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 the present study we found the Otub1/c-Maf axis could be a potential target. Otub1, an OTU family deubiquitinase, was found to interact with c-Maf by mass spectrometry. Otub1 abrogates c-Maf K48-linked polyubiquitination thus preventing its degradation and enhancing its transcriptional activity. Specifically, this deubiquitinating activity depends on its Lys71 and the N-terminus but independent UBE2O, a known E2 of c-Maf. Otub1 promotes MM cell survival and MM tumor growth. In contrast, silence of Otub1 leads to c-Maf degradation and c-Maf-expressing MM cell apoptosis. Therefore, the Otub1/c-Maf axis could be a 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, from which the generic cardiac glycoside lanatoside C (LanC) is found to prevent c-Maf de-ubiquitination and induces its degradation by disrupting the interaction of Otub1 and c-Maf. Consequently, LanC inhibits c-Maf transcriptional activity, induces c-Maf-expressing MM cell apoptosis, and suppresses MM growth and prolongs overall survival of model mice but without apparent toxicity. Therefore, the present study identifies Otub1 as a novel deubiquitinase of c-Maf and establishes that the Otub1/c-Maf axis is a potential therapeutic target for MM.

Background Immune checkpoint inhibitor-related cardiotoxicity is one of the most lethal adverse effects, and thus, the identification of underlying mechanisms for developing strategies to overcome it has clinical importance. This study aimed to investigate whether microbiota-host interactions contribute to PD-1/PD-L1 inhibitor-related cardiotoxicity. Methods A mouse model of immune checkpoint inhibitor-related cardiotoxicity was constructed by PD-1/PD-L1 inhibitor BMS-1 (5 and 10 mg/kg), and cardiomyocyte apoptosis and cardiotoxicity were determined by hematoxylin and eosin, Masson’s trichome and TUNEL assays. 16S rRNA sequencing was used to define the gut microbiota composition. Gut microbiota metabolites short-chain fatty acids (SCFAs) were determined by HPLC. The serum levels of myocardial enzymes (creatine kinase, aspartate transaminase, creatine kinase-MB and lactate dehydrogenase) and the production of M1 factors (TNF-α and IL-1β) were measured by ELISA. The colonic macrophage phenotype was measured by mmunofluorescence and qPCR. The expression of Claudin-1, Occludin, ZO-1 and p-p65 was measured by western blot. The gene expression of peroxisome proliferator-activated receptor α (PPARα) and cytochrome P450 (CYP) 4X1 was determined using qPCR. Statistical analyses were performed using Student’s t-test for two-group comparisons, and one-way ANOVA followed by Student–Newman–Keul test for multiple-group comparisons. Results We observed intestinal barrier injury and gut microbiota dysbiosis characterized by Prevotellaceae and Rikenellaceae genus depletion and Escherichia-Shigella and Ruminococcaceae genus enrichment, accompanied by low butyrate production and M1-like polarization of colonic macrophages in BMS-1 (5 and 10 mg/kg)-induced cardiotoxicity. Fecal microbiota transplantation mirrored the effect of BMS-1 on cardiomyocyte apoptosis and cardiotoxicity, while macrophage depletion and neutralization of TNF-α and IL-1β greatly attenuated BMS-1-induced cardiotoxicity. Importantly, Prevotella loescheii recolonization and butyrate supplementation alleviated PD-1/PD-L1 inhibitor-related cardiotoxicity. Mechanistically, gut microbiota dysbiosis promoted M1-like polarization of colonic macrophages and the production of proinflammatory factors TNF-α and IL-1β through downregulation of PPARα-CYP4X1 axis. Conclusions Intestinal barrier dysfunction amplifies PD-1/PD-L1 inhibitor-related cardiotoxicity by upregulating proinflammatory factors TNF-α and IL-1β in colonic macrophages via downregulation of butyrate-PPARα-CYP4X1 axis. Thus, targeting gut microbiota to polarize colonic macrophages away from the M1-like phenotype could provide a potential therapeutic strategy for PD-1/PD-L1 inhibitor-related cardiotoxicity. Graphical abstract

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c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis. Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival; therefore, the USP5/c-Maf axis could be a potential target for myeloma therapy. As a concept of principle, the present study established a USP5/c-Maf-based luciferase system that was used to screen an FDA-approved drug library. It was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-Maf-expressing myeloma cells. Moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. Further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the USP5/c-Maf axis. Mebendazole downregulated USP5 expression and disrupted the interaction between USP5 and c-Maf, thus leading to increased levels of c-Maf ubiquitination and subsequent c-Maf degradation. Mebendazole inhibited c-Maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-Maf downstream genes. In summary, this study identified mebendazole as a USP5/c-Maf inhibitor that could be developed as a novel antimyeloma agent.