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.

Ring finger protein 6 (RNF6) is a key oncogene in both prostate cancer and leukemia, but its role is elusive in breast cancer. In the present study, we found that RNF6 was overexpressed in more than 70% of breast cancer tissues and it was associated with overall survival. RNF6 increased breast cancer cell proliferation, migration and reduced cell sensitivity to doxorubicin. Further studies showed that RNF6 was closely associated with increased expression of estrogen receptor, a critical factor in the development of breast cancers. RNF6 was found to induce ERα expression and increased its stability. In doxorubicin-resistant breast cancer cells, RNF6 was found to be elevated in association with increased ERα and anti-apoptotic Bcl-xL, but not pro-apoptotic Bim-1. In consistence with this finding, overexpression of ERα led to increased Bcl-xL but had no effects on Bim-1. Therefore, this study demonstrated that there exists an RNF6/ERα/Bcl-xL axle in breast cancer which promotes cancer cell proliferation and survival. Targeting the RNF6/ERα/Bcl-xL axle could be a promising strategy in the treatment of breast cancer.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is a key player in tumorigenesis of non-small cell lung cancer (NSCLC) and was recently found to be inactivated by tripartite motif containing 25 (TRIM25)-mediated K63-linked polyubiquitination. However, the deubiquitinase (Dub) coordinate TRIM25 in PTEN ubiquitination is still elusive. In the present study, we found that this K63-linked polyubiquitination could be ablated by the ubiquitin-specific protease 10 (USP10) in a screen against a panel of Dubs. We found using coimmununoprecipitation/immunoblotting that USP10 interacted with PTEN and reduced the K63-linked polyubiquitination of PTEN mediated by TRIM25 in NSCLC cells. Moreover, USP10, but not its inactive C424A deubiquitinating mutant or other Dubs, abolished PTEN from K63-linked polyubiquitination mediated by TRIM25. In contrast to TRIM25, USP10 restored PTEN phosphatase activity and reduced the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate, thereby inhibiting AKT/mammalian target of rapamycin progrowth signaling transduction in NSCLC cells. Moreover, USP10 was downregulated in NSCLC cell lines and primary tissues, whereas TRIM25 was upregulated. Consistent with its molecular activity, re-expression of USP10 suppressed NSCLC cell proliferation and migration, whereas knockout of USP10 promoted NSCLC cell proliferation and migration. In conclusion, the present study demonstrates that USP10 coordinates TRIM25 to modulate PTEN activity. Specifically, USP10 activates PTEN by preventing its K63-linked polyubiquitination mediated by TRIM25 and suppresses the AKT/mammalian target of rapamycin signaling pathway, thereby inhibiting NSCLC proliferation, indicating that it may be a potential drug target for cancer treatment.

The transcription factor c-MAF could be polyubiquitinated and subsequently degraded in the proteasomes. Theoretically, any lysine residues in c-MAF could be ubiquitinated. In the present study, we tried to find out the specific lysine residue(s) mediating c-MAF ubiquitination. Through a series of mutational screens from lysine (K) to arginine (R), we found that any single lysine mutation (K to R) failed to prevent c-MAF ubiquitination, and any single lysine residue alone could not mediate c-MAF ubiquitination, which indicated that multiple lysine residues were required for c-MAF ubiquitination. Bioinformatics and computing analyses revealed that K85 and K350 could mediate c-MAF ubiquitination, which was confirmed by the cell-based assays. However, this duo was not the only pair because the K85R/K350R mutant could also be ubiquitinated. Functionally, both M12 (K85/K350) and W12 (K85R/K350R) mutants increased cyclin D2 promoter-driven luciferase activity, but they were less potent than the lysine-free counterpart (M14). In addition, M14 induced a higher level of expression of cyclin D2 at both mRNA and protein levels. Therefore, we demonstrated that c-MAF ubiquitination is mediated by multiple lysine residues, of which K85 and K350 were sufficient but not the only residues in mediating c-MAF ubiquitination. Moreover, c-MAF was found to be degraded by lysosomes. This study added a novel insight for c-MAF ubiquitination and degradation, suggesting that c-MAF stability is strictly regulated.

