assay.The apoptosis of Ph positive cell lines was detected by flow cytometry.The cell cycle of leukemia cells line was detected by DNA staining and permeabilization solution staining combined with flow cytometry.9.We analyze the transcriptomic and GEO DNA microarray data to explore the mechanism of CD146 in Ph positive leukemia.Results1.The flow and immunohistochemical showed that the percentage of CD 146 positivity was higher in Ph-positive acute B-lymphoblastic leukemia(Ph+ B-ALL)and chronic myeloid leukemia-blastic crisis(CML-BC)patients.The results of real-time PCR(RT-PCR)suggested that the expression of CD 146 was significantly higher in Ph+B-ALL than in other malignant hematological diseases and healthy donors(P<0.05).The expression of CD 146 was significantly higher in CML-BC than in the CML-chronic phase(CML-CP)(P=0.018).The expression of CD 146 in healthy donors’ hematopoietic stem/progenitor cells and mature cells was extremely low,both<2%.2.Acute leukemia RN A-seq database in our center showed that the expression of CD146 in acute lymphoblastic leukemia(ALL)was higher than acute myeloid leukemia(AML)and mixed phenotype acute leukemia(MPAL)(P<0.0001).In ALL,the expression of CD 146 was higher in Ph+B-ALL than in Ph-negative acute B-lymphoblastic leukemia(Ph-B-ALL)(P=0.04),Ph-like acute B-lymphoblastic leukemia(Ph like B-ALL)(P=0.01)and acute T-lymphoblastic leukemia(T-ALL)(P=0.02).CML RNA-seq database showed the expression of CD 146 was slightly higher in CML-BC than in CML-CP(P>0.05).The GEO database showed the expression of CD 146 was higher in adult acute B-lymphoblastic leukemia(B-ALL)than other hematologic malignancies.In ALL,the expression of CD 146 was higher in Ph+B-ALL than in Ph-B-ALL(P=0.01)and T-ALL(P=0.03).The pediatric B-ALL database showed the expression of CD 146 was higher in Ph+B-ALL than in Ph-BALL(P=0.0001).The CML database showed the expression of CD146 was significantly higher in CML-BC than CML-CP(P<0.0001)and CML-accelerated phase(CML-AP)(P=0.0029).3.We enrolled 87 de novo Ph+B-ALL patients in our center.The follow-up deadline is December 31,2021.In these patients,differences were found between these two different groups in terms of platelets(P=0.005)and OS(P=0.03).No significant differences were found between the two different groups including gender,age,leukocyte level,hemoglobin level,bone marrow blast percentage,fusion genes,chromosomal abnormalities,type of TKIs used for induction therapy,RFS and EFS(P>0.05).We found 77 Ph+B-ALL patients in GEO database.In these patients,the OS of patients in the high CD 146 expression group was significantly inferior to that of the low expression group(P=0.04).Besides,the expression of CD 146 was higher in relapse than in initial diagnosis in B-ALL(P=0.01).4.Compared to control mice,the proportion of hematopoietic stem cells(LSK)in CD 146 knockout mice was higher(P=0.01),the proportion of multipotential progenitor cells(MPP)was higher(P=0.03),and the proportion of megakaryocyte-erythroid progenitor cells(MEP)was higher(P=0.01).No significantly difference in the proportion of mature cells between the two genotypes of mice(P>0.05).In B lymphocyte development process,the proportion of progenitor B cells(pro-B)in bone marrow was significantly higher in CD146 knockout mice than in control mice(P=0.0018),and the proportion of immature B cells(Immature-B)was lower than in control mice(P=0.04).No significantly difference in the proportion of precursor B cells(pre-B)and recirc B cells between the two genotypes of mice(P>0.05).No significantly difference in the proportions of total B cells of the spleen and lymph nodes between the two genotypes of mice(P>0.05).Neither CD 146 knockout mice nor control mice developed leukemia.5.CD146 knockout delayed leukemogenesis