【作者】 王伟；

【导师】 戴云；

【作者基本信息】 吉林大学 ， 肿瘤学， 2023， 博士

【摘要】 研究目的:耐药是当前多发性骨髓瘤(MM)临床治疗的最主要难题,但由于其确切的机制不明,迄今仍缺乏有效的干预手段。Mcl-1是Bcl-2家族抗凋亡蛋白之一,其过表达与MM发生发展及耐药密切相关。Mcl-1属于一种快速代谢的短寿命蛋白,其半衰期约2-3小时,因其周转快,故维持其细胞内高水平以保证MM细胞生存,有赖于基因转录机制的持续活化。细胞内基因转录受P-TEFb(CDK9/cyclin T1/2复合体)调控,其通过磷酸化RNA-Pol II的C末端(CTD),激活该酶启动转录并介导转录延伸。在多种肿瘤(包括MM)细胞中,P-TEFb复合体不同亚基及其负性调节因子常见基因突变或表达异常,导致基因转录的持续激活,以维持肿瘤细胞的生存和快速增殖,并抵抗环境应激(包括化疗、放疗等),故肿瘤细胞被认为具有转录依赖性(或称转录成瘾性)。本课题通过对P-TEFb复合体及其负性调控因子的研究,探讨了P-TEFb复合体介导的转录依赖对MM发生、发展(特别是耐药)的影响,并提出了通过靶向P-TEFb转录调控Mcl-1表达或直接靶向Mcl-1蛋白抗凋亡功能、联合蛋白酶体抑制剂硼替佐米(Bortezomib,Btz;MM一线治疗药物)或Bcl-2抑制剂维奈托克(Venetoclax,又称ABT-199;已获批用于血液肿瘤治疗)的双靶点干预策略(dual-targeted therapy,DTT),以克服MM耐药。研究方法:1.利用R2:Genomics Analysis and Visualization Platform平台,通过MM转录组学数据库,分析MM患者中目的基因的表达水平,包括组成P-TEFb复合体相关基因CDK9和CCNT1/2(编码cyclin T1/T2)及其负调节基因HEXIM1/2等;KEGG分析目的基因相关的信号通路和功能;Kaplan–Meier曲线分析CDK9、CCNT1/2、HEXIM1/2等基因的表达水平与总生存率(OS)的相关性。2.RNA-seq和Western blot验证CDK9和cyclin T1/2及负调节因子(如HEXIM1/2等)在MM患者临床样本中的表达情况;sh RNA敲低实验分析CDK9和cyclin T1对RNA Pol II活性及Mcl-1表达的影响;免疫共沉淀实验分析CDK抑制剂Dinaciclib(又称SCH727965)等靶向P-TEFb(CDK9/cyclin T1复合体)的作用机制,及其对下游Mcl-1表达的影响。3.Western blot和流式细胞术分析CDK9特异性抑制剂Bay-1143572(Atuveciclib)通过抑制Mcl-1转录对不同MM细胞系(包括Btz耐药细胞系)的杀伤作用及其机制;应用荷瘤小鼠动物模型及小动物成像系统、免疫组化、Western blot实验等验证已进入临床试验的CDK抑制剂对MM细胞移植瘤的体内作用及其机制。4.流式细胞术分析CDK9抑制剂Bay-1143572联合Bcl-2抑制剂ABT199的抗MM作用;Western blot分析并验证两种抑制剂的协同机制;通过荷瘤小鼠模型及小动物成像系统验证两种抑制剂联合治疗对小鼠生存的影响;流式细胞术和Western blot分析CDK9抑制剂Bay-1143572与Btz联合应用的协同作用及其分子机制。5.RNA-seq分析Bcl-2凋亡蛋白家族成员相关基因MCL1、BCL2、BCL2L1(编码Bim)等在MM患者临床样本中的表达情况;流式细胞术分析Mcl-1抑制剂S63845对不同MM细胞系的杀伤作用;通过Western blot和免疫共沉淀实验分析S63845联合Btz的协同作用及其分子机制。6.Western blot分析比较Mcl-1、Bcl-2、Bim等在不同MM细胞系中的表达情况;流式细胞术、Western blot和免疫共沉淀检测Mcl-1抑制剂S63845联合ABT-199对MM细胞的杀伤作用及其机制。7.选择GEO数据库中RRMM患者骨髓CD138+细胞单细胞测序数据集(GSE161801),通过与健康供者比较,排除正常细胞,并排除NK细胞、单核细胞等细胞成分,获得MM细胞,进而对其进行分亚群;基于这些亚群,分析转录机制相关基因差异表达,获得存在转录机制异常的亚群,GO分析发现RNA Pol II通路异常亚群,进一步GO分析其相关功能;基于每个亚群Top 5转录机制相关差异表达基因,比较其在不同亚群中表达的情况。研究结果:1.转录组分析基因表达谱(GEP)发现,在意义未明单克隆免疫球蛋白血症(MGUS)和冒烟型骨髓瘤(SMM)向MM发生过程中,CDK9和CCNT1/2表达逐步升高,而HEXIM1/2表达则逐渐下降;与正常供者相比,初诊MM(NDMM)和复发难治MM(RRMM)患者中CDK9表达显著上调,而HEXIM1则下调;CDK9和CCNT1表达水平越高,MM患者的总生存期越短,呈正相关;相反,HEXIM1/2的表达则与患者的总生存期短(预后不良)呈负相关。KEGG分析显示,CDK9表达相关基因富集的主要信号通路包括spliceosome、OXPHOS、proteasome、RNA polymerase、DNA replication等,与CCNT1相似,与CCNT2则明显不同,而与HEXIM1/2则相反。提示MM细胞中CDK9和cyclin T1可能是构成P-TEFb的主要成分,而HEXIM1可能是其主要的负调节因子。RNA-seq分析显示,在MM患者临床样本中,CDK9表达显著上调,CCNT1在多数患者中上调,而HEXIM1则在部分患者中下调。进一步证实了上述推测。2.在MM细胞中,敲减CCNT1(编码cyclin T1)后,磷酸化CTD(p-CTD)和Mcl-1表达下调,但不显著影响CDK9表达;敲减CDK9后,不仅p-CTD和Mcl-1表达下调,且cyclin T1明显下调,提示与CDK9结合可能增加cyclin T1蛋白稳定性;CDK抑制剂Dinaciclib显著抑制p-CTD并下调Mcl-1,但不影响CDK9蛋白水平及磷酸化;免疫共沉淀发现,Dinaciclib处理明显减少CDK9与cyclin T1的结合,但增加CDK9或cyclin T1与HEXIM1的结合,提示Dinaciclib可能通过P-TEFb解聚而抑制其活性。3.CDK9特异性抑制剂Bay-1143572剂量依赖性抑制p-CTD并下调Mcl-1表达,诱导MM敏感细胞株(U266)及其Btz耐药细胞株(PS-R)凋亡,伴PARP剪切增加;与此相同,多种已进入临床试验的CDK抑制剂Dinaciclib、Flavopiridol(又称Alvocidib)和Seliciclib均能通过抑制p-CTD和Mcl-1表达,显著诱导MM细胞凋亡;在皮下移植瘤小鼠模型中,CDK抑制剂处理能显著抑制肿瘤生长,并与抑制p-CTD和下调Mcl-1有关;在经尾静脉注射MM细胞建立的骨髓归巢小鼠模型中,CDK抑制剂处理明显抑制肿瘤生长,免疫组化染色证实药物干预组小鼠骨髓切片中CD138阳性MM细胞显著减少。提示CDK抑制剂或特异性CDK9抑制剂,靶向P-TEFb,通过下调Mcl-1,发挥抗MM作用。