Molecular Mechanism Of Promoting The Proliferation Of Acute Myeloid Leukemia Cells Through Negative Regulation Of Notch1 By Annexin A1

Acute myeloid leukemia(AML)is a type of heterogeneous leukemia,which is the result of clonal transformation of hematopoietic precursors through the acquisition of chromosomal rearrangements and multiple gene mutations.At present,the use of anticancer drugs and allogeneic or autologous stem cell transplantation are the main clinical treatment means.Conventional chemotherapy drugs are accompanied by highdose toxic and side effects.And over time,there will be tumor resistance.Therefore,the development of new therapeutic targets and drugs is an urgent scientific problem to be resolved in AML treatmentAnnexin A1(ANXA1),a potent endogenous immunomodulatory protein,is abnormally expressed in a variety of cancer types,while the functional role of ANXA1 in AML remains unclear.Previous studies of our research group have found that ANXA1 is highly expressed in tumor samples from patients with AML and in AML cell lines.By constructing ANXA1 knockdown cell lines,we found that the proliferation of AML cells was significantly inhibited,and the cell cycle was arrested in the G0/G1 phase,without significant effect on cell apoptosis.This paper intends to deeply explore the molecular mechanism of ANXA1 promoting the proliferation of AML cells.We first examined the formyl peptide receptor(FPR)signaling pathway,as well as the protein expression level of some classical proliferation-related signaling pathways,and found no significant changes.The abnormal regulation of the Notch pathway has involved a variety of hematological and solid malignancies,and Notch1 inhibited the proliferation of AML cells in AML.We also detected that the proteins in Notch signaling pathway were more enriched than other signaling pathways in ANXA1 knockdown cell lines through proteomics,and then GSEA analysis revealed the enrichment of Notch signaling pathway,and c Bioportal database indicated that the m RNA levels of ANXA1 and Notch1 were negatively correlated.Next,our previous studies detected that the fluorescence co-localization of Notch1 and ANXA1 in KG1 a and HL60 cells,and further,the fluorescence co-localization no longer existed in ANXA1 knockdown KG1 a and HL60 cell lines.We further used coimmunoprecipitation(co-IP)experiments to reveal that Notch1 can be immunoprecipitated by ANXA1 specific antibody in KG1 a and HL60 cells,but after ANXA1 was knocked down in KG1 a and HL60 cells,Notch1 cannot be immunoprecipitated.Then,through the pull down experiment,we found that the fulllength and C-terminal domains of ANXA1 fused to express the GST tag could bind to Notch1,while its N-terminal domain cannot bind to it.In addition,the Notch1 protein pulled down in ANXA1 knockdown cell strains were more than that of in sh Control cell strains.Furthermore,Forte-bio protein interaction analysis revealed that the 2029-2052 amino acid fragment of Notch1 can interact with GST-ANXA1 fusion protein.We further used the proteasome inhibitor MG132 to treat parental KG1 a and HL60 cells,and the results showed that the protein level of intracellular Notch1 was significantly increased,and this phenomenon did not exist in ANXA1 knockdown KG1 a and HL60 cell lines.In addition,we stably overexpressed and knocked down Notch1 protein in KG1 a and HL60 cells,and found that in Notch1 overexpressing cell lines,the protein level of p15 increased significantly,the cell cycle was arrested in G0/G1 phase,and the cell proliferation was inhibited.We then knocked down the expression of p15 in ANXA1 knockdown KG1 a and HL60 cell lines,and found that the proportion of G0/G1 phase cells decreased significantly,and the cell proliferation viability was also significantly enhanced.Finally,we established an simulate human AML model in B-NDG mice via intravenous injection of KG1a-sh ANXA1 cells and KG1a-sh ANXA1 cells in vivo,and found that the survival of transplanted mice with KG1a-sh ANXA1 cells was significantly prolonged.Blood samples from AML patients and normal subjects were detected by immunocytochemistry and found,ANXA1 was significantly higher in AML patient samples,while Notch1 and p15 were rarely expressed.In normal samples,ANXA1 was almost not expressed,while Notch1 and p15 were highly expressed.In conclusion,based on our previous studies,this paper further revealed that ANXA1 interacts with Notch1 to promote the degradation of Notch1 in a proteasomal dependent manner,thereby down-regulating the expression level of cyclin-dependent kinase inhibitor p15 and promoting the molecular mechanism of AML cell proliferation,and found that the C-terminal domain of ANXA1 and the 2029-2052 amino acid fragment of Notch1 mediate their interactions.Our findings provide the possibility for the clinical development of new drugs based on the ANXA1 as the target for the treatment of AML.