NEDD8 Inhibitior MLN4924 Overcomes CKS1B Induced Drug Resistance By Upregulation Of P21 In Multiple Myeloma

Research backgroundMultiple myeloma is a neoplastic plasma cell disorder that is characterized by clonal proliferation of malignant plasma cells in the bone marrow microenvironment,monoclonal protein in the blood or urine,and associated organ dysfunction.MM is now the second most common hematological malignancy and accounts for approximately 13%of hematologic cancers[1].In the past decade,the introduction of autologous stem cell transplantation and the availability of agents such as thalidomide,lenalidomide,and Bortezomib have changed the management of myeloma and extended overall survival.In patients presenting at an age under 60 years,10-year survival is approximately 30%.In particular,Bortezomib,which targets the 26S proteasome subunit ?5,has induced a high level of positive response rates[2,3].However,toxicities associated with global proteasomal inhibition and resistance to Bortezomib in MM are major concerns,prompting the further development of novel therapies.These improved inhibitors are designed to more specifically target critical factors in protein turnover and are now part of ongoing clinical trials.One such molecule,MLN4924,is an inhibitor of NEDD8-activating enzyme(NAE),preventing the conjugation of the small ubiquitin-like protein NEDD8(neural precursor cell expressed developmentally down-regulated protein 8)to cullin-RING ubiquitin E3 ligases(CRL).This inhibitor has shown promise against MM in vitroby inhibiting the degradation of skpl/cullin-1/F-box SKP2(SCFSkP2)substrates including p27 and p21[4].CDC28 kinase subunit 1(CKS1B)is a necessary cofactor of the CRL complex,SCFSkp2,which regulates cellular entry into S phase and possesses anti-apoptotic activity through p27-dependent and-independent pathways,and is a critical factor in MM drug resistance[5].CKS1B is an essential protein for normal cell division and growth[6],and is expressed at a high level in various cancer tissues including hepatocellular carcinoma[7],colon[8],lung[9],oral squamous cell carcinoma[10],breast cancer[11],and others.In MM,amplification of region lq21,which includes the CKS1B gene,identifies a subpopulation with poor prognosis,an aggressive clinical course,and few therapeutic optiornsC12,13].CKS1B amplification is also associated with transformation from both the benign state of monoclonal gammopathy of undetermined significance(MGUS)to MM and further progression to plasma cell leukemia[14].Previously,we identified CKS1B as one of 70 high-risk genes inversely associated with survival in newly diagnosed MM[15]and high nuclear expression of CKS1B is an adverse prognostic factor for relapsed/refractory MM patients[16].These findings provide compelling evidence that CKS1B represents a strong candidate target gene for therapy.NEDD8 bind to Cullin-1 induce Neddylated SCFskp2,which represents the activated complex essential for the ubiquitination of CRL substrates[17].In this study,we test the efficacy of MLN4924 in MM cells overexpressing CKSIB.We explore how MLN4924 affects cell viability,cell colony formation and senescence in CKS1B overexpressing MM cells.Our study identifies a novel function for MLN4921 in the upregulation of p21 expression,which is independent of the CKS IB-mediated SCFSkp2 complex formation and NEDD8-dependent SCFSkp2 activation,resulting in killing MM cells.Objective1.Explore MM cells overexpressing CKS1B are resistant to Bortezomib but sensitive to MLN4924;2.Explore CKS1B regulates neddylation-related signaling;3.Verify MLN4924 overcomes CKS1 B-induced drug resistance by inhibiting p21 ubiquitin-mediated degradation;4.Further verify p21 is necessary for MLN4924 sensitivity in CKS1B overexpressing MM cells;5.Dysregulation of the neddylation pathway is associated with drug resistance and poor prognosis in MM clinically.Contends and methods1.Explore MM cells overexpressing CKS1B are resistant to Bortezomib but sensitive to MLN4921)Constructed pCMV/AIG-CKSIB-GFP plasmid with the insertion of CKS IB gene sequence through molecular cloning techniques,packed lentivirus using calcium phosphate technology,transfected and achieved CKS 1B stably overexpressing MM cells of OCI-My5 and ARP1;2)Tested virus transfection efficiency by flow cytometry,and verified CKS 1B were stably overexpressed in MM OCI-MY5 and ARP1 cells using western blot;3)Explored the drug function of MLN4924 or Bortezomib on CKS 1B overexpressing cells of MM OCI-My5 and ARP1 via Growth curve;4)Explored the clonogenic formation capacity of CKS IB overexpressing cells OCI-My5 and ARP1 treated with different MLN4924 or Bortezomib via clonogenic formation assay;5)Explored the happen of cell senescence for CKS 1B