Significance And Mechanism Of KDR Q472H Mutation In Multiple Myeloma Tumor Cells

Multiple myeloma(MM)is a terminal differentiated plasma cell malignant tumor,which is the second common malignant tumor of adult hematology.With the aging of society,the incidence rate of MM has been increasing,and the onset age has been trended to be younger.The effective combinational chemotherapy can make the initial treatment response rate of MM reach 95%,but,then almost 100% MM patients relapse and obtain drug resistance.With the in-depth study of MM,the identification of MM driven gene mutations has brought great promise for individualized treatment.Understanding the biological behavior caused by specific mutations and their undergrounded mechanisms will help to formulate individualized treatment plans for such patients.Studies have shown that although the mutation frequency of MM is high,there is a lack of universal driving mutations,suggesting that it may be the deregulation of key pathways rather than single gene mutation that play an important role in MM.In addition,several studies have shown that the key to these mutations in MM is the deregulation of MAPK pathway.Through previous studies based on whole exon sequencing,we found that KDR Q472 H mutation existed in tumor cells of newly treated or recurrent MM patients.The VEGFR2 protein encoded by KDR is one of the upstream proteins of MAPK pathway;Moreover,several studies have found that KDR Q472 H germline mutation can enhance the microvessel density of tumor tissue and is associated with poor prognosis.Therefore,we speculate that KDR Q472 H mutation may affect the biological behavior of MM.In order to explore the possible significance and mechanism of KDR Q472 H mutation in MM tumor cells,we conducted the following two parts of research:Part 1 Significance of KDR Q472 H mutation in MM tumor cellsObjective: To clarify the significance of KDR Q472 H mutation in MM tumor cellsMethods: 1.Construction of KDR wild type and KDR Q472 H eukaryotic expression plasmids;2.Construction of VEGFR2 wild-type and VEGFR2 Q472 H stable cell lines by lentivirus transfection;3.Using Western blot to detect the expression of related proteins in these two cells;4.Using flow cytometry to detect the cell death rate of these two cells treated with different drugs;5.Verify the results of in vitro experiments through animal experiments.Results: 1.KDR wild type and KDR Q472 H eukaryotic expression plasmids are successfully constructed,and the gene sequences are verified to be correct by sequencing;2.The transfection efficiency of both cells are more than 90% by fluorescence microscope and flow cytometry.The expression of VEGFR2 protein is verified by Western blot;3.The levels of phospho-VEGFR2 and phosphor-p44 / 42 MAPK protein increase in MM cells with KDR Q472 H mutation;4.In vitro,KDR Q472 H mutation enhanced the drug resistance of MM cells to proteasome inhibitors;Carfilzomib or Ixazomib combined with MEK1 / 2 inhibitor can reverse the drug resistance of MM cells with KDR Q472 H mutation to a certain extent;5.In animal experiments,KDR Q472 H mutation enhanced the drug resistance of tumor bearing mice to proteasome inhibitors;Conclusion: KDR Q472 H mutation can enhance the drug resistance of myeloma cells to proteasome inhibitors.Carfilzomib or Ixazomib combined with MEK1 / 2inhibitors can reverse the drug resistance of MM cells with KDR Q472 H mutation to a certain extent.Part 2 Mechanism of drug resistance in MM with KDR Q472 H mutationObjective: To explore the mechanism of drug resistance in MM with KDR Q472 H mutationMethods: 1.Sequencing and comparing the constructed KDR wild type and KDR Q472 H MM cells at the transcriptome level,and verifing the protein expression by Western blot;2.Knocking down the expression of AIM2 in MM cells with KDR Q472 H by sh RNA,then using flow cytometry to detect the cell death rate of myeloma cells treated with proteasome inhibitor and MEK1 / 2 inhibitor;3.Using Western blot to detect the expression of related proteins in KDR Q472 H MM cells with AIM2knockdown;4.Using RT-q PCR to detect the changes of AIM2 m RNA level after treatment with MEK1 / 2 inhibitor;5.Using flow cytometry to detect the cell death rate of KDR Q472 H MM cells treated with VX-765;6.Using Western blot to verify the expression of related proteins in subcutaneous transplanted tumor tissues of mice.Results: 1.KDR Q472 H mutation can increase the m RNA and protein levels of AIM2 in MM cells;2.Knockdown of AIM2 in MM cells with KDR Q472 H mutation can reverse the resistance to BTZ and MEK1 / 2 inhibitors;3.Knockdown of AIM2 in MM cells with KDR Q472 H mutation can reduce the level of phosphor-p44/42MAPK;The drug resistance of MM with KDR Q472 H mutation is dependent on AIM2-MAPK pathway;4.MEK1 / 2 can regulate the m RNA and protein level of AIM2 to a certain extent;5.The drug resistance of MM with KDR Q472 H mutation is not related to inflammasome pathway;6.The expression of AIM2 and phosphorp44/42 MAPK in subcutaneous transplanted tumor tissue of mice were consistent with those in vitro.Conclusion: The drug resistance of MM with KDR Q472 H mutation is AIM2-MAPK pathway dependent,inflammasome pathway independent,and the expression of AIM2 is regulated at the transcriptional level.