Study On The Mechanism Of SAE1 Promoting The Cellular Proliferation Via SUMOylation Pathway And Drug Intervention Treatment In Multiple Myeloma

Background and ObjectiveMultiple myeloma(MM)is a hematological malignancy with clonal proliferation of plasma cells,which is characterized by secreting a large amount of monoclonal immunoglobulins and accompanied by osteolytic lesions,hypercalcemia,anemia and other complications.Patients treated with existing drugs are inevitably relapsed due to drug-resistant,including those most who can’t benefit from monoclonal antibodies and Chimeric antigen receptor T cells(CAR-T).Therefore,it is urgent to develop new drugs and discover new targets.This project screened out SAE1(Small Ubiquitin-related Modifier Activating Enzyme Subunit 1)as a potential molecular targeting colchicine based on protein chip and gene expression profiling library.Lentivirustransfected MM cell lines stably expressing SAE1-OE or shRNA(SAE-KD)were constructed in both ARP1 and H929 cells,and their functions were verified by cell proliferation,cycle apoptosis and other related phenotype experiments.Then,high-throughput mass spectrometry coupled with coimmuneprecipitaition(Co-IP/MS)was used to screen the possible proteins and donwstream signaling pathways regulated by SAE1.Finally,the pharmacological effects of colchicine in the treatment of myeloma were studied in animal models.This study intends to provide new molecular targets and drug intervention schemes for the diagnosis and treatment of MM.Methods1.By labeling colcemid as a probe and co-incubating with protein chip(HuProt_V4),a new potential molecular target SAE1 was initially screened out,and further verified by microthermophoresis assay(MST).Gene library was used to verify the correlation between SAE1 gene expression and MM patients.2.Lentiviral transfection system was used to construct SAE1 overexpression(SAE1-OE)and shRNA mediated knockdown(SAE1-KD)MM cell lines,and the transfection efficiency was verified by fluorescence microscopy and WB(Western blot)experiments.CCK-8 proliferation assay and soft agar cloning assay were used to detect the proliferation of SAE1-OE cells and SAE1-KD cells.Flow cytomytry of apoptosis and Brdu/7-ADD staining assays were used to detect the effect of overexpression and knockdown of SAE1 on cell mitotic cycle and apoptosis.3.Co-IP/MS experiments were performed in ARP1 WT and SAE1-OE cells using SAE1 and SUMO1 antibodies to search for potential downstream targets regulated by SAE1;Co-IP experiments verified the interaction between SAE1 and target proteins.4.Colchicine dosing treatment(20 nM)MM cells;molecular docking to search for potential binding sites of colchicine and SAE1;overlapping PCR technology to construct binding site mutant plasmids;Expression and purification of SAE1 wild-type and point mutant proteins;MST experiment verified the ability of colchicine to bind to SAE1 wild-type and point mutant proteins5.Three different mouse models:5TMM3VT myeloma model,immunodeficient mouse MM cell xenograft tumor model,and human patient-derived tumor xenograft model,were used to confirmed the therapeutic effect of colchicine targeting SAE1.Results1.The incubation of protein with chip labeled Demecolcine probe showed that colchicine could bind to SAE1;MST experiment demonstrated that both colchicine and its derivative Demecolcine had interaction with SAE1.The gene profiling analysis of MM clinical database,it was found that SAE1 is highly expressed in MM patients,and SAE1 overexpression is closely related to the poor prognosis of patients(TT2:p<0.01;APEX:p<0.05).2.WB confirmed that SAE1 overexpression/knockdown stably transfected MM cell line was successfully constructed;proliferation,cycle and apoptosis experiments showed that SAE1 overexpression promoted cell proliferation by affecting cell cycle distribution,while knockdown of SAE1 had the opposite trend.3.The high-throughput Co-IP/MS results of SAE1 and SUMO1 showed that overexpression of SAE1 could change various signaling pathways,among which the PI3K-Akt signaling pathway was the most enriched.The expression of PI3K-Akt signaling pathway showed that overexpression of SAE1 did not change the expression level of total Akt,but enhanced the phorsphored level of p-Akt and S-Akt proteins;knockdown of SAE1 reduced the expression of p-Akt and S-Akt.4.The treatment of colchicine can inhibit the cell cycle progression and proliferation rate of SAE1-OE cells;molecular docking predicted the potential binding of colchicine with E74 site of SAE1;SAE1-E74A mutant plasmid was successfully constructed by overlapping PCR technology;and mutation of E74A weakened the binding ability between the two.5.Animal model experiments confirmed that SAE1 overexpression can promote the proliferation of MM cells in vivo,and colchicine treatment significantly prolongs the survival of mice and inhibits tumor growth.ConclusionIn this study of SAE1 function in MM cells,we found that SAE1 could positively regulate the PI3K-Akt pathway via enhancing the SUMOylation and phosphorylation level of Akt,and over-activation of the PI3K-Akt signaling pathway further regulates cell cycle progression,and promotes the proliferation of MM.The new use of an old drug,colchicine,inhibits the proliferation of MM cells in vitro and in vivo.This study provides novel molecular targets and drug references for the treatment of MM patients.