4.CDK9抑制剂Bay-1143572和ABT199联合应用,显著提高对MM细胞(包括Btz耐药株)的杀伤作用;Bay-1143572处理阻断ABT199介导的Mcl-1上调,促进细胞凋亡;C57BL/Ka Lw Rij小鼠尾静脉注射5TGM1细胞建立的模型中,CDK抑制剂和ABT199联合治疗,明显降低肿瘤负荷,并延长动物生存;与此相似,Bay-1143572与Btz联合,也表现出显著的协同作用,并抑制p-CTD和Mcl-1的表达。5.RNA-seq分析MM患者临床样本显示,Bcl-2家族成员抗凋亡基因(MCL1和BCL2)呈显著高表达,而促凋亡基因BCL2L1则呈低表达,提示MM细胞对Mcl-1和Bcl-2的依赖性;Mcl-1抑制剂S63845对MM敏感细胞株(U266)及其耐药株(PS-R)具有杀伤作用,且后者可能更为敏感;与髓内MM细胞株(KMS28-BM)对S63845高度敏感相反,其相应的髓外MM细胞株(KMS28-PB)则对S63845几乎无反应,提示MM髓外病变可能对Mcl-1抑制剂不敏感;进一步研究发现,S63845主要通过阻断Mcl-1和Bim的结合,释放并激活Bim,诱导MM细胞凋亡;与CDK抑制剂相似,S63845和Btz联合应用,对MM敏感细胞株(U266)及其Btz耐药系(PS-R),均显示出明显的协同作用,显著提高Btz的抗MM作用,并可能克服耐药。6.与MM患者临床样本相符,MM细胞系中抗凋亡蛋白Bcl-2和Mcl-1呈高表达,而促凋亡蛋白Bim也呈现高水平,提示Bim通过与Mcl-1和Bcl-2结合而失活,但可能使MM细胞处于凋亡待激(priming)状态,对凋亡敏感;MM细胞对Bcl-2抑制剂不敏感,可能与ABT-199抑制Bim与Bcl-2结合,但Bim转而与Mcl-1结合有关;免疫共沉淀和Western blot分析显示,Mcl-1抑制剂S63845则能阻断Bim与Mcl-1结合,可显著促进ABT199诱导的MM细胞凋亡,从而发挥协同抗MM作用。7.RRMM患者骨髓CD138+细胞单细胞转录组分析显示,同一患者中存在多个亚群(亚克隆;n=27),其差异基因表达各不相同,验证了MM的克隆异质性,其中半数以上亚群存在显著的转录机制相关基因表达异常,但其涉及的转录调控环节有所不同,并发现一个与RNA Pol II通路异常密切相关的亚群,其可能参与内质网应激、蛋白质翻译、B细胞激活等已知与MM生物学紧密相关的功能。因此,提示MM细胞的转录依赖性存在克隆异质性。结论:1.Mcl-1与MM一线药物蛋白酶体抑制剂(如Btz等)获得性耐药密切相关,Mcl-1蛋白半衰期短,故维持其在MM细胞中的高水平有赖于P-TEFb介导的转录机制的持续激活,即转录依赖性(成瘾性),可能参与MM耐药。2.MM细胞中转录机制的持续激活主要与CDK9和cyclin T1高表达及其相互结合(形成P-TEFb复合体)有关。3.单细胞转录组分析,提出MM细胞转录依赖性存在克隆异质性,并发现一个RNA Pol II通路异常的亚克隆,其可能与内质网应激、蛋白质翻译、B细胞激活等功能有关。4.CDK抑制剂(如Dinaciclib)能使P-TEFb(CKD9与cyclin T1复合体)解离,并促进CDK9或cyclin T1与HEXIM1结合,从而抑制P-TEFb活化,导致Mcl-1表达下调,代表了CDK抑制剂的一种新的作用机制。5.P-TEFb及其下游Mcl-1作为MM的分子靶点,应用泛CDK或CDK9特异性抑制剂靶向P-TEFb下调Mcl-1、或直接靶向Mcl-1蛋白的抗凋亡功能,联合Bcl-2抑制剂ABT-199或Btz,协同诱导MM细胞凋亡,提示双靶点干预策略,能显著增强抗MM作用,并有助于克服MM耐药。更多还原

【Abstract】 Background and ObjectiveDrug resistance is the major challenge in the clinical management of patients with multiple myeloma(MM).However,the effective interventions are still lacking,largely because the mechanism(s)underlying drug resistance remains virtually uncertain.Among numerous candidate targets involving drug resistance,the anti-apoptotic Mcl-1(one key member of the Bcl-2 family)overexpression is closely related to drug resistance of various cancer types,including MM.Mcl-1 is a kind of fast turnover and thus short-lived protein with a half-life of approximately 2-3 hours.Thus,powerful transcription machinery is required to ensure de novo synthesis of a large amount of Mcl-1 protein for maintaining its high levels in MM cells that are featured by highly efficient protein turnover via ubiquitin-proteasome system(UPS).The transcription of m RNA is primarily governed by positive transcription elongation factor b(P-TEFb,a complex of CDK9/cyclin T1 and/or T2),which functions to promote the initiation and particularly elongation of m RNA transcription by phosphorylating the C-terminal domain(CTD)of RNA polymerase II(Pol II)at serine 2 and 5.In this context,genes encoding the subunits of the P-TEFb complex are often overexppressed while genes encoding P-TEFb negative regulators are frequently mutated or down-regulated in various malignant cells,therefore resulting in constitutive activation of P-TEFb to ensure highly active transcription for satisfy demand for those fast turnover proteins(e.g.,Mcl-1)essential for survival and rapid growth of tumor cells(such as MM cells),especially under environmental stress and on