Study On The Mechanism Of CBL Inducing The Migration And Stemness Enhancement Of Multiple Myeloma Cells

Background and Objective:Multiple myeloma(MM)is a hematological tumor originating from plasma cells of bone marrow.It is characterized by abnormal proliferation of plasma cells,accompanied by monoclonal immunoglobulin or light chain(M protein).The treatment of MM mainly includes autologous stem cell transplantation,proteasome inhibitors,immunomodulators,monoclonal antibodies,chimeric antigen receptor T cells(CAR-T)and so on,however,MM patients remain incurable due to drug resistance and relapse.Based on the potential of microfluidics chip to screen cell morphologyspecific molecule targets for tumor cells.This study takes advantage of microfluidic chip sorting technology to screen out one potential oncogenic drug targets for MM,Casitas B-lineage lymphoma(CBL),and aims to elucidate the molecular regulatory role of CBL in the increased migration and stemness of MM cells which would further provide a new molecular target for clinical diagnosis,treatment and drug discovery of MM.Methods:1.We screened flexible deformed MM cell subgroups using microfluidic chip sorting technology.And then we used CCK-8 assay to verify the effect of sorting on MM cell proliferation as well as Transwell migration assay to characterize the migration of sorted subgroup cells.Then,some differentially expressed genes including CBL were screened by transcriptomic RNA sequencing in MM cells with superior proliferation and migration compared to control.2.Lentivirus was used to package CBL overexpression(OE)plasmid and transfected into CAG cells and ARP1 MM cell lines followed by puromycin selection to construct CBL-OE MM cells stably,overexpressing CBL(ARP1 CBL-OE and CAG CBL-OE).Western blot(WB)was used to confirm the successful construction.CCK-8 method and soft agar cloning experiment were used to verify the proliferation effect and Transwell migration assay was used to verify the altered migration between CBL-OE and wild-type cells.Aldehyde Dehydrogenase(ALDH)activity was detected to determine their stemness.Flow cytometry was used to re-verify the changed microfluidic chip outflow of overexpressed cells.3.We used co-immunoprecipitation combined with protein spectrometry(Co-IP/MS)to screen the downstream targets of CBL and pathways involved in MM cell migration and stemness enhancement.Finally,the interaction between CBL and downstream protein and its downstream pathway were verified by WB,nucleo-cytoplasmic separation experiment and immunofluorescence assay.4.Considering that CBL is the member of E3 ubiquitin ligase,we hypothesized that CBL might influence downstream pathways through the ubiquitin mediated of target proteins.And then we used ubiquitination experiment and half-life assay to verify the ubiquitination degradation of TJP2 by CBL.Results:1.The microfluidic chip sorting technology screened subgroup of flexible deformed MM cells;The CCK-8 assay confirmed that the sorted MM cells were significantly enhanced in cell proliferation;Transwell migration assay confirmed that the sorted MM cells were significantly enhanced in cell migration;then transcriptomic RNA-sequencing was performed on MM cells with proliferation and migration advantages to screen out differentially expressed genes including CBL gene,which is related to malignancy of MM cells..2.The results of WB confirmed that ARP1 CBL-OE and CAG CBL-OE cells were successfully constructed;CCK-8 assay and soft agar cloning experiments showed that overexpression of CBL could significantly promote the proliferation of MM cells.It was observed by Transwell migration assay that overexpression of CBL could enhance cell migration.ALDH experiments showed that overexpression of CBL could promote stemness of MM cells.Microfluidic chip technology verified that the flow rate of CBL-OE cells was significantly increased comparing to control groups.Mouse models of xenograft tumors show that overexpression of CBL can promote the growth of MM cells in vivo.3.Co-IP/MS results showed that the overexpression of CBL could increase β-catenin entry and activate the Wnt/β-catenin signaling pathway;further analysis screened out the downstream TJP2(Tight Junction Protein 2).WB showed that CBL activated the β-catenin pathway by downregulating TJP2 and promoting the increase of β-catenin entry into the nucleus.4.Co-IP assay and immunofluorescence assay showed that CBL interacts with TJP2,and the ubiquitination results showed that CBL ubiquitinated TJP2 and shorten the half-life of TJP2.WB results showed that overexpression of CBL could ubiquitinate TJP2 and promote GSK3βphosphorylation,which further reduced the phosphorylation of the GSK3β complex on β-catenin,thus increasing the nucleus β-catenin level and activating the β-catenin.Conclusion:Microfluidic chip technology has been applied to drug target discovery and to screen out potential MM drug target CBL in this study.Our results demonstrate that the overexpression of CBL promotes ubiquitination and degradation of TJP2,and enhances GSK3 β phosphorylation which further reduces the GSK3β complex to phosphorylate β-catenin,as well as increases the βcatenin level and nucleus transportation,thereby activating β-catenin pathway.