| Multiple myeloma is a hematologic malignancy characterized by the clonal proliferation of bone marrow plasma cells.The number of patients with MM accounts for 10% of all hematologic neoplasms and is the second most common hematologic malignancy.Although new therapies and drugs for MM have resulted in significantly longer median survival,the disease remains incurable at this time.Patients with effective initial therapy continue to face disease recurrence and progression,requiring multiple lines of follow-up therapy,and the depth and durability of response decreases with each salvage therapy.This is closely related to the tumor heterogeneity of MM,whose complex genetic abnormalities and clonal evolution are the underlying causes of MM pathogenesis and incurability.This study explored the tumor heterogeneity of MM from both clinical and basic perspectives,respectively.The clinical part focuses on the impact of 1q gain(+1q)on MM prognosis in the new drug era and risk stratification of MM patients with +1q.The basic part aims to discuss the role of IKBKB R536 Q mutation in MM cells and its mechanism.Part 1 Clinical heterogeneity of 1q gain high-risk multiple myelomaBackground and objective:MM is a highly heterogeneous malignant plasma cell tumor,and although the clinical application of novel drugs,autologous stem cell transplantation,and immunotherapy has led to significant improvements in patient outcomes and survival,25% of patients with high risk MM still have a poor prognosis with a predicted survival time of less than 3 years.Cytogenetic abnormalities and response to therapy are the most powerful predictors of HRMM,and currently known CA with poor prognosis include +1q,del(17p),t(4;14),t(14;16),and t(14;20).Of these,there is still controversy as to whether +1q is an independent risk factor.Some studies have shown that the poor prognosis of +1q depends on its copy number and only when the copy number is greater than 3 is a risk factor,while others have questioned the prognostic value of +1q depending on its concurrent HRCA.In addition to this,the prognostic heterogeneity of +1q patients and the difficulty of existing clinical treatment strategies to overcome the poor prognosis of +1q patients are issues that remain to be addressed.Therefore,studies are needed to fully understand the actual prognostic value of +1q and to establish a prognostic risk model for +1q NDMM patients,so as to more accurately distinguish +1q patients with different prognoses and to guide clinical practice for stratified diagnosis and treatment.Methods:A total of 934 NDMM patients treated with new agents from November 2009 to November 2019 were selected as the research subjects.The clinical characteristics and prognosis of NDMM with +1q were analyzed,and a prognostic risk model of patients with +1q was established for risk stratification.Results:1.Of the 934 patients in this study,+1q was found in 496 cases,accounting for 53.1% of all patients.Compared with patients without +1q,+1q patients showed more advanced diseases(ISS III: 56.9 vs 49.5%,P=0.025;R-ISS III: 33.6 vs 24.3%,P=0.004),larger tumor burden(higher bone marrow plasma cell ratio(P=0.008)and lactate dehydrogenase(P<0.001)),and more cases with other CA(del(13q): 55.8 vs 37.0%,P<0.001;t(4;14): 17.7 vs 11.9%,P=0.031;t(14;16): 3.9 vs 0.6%,P=0.004).2.Survival was significantly shorter in patients with +1q than in patients without +1q,with median PFS of 18.3 and 24.4 months and median OS of 36.5 and 46.7 months,respectively(P values were 0.0001 and 0.0003,respectively).Multivariate analysis showed that +1q was strongly associated with an increased risk of disease progression(HR=1.281,95%CI 1.052~1.561,P=0.014)or death(HR=1.400,95%CI 1.097~1.787,P=0.007),independent of other baseline prognostic factors.PFS and OS(m PFS: 21.9 vs 30.9ms,P=0.0081;m OS: 45.6 vs 58.6ms,P=0.0067)of +1q patients without other CA were significantly shorter than those of non-HRCA patients,suggesting that +1q itself is a poor prognostic CA.3.Concurrent HRCA further shortened the survival of patients with +1q(m PFS: 16.1 vs 21.9ms,P=0.0294;m OS: 29.4 vs 45.6ms,P=0.0381).Concurrence of t(14;16)led to the worst prognosis in patients with +1q,with a shortened median PFS(P<0.0001)and OS(P=0.0004)of 16.9 and 22.7 months,respectively.Concurrence of del(17p)also resulted in a shortened survival(m PFS: 13.0 vs 21.9ms,P=0.0221;m OS: 23.3 vs 45.6ms,P=0.0046)for patients with +1q.4.The results of multivariate analysis affecting PFS and OS in +1q patients showed that ISS stage III,elevated LDH,hypercalcemia and t(14;16)were independent prognostic factors,and a prognostic risk model was established by weighting them according to cox regression coefficient.