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4-anilinoquinazoline-containing inhibitors of the epidermal growth factor receptor (EGFR) are widely used in non-small cell lung cancer patients with mutated EGFR, but they are less effective in multiple myeloma (MM), a fatal malignancy derived from plasma cells. The present study designed a series of novel compounds by conjugating a peroxide bridge to the 4-anilinoquinazoline pharmacophore. Further studies showed that these agents such as 4061 and 4065B displayed potent activity to induce MM cell apoptosis by upregulating pro-apoptotic p53 and Bax while downregulating pro-survival Bcl-2. The mechanistic analysis revealed that both 4061 and 4065B inhibited IGF1-R, AKT and mTOR activation in a concentration dependent manner but had no effects on the expression of their total proteins, suggesting the conjugates of endoperoxide and 4-anilinoquinazoline may exert its anti-myeloma activity by targeting the IGF1-R/AKT/mTOR pathway.

TMEPAI (transmembrane prostate androgen-induced protein, also called prostate transmembrane protein, androgen-induced 1 (PMEPA1)) is a type I transmembrane (TM) protein, but its cellular function is largely unknown. Here, studying factors influencing the stability of c-Maf, a critical transcription factor in multiple myeloma (MM), we found that TMEPAI induced c-Maf degradation. We observed that TMEPAI recruited NEDD4 (neural precursor cell expressed, developmentally down-regulated 4), a WW domain-containing ubiquitin ligase, to c-Maf, leading to its degradation through the proteasomal pathway. Further investigation revealed that TMEPAI interacts with NEDD4 via its conserved PY motifs. Alanine substitution or deletion of these motifs abrogated the TMEPAI complex formation with NEDD4, resulting in failed c-Maf degradation. Functionally, TMEPAI suppressed the transcriptional activity of c-Maf. Of note, increased TMEPAI expression was positively associated with the overall survival of MM patients. Moreover, TMEPAI was down-regulated in MM cells, and re-expression of TMEPAI induced MM cell apoptosis. In conclusion, this study highlights that TMEPAI decreases c-Maf stability by recruiting the ubiquitin ligase NEDD4 to c-Maf for proteasomal degradation. Our findings suggest that the restoration of functional TMEPA1 expression may represent a promising complementary therapeutic strategy for treating patients with MM., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The deubiquitinase USP5 stabilizes c-Maf, a key transcription factor in multiple myeloma (MM), but the mechanisms and significance are unclear. In the present study, USP5 was found to interact with c-Maf and prevented it from degradation by decreasing its polyubiquitination level. Specifically, the 308th and 347th lysine residues in c-Maf were critical for USP5-mediated deubiquitination and stability. There are five key domains in the USP5 protein and subsequent studies revealed that the cryptic ZnF domain and the C-box domain interacted with c-Maf but the UBA1/UBA2 domain partly increased its stability. Notably, MafA and MafB are also members of the c-Maf family, however, USP5 failed to deubiquitinate MafA, suggesting its substrate specificity. In the functional studies, USP5 was found to promoted the transcriptional activity of c-Maf. Consistent with the high level of c-Maf protein in MM cells, USP5 was also highly expressed. When USP5 was knocked down, c-Maf underwent degradation. Interestingly, USP5 silence led to apoptosis of MM cells expressing c-Maf but not MM cells lacking c-Maf, indicating c-Maf is a key factor in USP5-mediated MM cell proliferation and survival. Consistent with this finding, WP1130, an inhibitor of several Dubs including USP5, suppressed the transcriptional activity of c-Maf and induced MM cell apoptosis. When c-Maf was overexpressed, WP1130-induced MM cell apoptosis was abolished. Taken together, these findings suggest that USP5 regulates c-Maf stability and MM cell survival. Targeting the USP5/c-Maf axis could be a potential strategy for MM treatment.

Background: UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown., Methods: Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively., Results: UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway., Conclusions: UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.

The mammalian target of rapamycin (mTOR) is extensively involved in multiple myeloma (MM) pathophysiology. In the present study, we reported a novel small molecule SC06 that induced MM cell apoptosis and delayed MM xenograft growth in vivo. Oral administration of SC06 to mice bearing human MM xenografts resulted in significant inhibition of tumor growth at doses that were well tolerated. Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK. Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex. SC06 also suppressed the phosphorylation of 4E-BP1 and P70S6K, two typical substrates in the mTORC1 signaling pathway. Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies. Therefore, given the potency and low toxicity, SC06 could be developed as a potential anti-MM drug candidate by disrupting the mTOR signaling. [ABSTRACT FROM AUTHOR]