The Role And Molecular Mechanism Of SUMO Modification Of ERG In The Development Of Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a type of severe hematological malignant tumor caused by canceration of immature myeloid hematopoietic stem cells.It is the most common type of leukemia,with low survival rate and poor prognosis.ERG(ETS-related gene)is a transcription factor of the ETS family.It plays a vital role in regulating the normal function of hematopoietic stem cells and maintaining the number of platelets in peripheral blood in the hematopoietic system,but its role in the pathogenesis of AML and the specific control mechanism is still unclear.SUMOylation is a type of post-translational modification of protein similar to ubiquitination,which plays an important regulatory role in maintaining normal life activities of cells.SUMOylation abnormalities are closely related to the occurrence and development of many human diseases,including hematological tumors.Whether ERG protein is modified by SUMO and the role of ERG SUMOylation in AML is still unknown.This paper has carried out preliminary research around this scientific question.Using the experimental techniques such as vector construction,lentivirus preparation and leukemia cell infection,ERG protein was stably knocked down or overexpressed in leukemia cells.MTT method and cell count were used to detect whether ERG affected the proliferation ability of AML cells.The results showed that compared with the control group,the proliferation rate of ERG knockdown cells was significantly slower,while the proliferation rate of ERG overexpression cells was significantly faster.Next,the effect of ERG on the differentiation of AML cells was further examined,and the differentiation inducer phorbol ester(PMA)was used to induce cell differentiation,which was detected by flow cytometry.The results showed that the expression of CD11b,a marker of surface differentiation of ERG overexpressing cells,was significantly lower than that of control cells,indicating that ERG inhibited the differentiation of AML cells.The above results indicate that ERG promotes the proliferation of AML cells and inhibits the differentiation of cells.We predicted from the database that ERG protein is a potential target protein for SUMOylation.Using protein immunoprecipitation(Co-IP),it was detected that ERG was mainly modified by SUMO2.Immunofluorescence experiments found that ERG and SUMO2 were co-localized in the nucleus.This result further verifies that SUMO2 and ERG combine with each other and accompany with modification process.Next,we confirmed PIAS4 as the major E3 ligase mediating SUMO modification of ERG through co-IP experiments,and the co-localization of ERG and PIAS4 further verified this conclusion.While the specific protease that mediates ERG de-SUMOylation mainly SENP2.Next,we constructed the SUMOylation site mutation vector of ERG,and further confirmed the 37th,74th and 289th lysine residues of ERG protein as the SUMOylation site by protein Co-IP experiment.The above results indicate that ERG mediates its SUMOylation through E3 enzyme PIAS4,and achieves SUMOylation modification through SENP2,and the SUMOylation modification sites of ERG protein include K37,K74 and K289.SUMO modification can regulate the stability of the protein,thereby affecting its function.Appropriate concentration of cycloheximide(CHX)can inhibit the synthesis of cellular proteins.It was found by immunoblotting that the SUMOylation of ERG can inhibit its ubiquitination modification,thereby enhancing its stability.PML can form PML bodies in the nucleus,recruit proteins and coordinately regulate the SUMOylation of proteins.We tested the role of PML in the SUMOylation of ERG through Co-IP experiments,and found that PML can promote the SUMOylation of ERG and Immune fluorescence experiments show that ERG and PML co-localize in the nucleus.AS2O3 has been used clinically APL treatment,the experiment further shows that AS2O3 can reduce the expression of ERG protein in AML cells and promote the expression of differentiation marker CD11b,so ERG may play a role in AS2O3 treatment of leukemia.Finally,we used ERG SUMOylation site mutant lentiviral expression vector to transfect cells,and successfully constructed a SUMOylation site mutant ERG stable transfected cell line.First,through cell counting experiments,it was found that the ERG mutation at the SUMOylation site can significantly inhibit the proliferation of leukemia cells.We also used PMA to induce cell differentiation.Through flow cytometric screening,we found that the expression of CD11b in ERG leukemia cells with SUMO mutations was significantly up-regulated.These results indicate that the SUMO mutation of ERG inhibits the proliferation of leukemia cells and promotes the differentiation of leukemia cells.In conclusion,the role of SUMOylation of ERG in acute myeloid leukemia and its possible molecular mechanism were preliminarily studied in this paper,and it was found that ERG could promote the proliferation of AML cells and inhibit cell differentiation.The E3 enzyme PIAS4 mediates the SUMOylation of ERG protein,while SENP2 specifically removes the sumo-modification of ERG protein.The sites of SUMo-modification of ERG protein include K37,K74 and K289.SUMOylation inhibited the ubiquitination degradation of ERG to improve the stability of ERG,PML promoted the sumoylation of ERG,and AS2O3 inhibited the expression of ERG in AML cells.The SUMO modified mutation of ERG inhibits the proliferation of AML cells and promotes cell differentiation.In this paper,the SUMOylationmechanism of transcription factor ERG was clarified,and the possible role of SUMOylationof ERG in the occurrence and development of AML was preliminarily discussed,providing a new idea for targeted therapy of AML.