+1q NDMM was divided into three groups: low,intermediate and high risk,with median OS of 59.8,34.8 and 18.9 months,respectively(P<0.0001 for pairwise comparison).Similar results were obtained by external validation of Co MMpass database.The median OS of low,intermediate and high risk +1q patients in this cohort was not reached,55.5 and 21.6 months,respectively(intermediate vs low risk: P=0.0363;high vs intermediate risk: P=0.0186;high vs low risk: P=0.0006).The C index of this prediction model was 0.6768(95%CI 0.6358~0.7178),and the C index of external validation was 0.6139(95%CI 0.5233~0.7046).5.The incidence of early relapse was significantly higher in patients with +1q than in patients without +1q(ER12: 27.3 vs 16.7%,P=0.002;ER24:59.2 vs 42.9%,P<0.001)and +1q was an independent risk factor for ER24(OR=4.03,95%CI 1.34~12.12,P=0.013).Risk stratification of +1q patients using the prognostic risk model revealed that the risk of ER decreased with decreasing grade of risk stratification(ER12: low vs intermediate risk OR=0.503,95%CI 0.285~0.889,P=0.013;intermediate vs high risk OR=0.451,95%CI 0.273~0.747,P=0.023;ER24: low vs intermediate risk OR=0.792,95%CI 0.618~1.014,P=0.054;intermediate vs high risk OR=0.662,95%CI 0.540~0.812,P=0.032).6.The rate of undetectable MRD(MRD-)was significantly lower in patients with +1q than in patients without +1q(41.9 vs 62.5%,P=0.004).PFS and OS(m PFS: 35.1 vs 11.6ms,P<0.0001;m OS: not reached vs 25.4ms,P<0.0001)were significantly prolonged in +1q patients who achieved MRD-.Patients with +1q were divided into low and intermediate/high risk groups according to the prognostic risk model,and survival differences were compared with those without +1q under different treatment conditions.The results found that two-drug induction therapy with proteasome inhibitors and immunomodulatory drugs or ASCT could overcome the adverse effect of OS in +1q low risk patients,but did not significantly improve the prognosis of +1q intermediate/high risk patients.However,achieving MRD-significantly prolonged PFS(m PFS: 31.4 vs 11.4ms,P=0.0008)and OS(m OS: 47.2 vs 23.6ms,P=0.0028)in intermediate/high risk +1q patients.Conclusions:1.+1q has a high incidence in Chinese patients,which is an independent adverse prognostic factor affecting NDMM in the era of new agents.2.MM with +1q showed high heterogeneity in baseline characteristics,treatment response and outcome,which may be related to the diversity of concurrent CA.3.ISS stage III,elevated LDH,hypercalcemia,t(14;16)are independent predictors of survival in +1q patients,and a prognostic risk model based on these four factors can further stratify the risk of +1q patients.4.The prognostic risk model for +1q patients is helpful to predict early relapse.5.MRD-,especially sustained MRD-,might circumvent the poor outcome of +1q patients.Part 2 The role and mechanism of IKBKB gene mutation in multiple myelomaBackground and objective:MM,as a highly heterogeneous plasma cell tumor,is characterized by malignant proliferation and extensive infiltration of plasma cells in bone marrow,resulting in tissue and organ damage.MM was once considered to be an incurable disease with a short overall survival.With the understanding of its tumor heterogeneity and the widespread clinical application of new therapeutic strategies,the prognosis of MM has been significantly improved.However,the effect of MM treatment varies greatly between individuals,and the vast majority of patients are still incurable,and the problem of relapse and drug resistance is becoming more prominent.This is a combination of the diversity of MM cytogenetic alterations,genomic instability,and the interaction between tumor cells and the microenvironment.Although many studies have shown that the frequency of MM gene mutation is high,the exact driving mutation has not been found.Existing studies have found that RAS/MAPK and NF-κB pathways are the most frequently mutated pathways in MM.The presence of NF-κB activating mutations in MM has been confirmed by several studies on tumor genomes.In previous studies based on whole-exome sequencing,we found that 35.9%(14/39)of all MM patients(including NDMM and RRMM)had at least one NF-κB related gene mutation,and multiple RRMM patients had IKBKB R536 Q mutations.IKKβ encoded by IKBKB is a key kinase that activates the classical pathway of NF-κB signaling pathway.IKKβ is a three-layer structure consisting of a kinase domain(KD),a ubiquitin-like domain(ULD),and an α-helical scaffold/dimerization domain(SDD).The 536 amino