overexpressing cells OCI-My5 and ARP1 treated with different MLN4924 or Bortezomib by senescenceassay.2.Explore CKS1B regulates neddylation-related signaling1)Tested NEDD8(Cullin-1)expression in CKS1B knockdown or stably overexpressing MM cells OCI-My5 and ARP1;2)Tested the NEDD8(Cullin-1)expression in CKS1B stably overexpressing MM cells OCI-My5 and ARP1 treated with MLN4924.3.Verify MLN4924 overcomes CKS1B induced drug resistance by inhibiting p21 ubiquitin-mediated degradation1)Treated CKSIB overexpressing MM cells OCI-My5 and ARP 1 with different drug concentrations of MLN4924 or Bortezomib for 48 h;2)Tested P21,P27,P-RB,Cullin-1 expression in CKS1B overexpressing MM cells OCI-My5 and ARP1 treated with above drugs via western blot;3)Tested P21 mRNA expression level in CKS1B overexpressing MM cells OCI-My5 and ARP1 treated with above drugs by RT-PCR.4.Further verify p21 is necessary for MLN4924 sensitivity in CKS1B overexpressing MM cells1)Constructed pLentiCRISPR-P21sgRNAplasmid with P21 sgRNA gene insertion through using the CRISPR/CAS9 gene knock-out technology;2)Packed lentivirus using calcium phosphate technique,transfected and constructed P21 knock-out CKS1B overexpressing MM cells OCI-My5;3)Treated P21 knock-out CKS1B overexpressing MM cells OCI-My5 with different concentration of MLN4924 for 24 h,testified P21 expression in these cells via western blot;4)Treated P21 knock-out CKS1B overexpressing MM cells OCI-My5 with different concentration of MLN4924,tested the cell viability,clonogenic formation and senescence appearance in P21 knock-out CKS1B overexpressing MM cells OCI-My5 via the experiments of growth curve,clonogenic formation assay and senescence assay.5.Dysregulation of the neddylation pathway is associated with drug resistance and poor prognosis in MM clinically1)Examination of the mRNA expression profiles of neddylation pathway genes;2)Examination of NEDD8 expression in patients categorized as unresponsive or responsive to treatment with dexamethasone and Bortezomib;3)Examination of NEDD8 expression in patients categorized as unresponsive or responsive to treatment with Bortezomib or dexamethasone;4)Kaplan-Meier and long-rank test estimate overall survival in MM patients with varied expression of neddylation pathway genes;5)Kaplan-Meier and long-rank test estimate overall survival in MM patients with sorted CKS1B and neddylation enzyme gene expression profiles.Results1.MM cells overexpressing CKS1B are resistant to Bortezomib but sensitive to MLN4921)Constructed PCMV/AIG-CKS1B-GFP plasmid with the insertion of CKS1B gene sequence through molecular cloning techniques,used IRES reverse primer to test the plasmid gene sequence.The outcome of sequencing was right,and in the help of tool cell of HEK-293T to pack lentivirus using calcium phosphate technology,transfected and achieved CKS1B stably overexpressing MM cells of OCI-MY5 and ARP1;2)Tested virus transfection efficiency by flow cytometry,which defined the transfection efficiency of OCI-My5 and ARP1 came to about 95%,and verified CKS1B were stably overexpressed in MM OCI-My5 and ARP1 cells using western blot;3)Explored the drug function of MLN4924 or Bortezomib on CKS1B overexpressing cells of MM OCI-My5 and ARP1 via Growth curve;CKS1B overexpressing MM cells of OCI-My5 and ARP1 treated with 1uM MLN4924 or 5nM Bortezomib for 7 days,triplicate,results were expressed as means ± SD,results showed cells in empty vector group were sensitive to MLN4924 and Bortezomib,MM cells overexpressing CKS1B are resistant to Bortezomib but sensitive to MLN492;4)Explored the clonogenic formation of CKS1B overexpressing cells OCI-My5 and ARP I treated with different MLN4924 or Bortezomib via clonogenic formation assay;results showed that the clonogenic formation decreased significantly in empty vector,the clonogenic formation for CKS IB overexpressing MM cells decreased significantly in MLN4924 treatment than Bortezomib treatment group;5)Explored the happen of cell senescence for CKS1B overexpressing cells OCI-My5 and ARP1 treated with different MLN4924 or Bortezomib by senescence assay,results showed that the induced knock-down of CKS1B prompted cell senescence,and MLN4924 decreased cell senescence significantly in contrast to Bortezomib.2.CKS1B regulates neddylation-related signaling genes1)Testified NEDD8(Cullin-1)expression in CKS1B knockdown or overexpressing MM cells OCI-My5 and ARP1;results displayed that NEDD8(Cullin-1)expression was consistent with the changes of CKS1B in MM;2)Tested the NEDD8(Cullin-1)expression in CKS1B stably overexpressing MM cells OCI-My5 and ARP1 treated with MLN4924.The outcome was consistent with the MLN4924 of NAE inhibitor,and MLN4924 blocked NEDD8(Cullin-1)expression significantly.3.MLN4924 overcomes