Interferon-stimulated gene 15 (ISG15) is an important cytokine that has been reported in carcinogenesis. However, we found that ISG15 and de-ISGylase USP18 were induced by several anti-cancer agents, which was confirmed by both RT-PCR and immunoblotting assays. Further studies demonstrated that ectopic ISG15 and USP18 inhibited proliferation of myeloma, leukemia and cervical cancer cells. More importantly, ISG15 and USP18 induced cancer cell apoptosis. This finding was confirmed in a cervical xenograft model in which cervical cancer growth was suppressed by lentiviral ISG15. In the mechanistic study, ISG15 was found to disrupt the NF-κB signaling pathway by downregulating the expression of IKKβ and p65, phosphorylation of p65 and IκBα. Consistent with this finding, ISG15 suppressed the expression of NF-κB recognition element-driving luciferase and decreased the transcription of XIAP and Mcl-1, two typical genes regulated by NF-κB. Therefore, the present study demonstrated that ISG15 induces cancer cell apoptosis by disrupting the NF-κB signaling pathway. This study highlighted a novel role of ISG15 in tumor suppression.

Collagen fibrils represent a unique case of protein folding and self-association. We have recently successfully developed triple-helical peptides that can further self-assemble into collagen-mimetic mini-fibrils. The 35 nm axially repeating structure of the mini-fibrils, which is designated the d-period, is highly reminiscent of the well-known 67 nm D-period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo-identical repeating sequence units in the primary structure of the designed peptides that give rise to the d-period of the quaternary structure of the mini-fibrils. In this work, we characterize the self-assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple-helix domain of Col877 consists of three pseudo-identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self-associate laterally under fibril forming conditions to form mini-fibrils having the predicted d-period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self-assembly of collagen mini-fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen-mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.

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.

Chronic myelogenous leukemia (CML) is a clonal malignancy of hematopoietic stem cells featured with the fusion protein kinase BCR-ABL. To elicit the mechanism underlying BCR-ABL stability, we perform a screen against a panel of deubiquitinating enzymes (DUBs) and find that the ubiquitin-specific protease 7 (USP7) drastically stabilizes the BCR-ABL fusion protein. Further studies show that USP7 interacts with BCR-ABL and blocks its polyubiquitination and degradation. Moreover, USP7 knockdown triggers BCR-ABL degradation and suppresses its downstream signaling transduction. In line with this finding, genetic or chemical inhibition of USP7 leads to BCR-ABL protein degradation, suppresses BCR/ABL signaling, and induces CML cell apoptosis. Furthermore, we find the antimalarial artesunate (ART) significantly inhibits USP7/BCR-ABL interaction, thereby promoting BCR-ABL degradation and inducing CML cell death. This study thus identifies USP7 as a putative Dub of BCR-ABL and provides a rationale in targeting USP7/BCR-ABL for the treatment of CML.

The zinc finger ubiquitin ligase RNF6 has been proposed as a potential therapeutic target in several cancers, but understanding its molecular mechanism of degradation has been elusive. In the present study, we find that RNF6 is degraded via auto-ubiquitination in a manner dependent on its Really Interesting New Gene (RING) domain. We determine that when the RING domain is deleted (ΔRING) or the core cysteine residues in the zinc finger are mutated (C632S/C635S), the WT protein, but not the ΔRING or mutant RNF6 protein, undergoes polyubiquitination. We also identify USP7 as a deubiquitinase of RNF6 by tandem mass spectrometry. We show that USP7 interacts with RNF6 and abolishes its K48-linked polyubiquitination, thereby preventing its degradation. In contrast, we found a USP7-specific inhibitor promotes RNF6 polyubiquitination, degradation, and cell death. Furthermore, we demonstrate the anti-leukemic drug Nilotinib and anti-myeloma drug Panobinostat (LBH589) induce RNF6 K48-linked polyubiquitination and degradation in both multiple myeloma (MM) and leukemia cells. In agreement with our hypothesis on the mode of RNF6 degradation, we show these drugs promote RNF6 auto-ubiquitination in an in vitro ubiquitination system without other E3 ligases. Consistently, reexpression of RNF6 ablates drug-induced MM and leukemia cell apoptosis. Therefore, our results reveal that RNF6 is a RING E3 ligase that undergoes auto-ubiquitination, which could be abolished by USP7 and induced by anti-cancer drugs. We propose that chemical induction of RNF6 auto-ubiquitination and degradation could be a novel strategy for the treatment of hematological malignancies including MM and leukemia.

