Dual-Targeted Therapy Based On Transcription Addiction And Apoptosis-Regulatory Proteins Circumvents Drug Resistance In Multiple Myeloma

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.