acid is located in the SDD region.SDD can mediate IKKβ dimerization,and IKKβ dimerization is required for IKKβ activation.Therefore,we decided to explore the influence of IKBKB R536 Q mutation on the biological behavior of MM and the related mechanisms inducing its occurrence.Methods:1.The expressions of IKKβ and P-IKKα/β proteins in 9 MM cell lines U266,PSR,BM,PE,H929,OPM2,8226,DR and R10 R were detected by Western blot;2.IKBKB wild type,IKBKB R536 Q mutant and control stable cell lines were constructed by lentivirus transfection;3.The expressions of IKKβ and NF-κB signaling pathway and its upstream and downstream proteins were detected by Western blot;4.Cell counting method was used to record the proliferation rate of three kinds of stable cell lines in vitro.U266-EV,U266-IKBKB WT and U266-IKBKB R536 Q were injected subcutaneously into mice with severe immune deficiency to construct MM transplant tumor model,and the proliferation rate of the three kinds of stable cell lines in vivo was compared;5.Flow cytometry and CCK-8 method were used to determine the drug effects of three proteasome inhibitors,bortezomib,carfilzomib and ixazomib,on U266-EV,U266-IKBKB WT and U266-IKBKB R536 Q cell lines;6.Flow cytometry was used to detect the apoptosis of three stable cell lines treated with NF-κB pathway inhibitors(MLN120B,Bay 11-7082,Birinapant,Venetoclax)alone or in combination with bortezomib/ixazomib;7.Transcriptome sequencing was performed on U266-EV,U266-IKBKB WT and U266-IKBKB R536 Q cell lines,and the differentially expressed genes were compared between every two cell lines;8.The transcription and translation levels of the significantly up-regulated gene OLR1 in U266-IKBKB R536 Q cells were verified by q RT-PCR and Western Blot.Western Blot was used to detect the changes of OLR1-encoded protein LOX1 and NF-κB signaling pathway protein expression in three cell lines after treatment with NF-κB inhibitor Bay 11-7082.Results:1.U266-EV,U266-IKBKB WT and U266-IKBKB R536 Q were successfully constructed,which verified by fluorescence microscopy and flow cytometry.2.Western blot was used to detect IKKβ protein,NF-κB signaling pathway related P-IKKα/β,IKKγ,p65,P-p65 protein,upstream adaptor protein RIP,TRAF2,TRAF6 and NF-κB target gene c-IAP1 and Bcl-2 corresponding protein expression in U266-IKBKB WT and U266-IKBKB R536 Q cells were significantly up-regulated compared with U266-EV cells.Among them,the expression of adaptor proteins RIP,TRAF2,TRAF6 and phosphorylated proteins P-IKKα/β,P-p65 related to IKK complex activation in U266-IKBKB R536 Q cells was stronger than that in U266-IKBKB WT cells.3.The proliferation rates of U266-IKBKB WT and U266-IKBKB R536 Q cells in vivo and in vitro were significantly higher than those of U266-EV cells.4.The apoptosis rate of U266-IKBKB WT and U266-IKBKB R536 Q cells treated with carfilzomib was higher than that of U266-EV cells.In contrast,the IC50 values of U266-IKBKB WT and U266-IKBKB R536 Q cells against bortezomib and ixazomib were significantly higher than those of U266-EV cells.5.The specific IKKβ inhibitor(MLN120B)and c-IAP1 strong antagonist(Birinapant)could not improve the resistance of IKBKB overexpression and IKBKB R536 Q mutation to bortezomib/ixazomib,but NF-κB inhibitor(Bay 11-7082)and Bcl-2 selective inhibitor(Venetoclax)could reverse the resistance of U266 cells caused by IKBKB gene change to some extent.6.The mRNA and protein expression levels of OLR1 gene in U266-IKBKB R536 Q cells were significantly up-regulated.Patients with high OLR1 expression in MM had significantly shorter OS than those with low expression in MM(median OS were both not reached,P=0.0417).The expression levels of IKKβ,LOX-1,P-IKKα/β,P-p65 and Bcl-2 proteins in U266-IKBKB WT and U266-IKBKB R536 Q cells were decreased by Bay 11-7082.Conclusions:1.IKBKB overexpression and IKBKB R536 Q mutation can activate the NF-κB classical pathway,and IKBKB R536 Q mutation may lead to stronger activity of this signaling pathway.2.IKBKB overexpression and IKBKB R536 Q mutation increase the proliferation rate of MM cells in vivo and in vitro.3.IKBKB overexpression and IKBKB R536 Q mutation resulted in U266 cells resistant to bortezomib and ixazomib,and sensitive to carfilzomib.4.NF-κB inhibitor or Bcl-2 inhibitor combined with bortezomib/ixazomib can reverse the drug resistance of MM cells caused by IKBKB overexpression and IKBKB R536 Q mutation to a certain extent.5.The proliferation and drug resistance of MM cells caused by IKBKB R536 Q mutation may be related to the up-regulation of OLR expression. |
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