induced by BCR/ABL1 and prolonged the overall survival in mice transplantation(P<0.05).After transducing with P190,the liver weight of CD 146 knockout mice was lower than in control mice(P=0.02).The immunophenotype at onset showed that the proportion of LSK was lower in CD 146 knockout mice than in control mice(P=0.03);the proportion of MPP was lower than in control mice(P=0.035);and the proportion of short-term hematopoietic stem cells(STHSC)was lower than in control mice(P=0.03).No significantly difference in the proportion of hematopoietic progenitor cells(LK),granulocyte-mononuclear progenitor cells(GMP),common myeloid progenitor cells(CMP)and megakaryocyte-erythroid progenitor cells(MEP)between the two genotypes of mice(P>0.05).However,the mature cells in the two genotypes of mice were more heterogeneous at the onset of the disease,with mostly mixed lineage.6.The survival time of mice both CD 146 and BCR/ABL1 overexpression in BaF3 cells was significantly shorter than that of BCR/ABL1 overexpression alone(P=0.0019).In vitro cell proliferation assay showed that the proliferation ability of both CD146 and BCR/ABL1 overexpression in BaF3 cells was significantly higher than that of BCR/ABL1 overexpression alone(P<0.0001).7.It was showed that CD146 silencing was associated with reduced proliferation of K562 and SUP-B15 cells compared with control groups(P<0.05);CD 146 overexpression was associated with increased proliferation of MEG01 cells compared with control groups(P<0.05).K562-shCD 146 cells treated with imatinib exhibited higher apoptosis rates than control cells(P=0.009),while MEG01-CD 146 cells exhibited lower apoptosis rates than control cells by flow cytometry(P=0.0007).CD 146 silencing was associated with increased in G2M of K562 compared with control groups(P<0.05);CD 146 overexpression was associated with reduced G2M of MEG01 compared with control groups(P<0.05).8.The GO and KEGG analysis of Ph+B-ALL based on CD 146 expression showed that the up-regulated genes were enriched in stem cell development,cell proliferation,the enriched pathways were PI3K-Akt,MAPK,transforming growth factor-β(TGF-β)signaling pathway and cell adhesion molecule(CAM),etc.The down-regulated genes were enriched in B-lymphocyte differentiation and activation,and enriched pathways were hematopoietic cell lineage,etc.GSEA revealed that the pathways of tumor necrosis factorα(TNF-α),TGF-β,P53,IL-2-STAT5,WNT-β-CATENIN and KRAS were enriched.The analysis of Ph+B-ALL GEO database showed the up-regulated genes were enriched in PI3K-Akt signal pathway,extracellular matrix(ECM)receptor interaction pathway,Rapl signal pathway,cAMP signal pathway,TNF signal pathway,etc.Conclusions1.CD 146 was highly expressed in many types of leukemia,including ALL and CML,with the highest expression in ALL,especially in Ph+B-ALL,and was closely correlated with prognosis.During the progression of CML,CD 146 expression is significantly increased.2.The expression of CD 146 in healthy donors’ hematopoietic stem/progenitor cells and mature cells was extremely low.3.The proportion of partial hematopoietic stem/progenitor cells(LSK,MPP,MEP)was increased in CD146 knockout mice.No significantly difference in the proportion of mature cells between the two genotypes of mice.4.CD 146 knockout delayed the process of leukemogenesis induced by BCR/ABL1 and prolonged survival time in mice.CD 146 overexpression accelerated the process of leukemogenesis induced by BCR/ABL1 and shortened survival time in mice.5.CD146 silencing inhibited the proliferation of Ph+leukemia cell lines,promoted the apoptosis rates of the Ph+ leukemia cell line treated by imatinib.CD 146 overexpression promoted the proliferation of Ph+leukemia cell line,inhibited the apoptosis Ph+leukemia cell line.