The Discovery And Functional Analysis Of Otub1/c-Maf Axis Inhibitors In The Development Of Multiple Myeloma

The oncogenic transcription factor c-Maf has been proposed as an ideal therapeutic target for multiple myeloma(MM)but how to achieve it is still elusive.In our previous study we found Otub1,an OTU family deubiquitinase,interacted with c-Maf by mass spectrometry.Otub1 abrogated c-Maf K48-linked poly-ubiquitination thus preventing its degradation and enhancing its transcriptional activity.Therefore,Otubl/c-Maf axis could be a potential therapeutic target of MM.In order to explore this concept,we performed a c-Maf-recognition element-driven luciferase-based screen against FDA-approved drugs and natural products and found 3 potential inhibitors:Lanatoside C(LanC),Nanchangmycin(Nam),acevaltrate(AVT).We found that these three candidates could prevent c-Maf de-ubiquitination and induce its degradation.Consequently,these inhibitors inhibited c-Maf transcriptional activity,induced MM cell apoptosis,and suppressed MM xenograft tumor growth but without apparent toxicity.In addition,Nam was found to induce blood cancer cell apoptosis and delay leukemia xenograft tumor growth via promoting MDM2 ubiquitination and down-regulating MDM2 protein level which independent with p53 antitumor activity.In conclusion,the present study testified that Otub1 is an oncogene in MM pathogenesis,and Otub1/c-Maf is a therapeutic target of MM.Aims:To evaluate the function of Otub1 in the developmental stage of MM at a celluar level and in animals,and to perform a c-Maf-recognition element-driven luciferase-based screen against FDA-approved drugs and natural products.Then to testify the screening results and explore the function and mechanism of potential inhibitors,which might provide pharmacological basis for the application of Otubl/c-Maf inhibitors in the treatment of hematological malignancies.Methods:(1)GEO database analysis and RT-PCR method were applied to detect Otubl mRNA level in bone marrow cells from healthy donors and MM patients;At the same time,cell proliferation and apoptosis were detected after the over-expression and silence of Otubl,and these results were further verified in MM-disseminated mouse model.Furthermore,PROGgene database analysis was used to analyze the prognosis of MM patients influenced by Otub1(2)HEK293T cells steadily expressed MARE.Luci,c-Maf and Otublplasmids were incubated with FDA-approved drugs and natural products for 24 hours,followed by c-Maf luciferase activity detection to search for potential inhibitors.Then WB assay was applied to detect c-Maf and PARP protein level.Finally,MTT,RT-PCR,WB and Luciferase assay were adopted to testify cell proliferation and c-Maf transcriptional activity altered by candidate inhibitors,so as to find an appropriate working concentration of those inhibitors.(3)A panel of MM cell lines was treated LanC,Nam,and AVT respectively,followed by c-Maf protein level,mRNA level and ubiquitination assessment,cell proliferation and apoptosis detection by WB,MTT,RT-PCR and flow cytometry methods.Moreover,WB,IP and luciferase assay were used to examine cell apoptosis,c-Maf ubiquitination and transcriptional activity in Otubl over-expressed or knock down MM cells changed by LanC,Nam and AVT,in order to evaluate Otub1 anti-blood cancer activity.(4)Nude mice in MM xenograft model and disseminated model or leukemia xenograft model were detected with tumor growth rate,survival periods,body weight,blood biochemical indexes and related protein level including c-Maf,Otub1,etc.after the treatment of LanC,Nam and AVT.Furthermore,colony forming assay was used to detect CFC production altered by LanC,Nam and AVT.Results:(1)By analysis of two independent datasets,Otub1 expression was found steadily up-regulated from healthy donors to MGUS to SMM,similarly,RT-PCR revealed Otub1 mRNA in MM patients were higher than in healthy donors;Besides,over-expression of Otub1 promoted cell proliferation,in contrast,knockdown of Otub1 induced MM cell apoptosis.Moreover,Otubl promoted death of nude mice carrying MM cell line RPMI-8226 that expressed a high level of endogenous c-Maf,while the survival periods of nude mice carrying a low level of endogenous c-Maf was not altered by Otub1.(2)All three candidates could inhibit Otub1 mediated c-Maf transcriptional activity,down-regulate the mRNA and protein level of c-Maf down-stream genes.At the same time,the candidate compounds could inhibit MM cell proliferation,induce cell apoptosis,showing great anti-MM activity.(3)RT-PCR and ?P/?B assays revealed that LanC and AVT abrogated c-Maf K48-linked poly-ubiquitination,thus down-regulated c-Maf protein level,further induced MM cell apoptosis.Moreover,knock down Otubl weakened c-Maf transcriptional activity and MM cell apoptosis influenced by LanC and AVT,as the same time,over-expression of c-Maf antagonisted apoptosis induced by LanC and AVT.While Nam displayed excellent anti-tumor activity against almost all MM cell lines and a variety of leukemia cell lines through down-regulating MDM2 protein level as well as c-Maf.It is worth emphasizing that Otub1 was necessary in this process.(4)In vivo animal experiments and colony forming assay confirmed that LanC and AVT delayed tumor growth,prolonged the survival period of tumor-bearing mice and inhibited CFC production of blood stem cells.Nam suppressed MM and leukemia xenograft tumor growth and colony formation of bone marrow cells of MM and leukemia patients.In supplement,LanC,Nam and AVT showed no apparent toxicity.Results:Otub1 promoted MM pathogenesis via increasing c-Maf stability and transcriptional activity,and the screening we performed found three candidate inhibitors of Otubl based on the concept.These three candidates could promote c-Maf degradation,inhibit c-Maf transcriptional activity and induce MM cell apoptosis.LanC and AVT disrupted the interaction of c-Maf and Otub1,induced apoptosis of MM cells which expressed a high level of endogenous c-Maf and suppressed the growth of subcutaneous tumor.However,Nam could not only inhibit Otubl/c-Maf pathway,but also induced blood cancer cell apoptosis via promoting MDM2 ubiquitination,regulating Rb/E2F pathway and p21 protein level which was independent with p53 anti-tumor activity.