degrasyn and Multiple-Myeloma

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.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyanoacrylates; Drug Repositioning; Drug Synergism; Female; HEK293 Cells; Humans; Mebendazole; Mice, Inbred BALB C; Mice, Nude; Multiple Myeloma; Proof of Concept Study; Protein Binding; Proto-Oncogene Proteins c-maf; Pyridines; Ubiquitin-Specific Proteases; Ubiquitination; Xenograft Model Antitumor Assays

Usp9x was recently shown to be highly expressed in myeloma patients with short progression-free survival and is proposed to enhance stability of the survival protein Mcl-1. In this study, we found that the partially selective Usp9x deubiquitinase inhibitor WP1130 induced apoptosis and reduced Mcl-1 protein levels. However, short hairpin RNA-mediated knockdown (KD) of Usp9x in myeloma cells resulted in transient induction of apoptosis, followed by a sustained reduction in cell growth. A compensatory upregulation of Usp24, a deubiquitinase closely related to Usp9x, in Usp9x KD cells was noted. Direct Usp24 KD resulted in marked induction of myeloma cell death that was associated with a reduction of Mcl-1. Usp24 was found to sustain myeloma cell survival and Mcl-1 regulation in the absence of Usp9x. Both Usp9x and Usp24 were expressed and activated in primary myeloma cells whereas Usp24 protein overexpression was noted in some patients with drug-refractory myeloma and other B-cell malignancies. Furthermore, we improved the drug-like properties of WP1130 and demonstrated that the novel compound EOAI3402143 dose-dependently inhibited Usp9x and Usp24 activity, increased tumor cell apoptosis, and fully blocked or regressed myeloma tumors in mice. We conclude that small-molecule Usp9x/Usp24 inhibitors may have therapeutic activity in myeloma.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cyanoacrylates; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, Mantle-Cell; Male; Mice; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Pyridines; Ubiquitin Thiolesterase

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cyanoacrylates; Dimerization; Humans; Models, Molecular; Multiple Myeloma; Nitriles; Pyridines; Ubiquitin Thiolesterase

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,β-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P(∗∗) level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C(β) carbon were utilized in developing a model to explain the compound activity.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Humans; Janus Kinase 2; Mice; Mice, Nude; Models, Molecular; Multiple Myeloma; Signal Transduction; STAT3 Transcription Factor; Structure-Activity Relationship; Tyrphostins; Ubiquitin

c-Myc is a highly unstable transcription factor whose deregulation and increased expression are associated with cancer. Degrasyn, a small synthetic molecule, induces rapid degradation of c-Myc protein in MM-1 multiple myeloma and other tumor cell lines. Destruction of c-Myc by degrasyn requires the presence of a region of c-Myc between amino acid residues 316 and 378 that has not previously been associated with c-Myc stability. Degrasyn-induced degradation of c-Myc depends on proteasomes but is independent of the degron regions previously shown to be important for ubiquitin-mediated targeting and proteasomal destruction of the protein. Degrasyn-dependent c-Myc proteolysis is not mediated by any previously identified c-Myc regulatory mechanism, does not require new protein synthesis, and does not depend on the nuclear localization of c-Myc. Degrasyn reduced c-Myc levels in A375 melanoma cells and in A375 tumors in nude mice, and this activity correlated with tumor growth inhibition. Together, these results suggest that degrasyn reduces the stability of c-Myc in vitro and in vivo through a unique signaling process that uses c-Myc domains not previously associated with c-Myc regulation.

Topics: Animals; Cell Line, Tumor; Cyanoacrylates; Down-Regulation; Enzyme Activation; Female; HeLa Cells; Humans; Melanoma; Mice; Mice, Nude; Multiple Myeloma; Nitriles; Peptide Mapping; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; Pyridines; Xenograft Model Antitumor Assays