CKS1B-induced drug resistance by inhibiting p21 ubiquitin-mediated degradation1)Treated CKS1B overexpressing MM cells OCI-My5 and ARP1 with different drug concentrations of MLN4924 or Bortezomib for 48 h;2)Tested P21,P27,P-RB,Cullin-1 expressions in CKS1B overexpressing MM cells OCI-My5 and ARP1 treated with above drugs via western blot.Results displayed that Bortezomib and MLN4924 could induce the upregulation of P27 and downergulation of P-RB;MLN4924 induced upregulation of P21 significantly in contrast to Bortezomib,and it also blocked the neddylation of Cullin-1;3)Tested P21 mRNA expression level in CKS1B overexpressing MM cells OCI-My5 and ARPM treated with above drugs by RT-PCR,P21 mRNA expression was increased significantly in MLN4924 treatment than in Bortezomib treatment.4.Further verify p21 is necessary for MLN4924 sensitivity in CKS1B overexpressing cells1)Constructed pLentiCRISPR-P21sgRNA plasmid with P21sgRNA gene insertion through using the CRISPR/CAS9 gene knock-out technology,U6 promotor forward primer was used for gene sequence,the construct was right;2)Packed lentivirus using calcium phosphate technique,transfected and constructed P21 knock-out CKS1B overexpressing MM cells OCI-My5;screened the transfected cells with certain puromycin,got the specially puromycin resistant P21 knock-out CKS1B overexpressing MM cells;3)The basal P21 expression in MM is low,so treated P21 knock-out CKS1B overexpressing MM cells OCI-My5 with different concentration of MLN4924 for 24 h,testified P21 expression in these cells via western blot,results revealed that P21 expression in knock-out cells did not increase significantly,P21 knock-out in CKS1B overexpressing MM cells were successful;4)Treated P21 knock-out CKS1B overexpressing MM cells OCI-My5 with different concentration of MLN4924,tested the cell viability,clonogenic formation and senescence appearance in P21 knock-out CKS1B overexpressing MM cells OCI-My5 via the experiments of Growth curve,clonogenic formation assay,senescence assay.These results demonstrated that MLN4924 sensitivity on cell proliferation andsurvival,clonogenic capacity,and senescence in CKSIB-overexpressing cells is dependent upon p21 and a loss of p21 shifts CKSIB-overexpressing cells towards insensitivity to MLN4924.5.Dysregulation of the neddylation pathway is associated with drug resistance and poor prognosis in MM clinically1)Examination of the mRNA expression profiles of neddylation pathway genes.Gene expression profiling of patients demonstrated that,compare to healthy donors and MGUS patients,the mRNA expression of NAE1,UBA3,UBC12 and NEDD8 was elevated in plasma cells from patients newly diagnosed with MM;2)Examination of NEDD8 expression in patients categorized as unresponsive or responsive to treatment with dexamethasone and Bortezomib;results displayed NEDD8 was significantly elevated in patients exhibiting decreased response to clinical treatment;3)Examination of NEDD8 expression in patients categorized as unresponsive or responsive to treatment with Bortezomib or dexamethasone,NEDD8 expression was significantly elevated in patients exhibiting decreased clinical response to Bortezomib,but was not significantly altered in patients resistant to dexamethasone;4)Kaplan-Meier and long-rank test estimate overall survival in patients with varied expression of neddylation pathway genes;The expression of UBA3(p = 0.003),UBC12(p = 0.001),or CKS1B(p = 0.015)each demonstrated significantly poorer prognoses than patients with low expression of the same gene;5)Kaplan-Meier and long-rank test estimate overall survival in patients with sorted CKS1B and neddylation enzyme gene expression profiles;greatest overall survival was seen in(low/low)CKS1B-UBA3 or CKS1B-UBC12 populations than any other combined groups with different CKS1B and UBC12 or UBA3.ConclusionMM Cells overexpressing CKS1B were resistant to Bortezomib but sensitive to MLN4924.Treatment of CKS1B-overexpressing cells with MLN4924 decreased cell proliferation and growth,cell clonogenicity,and induced cellular senescence.MLN4924,but not Bortezomib,induced stabilization of p21 and upregulated P21 expression significantly,knockout of p21 resulted in loss of MLN4924 sensitivity on CKS1B overexpressing MM.MM patients exhibited increased expression of NEDD8 pathway genes relative to normal and MGUS patients plasma cells,MM patients with high NEDD8 expression had inferior outcomes.In conclusion,our data demonstrate cells with elevated CKS1B expression are resistant to Bortezomib but sensitive to MLN4924 and offer a mechanism through inhibiting degradation of P21 to stabilize p21 expression.These findings provide rationale for targeting the NEDD8 pathway in MM patients exhibiting elevated expression of CKS1B clinically.