various therapies(e.g.,chemotherapy and targeted therapy).Thus,these types of cancer such as MM may be considered to be transcription-dependent or-addictive.In this study,we have investigated the regulatory mechanisms of the P-TEFb complex and its negative regulators in MM,particularly involving drug resistance.Based on the regulation of Mcl-1 expression by P-TEFb and the anti-apoptotic function of Mcl-1,we also proposed a dual-targeted therapy(DTT)strategy combining CDK inhibitors that downregulate Mcl-1 via inhibiting P-TEFb or a Mcl-1 inhibitor that directly inhibits Mcl-1 with the proteasome inhibitor bortezomib(Btz,the first-line therapeutic agent for MM treatment)or Bcl-2 inhibitor ABT-199(also known as Venetoclax,which has been approved for treating various hematologic malignancies)to improve anti-MM efficacy and help overcome drug resistance.Methods1.Using the MM transcriptomic database available on the R2 Genomics Analysis and Visualization Platform,we analyzed the expression of transcription-regulatory genes in MM patients,including the P-TEFb complex-related genes CDK9,CCNT1/2(encoding cyclin T1 and T2,respectively)and its negatively regulatory genes HEXIM1/2.We further performed KEGG analysis on the signaling pathways and biological functions that are associated with their expression.Moreover,we also analyzed the correlation between the expression of CDK9,CCNT1/2,HEXIM1/2 and other P-TEFb-related genes and overall survival(OS)of MM patients.2.Using RNA-seq and Western blot analysis,we varified the expression of CDK9,cyclin T1/2 and their negative regulators HEXIM1/2 in primary samples of MM patients.The effects of CDK9 and cyclin T1 on the activation of RNA Pol II and expression of Mcl-1 were analyzed using genetic means(e.g.,sh RNA)or pharmacological pan-CDK inhibitors(e.g.,dinaciclib,also known as SCH727965)via Western blot via co-immunoprecipitation(co-IP)experiments.3.The novel CDK9-specific inhibitor Atuveciclib(or Bay-1143572)was then used to validate the effects of agents specifically targeting CDK9 on the activity of P-TEFb,expression of Mcl-1,and apoptosis detected by Western blot analysis and flow cytometry.We also examined the in vivo anti-MM effects and the underlying molecular mechanism of the clinically relevant CDK inhibitors in MM xenograft mouse models via immunohistochemistry and Western blot analysis.4.The synergistic interaction between the CDK9-specific inhibitor Bay-1143572 and the Bcl-2 inhibitor ABT199 and underlying molecular mechanism were examined in killing of MM cells detected by flow cytometry,Western blot analysis,and co-IP.The in vivo efficacy of this combination regimen was further validated in a C57BL/Ka Lw Rij mouse model established by tail vein injection of the murine MM cells(5TGM1)expressing luciferase using a small animal imaging system.Alternatively,the synergy and between Bay-1143572 and Btz was also tested by flow cytometry and Western blot analysis.5.Using RNA-seq analysis,the expression of various Bcl-2 family members(e.g.,MCL1,BCL2,and BCL2L1)in primary samples of MM patients was examined.Then,the anti-MM activity of the novel Mcl-1 inhibitor