The Mechanism And Influence Factors Of Targeting Ferroptosis To Overcome Drug Resistance In Multiple Myeloma

Background and objective:Multiple Myeloma(MM)is currently the second most common hematologic malignancy.Advances in genomics and proteomics have increased our understanding of the pathogenesis of multiple myeloma,helped to identify novel therapeutic targets,and provided new targeted therapies such as bortezomib(BTZ,the proteasome inhibitor)and lenalidomide(the novel immunomodulatory agent).On these bases,most patients with MM can achieve effective remission in the early stage.But in the late stage of the disease,almost all of them progress to relapse and drug resistance,which is the biggest challenge in the current treatment of MM.Therefore,the research on the mechanism of drug resistance of relapsed and refractory MM and the search for new targeted drugs to reverse drug resistance have become the main focus of clinical practice and study in MM.We used established acquired BTZ-resistant MM cell lines(PS-R)and BTZ-na?ve cell lines(U266)to extract their genomic DNA for sequencing of whole genome DNA and RNA sequencing.After comparative analysis,the expression of ferroptosis-related genes was different between the two cell lines,suggesting that ferroptosis may be related to MM drug resistance.We hope to further investigate whether ferroptosis is involved in the occurrence of MM drug resistance,whether targeted ferroptosis can reverse MM drug resistance,and further explore its mechanism and influencing factors.Methods:1.MTS assay was used to detect the effects of erastin,RSL3 and their combination with BTZ on proliferation of MM cell lines.2.Flow cytometry was performed to detect the effects of erastin,RSL3 on the death of MM cell lines,and the effects of ferrostatin-1,DFO and L-Nac on the above treatments.The effect of calcium chelators on RSL3 was also detected.The effects of RSL3 in combination with apoptosis inducer(RSL3)and autophagy inhibitor(spautin1,chloroquine),respectively,were also examined.3.BODIPY C11 staining was used to detect the level of lipid peroxidation.4.Western Blotting was used to detect the relative expression of ferroptosis-related proteins,NF-?B signaling pathway-related proteins and autophagy-related proteins.5.Lentiviral transfection was used to knock down GPX4 in MM cell lines or overexpress different isoforms of GPX4,so as to explore the role of GPX4 in RSL3-induced ferroptosis in MM.Immunofluorescence and laser confocal focus were performed to observe the intracellular localization of different subtypes of GPX4 around RSL3 treatment.6.Co-IP technique was applied to detect the level of post-translational modifications of GPX4.7.Flow cytometry was used to detect the expression of FPN1 on MM cell surface,WB was used to detect iron metabolizing-related proteins in MM cell lines,and ELISA was used to detect hepcidin secretion in MM cell lines.The effects of FPN1,hepcidin and IL6 on RSL3-induced ferroptosis were detected by flow cytometry.8.Lentiviral transfection was employed to knock down ferritin in MM cell lines,and the flow cytometry was used to detect the effect of Ferritin-knockdown on RSL3-induced ferroptosis.9.MM cells were co-cultured with THP1 derived macrophages,then the sensitivity of MM cells to RSL3 was observed by flow cytometry.Flow cytometry was used to detect the expression of ferroportin on the surface of macrophages,and recombinant Hepcidin and ferroportin antibodies were used to interfere with iron metabolism of macrophages to observe whether co-culture affected the sensitivity of MM cell lines to RSL3.10.MM cell lines overexpressing GPX4 and their control group were subcutaneously injected into the outer thigh of NCG mice,and then intraperitoneally injected with PBS or RSL3 to observe the effect of RSL3 on MM and the effect of GPX4 on ferroptosis in vivo.11.CD138-positive cells and CD138-negative cells were separated from bone marrow biopsy samples,peripheral blood samples or pleural fluid samples of MM patients and separately treated with RSL3.Flow cytometry was used to detect the difference in sensitivity to ferroptosis between primary MM cells and control cells,and WB was used to detect the changes of GPX4 in primary MM cells.12.The relationship between metabolism and ferroptosis in MM was investigated by measuring Seahorse XF mitochondrial pressure and Seahorse XF glycolysis pressure.Results:1.RSL3 had significant effects on inhibiting proliferation and inducing cell death in MM cell lines in a dose-dependent manner.And BTZ-resistant cell line(PS-R)was more sensitive than its parent line(U266).Ferroptosis inhibitor ferrostatin-1,iron chelating agent DFO or ROS scavenger L-Na C could completely block the cell death and lipid peroxidation induced by RSL3,which