S63845,alone or in combination with Btz,in multiple MM cell lines was tested by flow cytometry.The molecular mechanism for the synergitic interaction between S63845 and Btz was analyzed via Western blot and co-IP experiments.6.The expressions levels of Mcl-1,Bcl-2 and Bim in different MM cell lines were detected and compared by Western blot.The synergistic effect of the combination of Mcl-1 inhibitor S63845 and Bcl-2 inhibitor ABT-199 on killing MM cells was detected by flow cytometry,Western blot and co-IP.7.Using a GEO dataset(GSE161801)containing the single-cell RNA sequencing(sc RNA-seq)data of 20 refractory MM patients,we further analyzed the heterogeneity of MM cells in transcription addiction.Results1.Through the transcriptomic analysis on gene expression profile(GEP)of MM patients,we found that the expression of CDK9 and CCNT1/2 progressively increased during the development of MGUS and SMM to MM,while the expression of HEXIM1/2 gradually decreased,when compared with normal donors.Moreover,CDK9 expression was significantly up-regulated in patients with both newly diagnosed MM(NDMM)and relapsed refractory MM(RRMM),while HEXIM1 was markedly downregulated.Of note,the high levels of CDK9 and CCNT1 were associated with shorter OS of MM patients.In contrast,the expression of HEXIM1/2 negatively correlated with OS of MM patients.Furthermore,RNA-seq analysis validated up-regulation of CDK9 and CCNT1 in most primary samples of MM patients,but HEXIM1 was down-regulated at least in some patients.KEGG analysis revealed that CDK9-related genes were enriched for multiple crucial signal pathways,including spliceosome,OXPHOS,proteasome,RNA polymerase,DNA replication,etc.,which were similar to those for CCNT1 but not CCNT2,while opposite to those for HEXIM1/2.Together,these results suggest that CDK9 and cyclin T1 might be the primary components for forming the P-TEFb complex in MM cells,while HEXIM1 might represent its main negative regulator.2.In MM cells,knock-down of CDK9 by sh RNA markedly decreased the levels of cyclin T1,phosphorylated CTD(p-CTD)of RNA Pol II,and Mcl-1,while knockdown of CCNT1 down-regulated the expression of p-CTD and Mcl-1 but did not significantly affect CDK9 expression,suggesting that the binding with CDK9 might increase the stability of cyclin T1 protein.Consistently,the pan-CDK inhibitor dinaciclib also significantly inhibited p-CTD and down-regulated Mcl-1,without affecting the protein level and phosphorylation of CDK9.Notably,co-IP analysis revealed for the first time,to the best of our knowledge,that exposure to dinaciclib sharply diminished the binding of cyclin T1 with CDK9,while increased the binding of HEXIM1 to CDK9 and cyclin T1.Thus,these findings indicate that CDK inhibitors like dinaciclib could act to disassemble the P-TEFb complex,a novel mechanism of action for CDK inhibitors to target P-TEFb.They also suggest,though indirectly,that in addition to high levels of CDK9 and cyclin T1,the assembly of these subunits to form the P-TEFb complex might play an important role in maintaining the constitutive activation of P-TEFb and thus high efficiency of transcription machinery in MM cells.3.The CDK9-specific inhibitor Bay-1143572 effectively inhibited p-CTD,downregulated Mcl-1 expression in a dose-dependent manner,and induced apoptosis in drugnaive cells(U266)and their Btz-resistant counterparts(PS-R),with increased PARP cleavage.Similarly,various clinically relevant CDK inhibitors,including dinaciclib,flavopiridol(also known as alvocidib),and seliciclib,significantly blocked the expression of p-CTD and Mcl-1 and induced MM cell apoptosis in vitro.Their antiMM activity was further validated in vivo in a subcutaneous xenograft mouse model,in which administration of CDK inhibitors markedly inhibited tumor growth,in association with inhibition of p-CTD and down-regulation of Mcl-1.Furthermore,the treatment with CDK inhibitors sharply inhibited the growth of MM cells homing in the bone marrow in a mouse models established via tail vein injectiong of MM cells.Immunohistochemical staining confirmed that CDK inhibitor administration eliminated CD138-positive MM cells in the bone marrow.Together,these results argue that both pan-CDK inhibitors or CDK9-specific inhibitors are effective against MM cells(including those resistant to Btz)via down-regulation of Mcl-1 by targeting P-TEFb.4.Co-administration of the CDK9-specific inhibitor Bay1143572 significantly increased apoptosis induced by the Bcl-2 antagonist ABT199 in various MM cell lines,which were insensitive to ABT-199,including those resistant to Btz.Whereas exposure to ABT-199 up-regulated the protein levels of Mcl-1,this event was blocked by Bay-1143572.In a C57BL/Ka Lw Rij mouse model established by tail vein injection of5TGM1 cells,the combination of CDK inhibitors with ABT-199 dramatically reduced tumor burden and prolonged animal survival.Analogously,Bay-1143572 also significantly potentiated the anti-MM activity of Btz via inhibition of p-CTD and downregulation of Mcl-1.5.RNA-seq analysis revealed high expresion of the genes of the Bcl-2 family members(including MCL1 and BCL2)in MM patients.The Mcl-1 inhibitor S63845 displayed a marked lethal effect on both drug-naive U266 and the Btz-resistant PS-R cells,while the latter was even more sensitive than the former.However,unlike highly S63845-sensitive phenotype of KMS28-BM cell line derived from MM cells localized in the bone marrow,their counterparts(KMS28-PE)derived from exmedulatory lesions were almost completely insensitivie to S63845,suggesting independence of Mcl-1 for their survival of exmedulatory MM cells(e.g.,plasma cell leukemia and extramedullary plasmacytomas).Mechanistically,S63845 expsorue blocked the binding of the proapoptotic protein Bim to Mcl-1 and thus induced apoptosis of MM cells via releasing and activating Bim.Consistent with the findings on CDK9 inhibitors that downregulated Mcl-1 expression,co-administration of S63845 and Btz displayed a synergistic lethal effect on both drug-naive U266 cells and Btz-resistant PS-R cells,suggesting a promising approach to improve the anti-MM activity of Btz and even overcome drug resistance.6.Similar to the observations in primary samples of MM patients,the antiapoptotic proteins Bcl-2 and