validating the occurrence of ferroptosis.On the contrary,erastin and Buthionine-(S,R)-Sulfonylimine(BSO)have no obvious effect on proliferation and death in MM cell lines.2.Knockdown of GPX4 induced ferroptosis in MM cell lines and increased the sensitivity of MM cells to RSL3.WB experiments showed that GPX4 had two bands in MM,which were named GPX4 S and GPX4 L.The effect of RSL3 led to the down-regulation of GPX4 S and up-regulation of GPX4 L.Overexpression of GPX4 S decreased the sensitivity of MM cells to RSL3-induced ferroptosis and lipid peroxidation,while overexpression of GPX4 L did not affect the sensitivity of MM cells to RSL3.3.Unlike iron chelating agent,calcium chelating agent could not block ferroptosis induced by RSL3,but increased the sensitivity of MM cells to RSL3.4.RSL3 induced ferroptosis in MM accompanied by up-regulation of iron metabolism-related proteins such as FPN1.However,blocking FPN1 receptor or interfering with the function of FPN1 by hepcidin and IL6 had no significant effect on RSL3.5.Macrophages express FPN1 highly and are insensitive to RSL3.The sensitivity of MM cells to RSL3 was significantly reduced after co-culture with macrophages.Neutralization and closure of macrophages with hepcidin and FPN1 antibodies further reduced the sensitivity of MM cells to RSL3,while simultaneous closure of macrophages and MM cells with hepcidin and FPN1 did not further affect the sensitivity of MM cells to RSL3.6.Both RSL3 and erastin showed significant synergistic effects with BTZ in both parental and resistant cell lines.Ferrostatin-1,DFO or L-Nac had no significant effect on BTZ-induced apoptosis,nor on the synergistic effect of erastin with BTZ,but could inhibite the synergistic effect of RSL3 with BTZ to the level of BTZ-treatment alone.The assay of related proteins suggested that the synergistic effect on RSL3 and BTZ was achieved by the combination of RSL3-induced ferroptosis and BTZ-induced apoptosis.However,the synergistic effect of erastin and BTZ was not related to ferroptosis,but may be due to that RSL3 enhanced the inhibitory effect of BTZ on NF-?B signaling pathway.7.We found that RSL3 induced ferroptosis in MM cell lines while inducing up-regulation of SQSTM1/P62 and LC3 II,suggesting the activation of autophagy.Further studies showed that inhibition of autophagy increased the sensitivity of MM to RSL3.8.In vivo experiments confirmed that RSL3 could inhibit the growth of MM xenografted tumor,and GPX4 S overexpression resulted in resistance to RSL3.9.In patient samples,RSL3 could induce ferroptosis in CD138+cells,the down-regulation of GPX4 S and up-regulation of GPX4 L.10.Mitochondrial stress test showed that the ATP production dependent on mitochondrial respiration decreased in MM cells and MM cells overexpressing GPX4 L after RSL3 treatment,while the production of mitochondrial ATP was up-regulated in MM cells overexpressing GPX4 S.Glycolysis stress tests showed similar glycolytic production and glycolysis abilities in control and GPX4 L overexpressed MM cells,while GPX4 S was significantly lower than both.After RSL3 treatment,the glycolytic production and glycolysis capacity increased significantly in MM cells with GPX4 L overexpressed,but not in those with GPX4 S overexpressed.Conclusions:1.MM was insensitive to type I ferroptosis but highly sensitive to type II ferroptosis.Inducing type II ferroptosis can not only effectively kill MM and inhibit MM growth,but also has a more significant effect on drug-resistant MM,suggesting that inducing ferroptosis can help MM overcome drug resistance to BTZ.2.There exist different isoforms of GPX4,among which GPX4 S plays an important role in resistance to ferroptosis;knockdown of GPX4 spontaneously induced ferroptosis in MM and increased susceptibility to RSL3.3.The decrease of intracellular Ca2+ can increase the sensitivity of MM to ferroptosis.4.There were differences in iron metabolism between MM and macrophage,which resulted in different sensitivity to ferroptosis.The macrophages were significantly resistant to ferroptosis,and even partly protected MM cells from ferroptosis.5.The combination of ferroptosis inducer and BTZ can not only increase its efficacy,but also effectively overcome drug resistance.6.In MM,autophagy is a feedback protective mechanism of ferroptosis,which is activated upon the occurrence of ferroptosis.And inhibition of autophagy can increase the sensitivity of MM to ferroptosis.7.MM associated ferroptosis is accompanied by the reprogramming of energy metabolism from mitochondrial respiration to glycolysis metabolism.GPX4 S makes MM resist ferroptosis by inhibiting the reprogramming of energy metabolism(reducing the dependence of MM on glycolytic metabolism and making MM dependent on ATP produced by aerobic respiration).