Mcl-1 were highly expressed in MM cell lines,while surprisingly,the proapoptotic protein Bim also exhibited high protein levels.However,Bim was sequestered and inactivated by binding to Mcl-1 and Bcl-2,a mechanism for protecting MM cells from apoptosis.In this context,we considered that MM cells might be primed for death.Co-IP analysis showed that the Bcl-2 inhibitor ABT-199 disassociated the binding of Bcl-2 with Bim,the latter instead bound to Mcl-1,explaining the insentivity of MM cells ABT-199.In this context,co-administration of the Mcl-1 inhibitor S63845 with ABT-199 was able to relsease Bim from both Bcl-2and Mcl-1,leading to a highly synergistic anti-MM effect of this regimen combining these two agents on MM cells,including drug-resistant ones.7.Through bioinformatics analyses of the sc RNA-seq data,we obtained 27clusters(subclones)of MM cells,from which 14 clusters were identified to be associated with various abnormalities involving transcriptional machinery.Moreover,one unique cluster was further characterized as RNA Pol II-dependent subclone,in association with alterations in ER stress(or response to unfolded protein),cytoplasmic translation,and B cell activation,the events known to contribute to MM biology.Conclusions1.Mcl-1 is closely related to the acquired Btz resistance.As a rapid turnover protein,Mcl-1 maintains its high level in MM cells through constitutive activation of the P-TEFb-dependent transcription,thus considering MM as transcription-dependent or-addictive malignancy and P-TEFb as a potential target for overcoming drug resistance.2.In MM cells,the constitutive activation of P-TEFb likely stems from not only high expression of CDK9 and cyclin T1 but also assembly of these two main subunits to form the active P-TEFb complex.3.MM cells display marked clonal heterogeneity in transcriptiona addition,while a novel clone of MM cells likely depending upon RNA Pol II-mediated transcription machinery is identified for the the first time,to the best of our knwoledge,which may functionally contribute to the activation of ER stress(or unfolded protein response)and B cell activation pathway.4.The CDK inhibitor(such as dinaciclib)acts to disassemble the P-TEFb complex while promotes the binding of CDK9 and cyclin T1 to HEXIM1,thereby inhibiting the activity of P-TEFb,suppressing Mcl-1 expression,and promoting MM cell apoptosis.This may represent a novel mechanism of action for CDK inhibitors,providing an alternative basis for developing more effective and specific CDK9 inhibitors.5.Either down-regulation of Mcl-1 by CDK9 inhibition or direct disability of Mcl-1 by specific Mcl-1 inhibitors potentiates the anti-MM activity of Bcl-2 antagonists or the first-line therapeutic agents(e.g.,Btz)in a highly synergistic manner.High expression of both anti-(e.g.,Bcl-2 and Mcl-1)and pro-apoptotic(e.g.,Bim)Bcl-2family members makes MM cells more vulnerable to this dual-targeted therapy(DDT)strategy,which could improve the efficacy of anti-MM agents and likely overcome intrinsic resistance to Bcl-2 antagonists(e.g.,ABT-199)and acquired resistance to the first-line therapeutic agents in MM.更多还原