The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is emerging as a promising therapeutic target for multiple myeloma (MM). In the present study, we performed a virtual screen against 800,000 of small molecule compounds by targeting PI3Kγ. C96, one of such compounds, inhibited PI3K activated by insulin-like growth factor-1 (IGF-1), but did not suppress IGF-1R activation. The cell-free assay revealed that C96 preferred to inhibit PI3Kα and δ, but was not active against AKT1, 2, 3 or mTOR. C96 inhibited PI3K activation in a time- and concentration-dependent manner. Consistent with its inhibition on PI3K/AKT, C96 downregulated the activation of mTOR, p70S6K, 4E-BP1, but did not suppress other kinases such as ERK and c-Src. Inhibition of the PI3K/AKT signaling pathway by C96 led to MM cell apoptosis which was demonstrated by Annexin V staining and activation of the pro-apoptotic signals. Furthermore, C96 displayed potent anti-myeloma activity in a MM xenograft model in nude mice. Oral administration of 100 mg/kg bodyweight almost fully suppressed tumor growth within 16 days, but without gross toxicity. Importantly, AKT activation was suppressed in tumor tissues from C96-treated mice, which was consistent with delayed tumor growth. Thus, we identified a novel PI3K inhibitor with a great potential for MM therapy.

Magnetic iron oxide nanoparticles (NPs) are emerging as novel materials with great potentials for various biomedical applications, but their biological activities are largely unknown. In the present study, we found that ferroferric oxide nanoparticles (Fe3O4 NPs) induced autophagy in blood cells. Both naked and modified Fe3O4 NPs induced LC3 lipidation and degraded p62, a monitor of autophagy flux. And this change could be abolished by autophagy inhibitors. Mechanistically, Fe3O4 NP-induced autophagy was accompanied by increased Beclin 1 and VPS34 and decreased Bcl-2, thus promoting the formation of the critical complex in autophagy initiation. Further studies demonstrated that Fe3O4 NPs attenuated cell death induced by anticancer drugs bortezomib and doxorubicin. Therefore, this study suggested that Fe3O4 NPs can induce prosurvival autophagy in blood cells by modulating the Beclin l/Bcl-2/VPS34 complex. This study suggests that caution should be taken when Fe3O4 NPs are used in blood cancer patients.

We previously reported a PI3K inhibitor S14161 which displays a promising preclinical activity against multiple myeloma (MM) and leukemia, but the chiral structure and poor solubility prevent its further application.

MafA and c-Maf are close members of the Maf transcription factor family and indicators of poor prognosis of multiple myeloma (MM). Our previous study finds that the ubiquitin ligase HERC4 induces c-Maf degradation but stabilizes MafA, and the mechanism is elusive. In the present study, we find that HERC4 interacts with MafA and mediates its K63-linked polyubiquitination at K33. Moreover, HERC4 inhibits MafA phosphorylation and its transcriptional activity triggered by glycogen synthase kinase 3β (GSK3β). The K33R MafA variant prevents HERC4 from inhibiting MafA phosphorylation and increases MafA transcriptional activity. Further analyses reveal that MafA can also activate the STAT3 signaling, but it is suppressed by HERC4. Lastly, we demonstrate that lithium chloride, a GSK3β inhibitor, can upregulate HERC4 and synergizes dexamethasone, a typical anti-MM drug, in inhibiting MM cell proliferation and xenograft growth in nude mice. These findings thus highlight a novel regulation of MafA oncogenic activity in MM and provide the rationale by targeting HERC4/GSK3β/MafA for the treatment of MM.

The activated JAK2-STAT3 signaling pathway is a high risk factor for multiple myeloma (MM), a fatal malignancy of plasma cells. In the present study, SC09, a potential inhibitor of cholesterol absorption, was identified in a STAT3-targeted drug screen. SC09 suppressed the activation of STAT3 in a time-course and concentration-dependent manner but did not affect its family members STAT1 and STAT5. SC09 inhibited STAT3 transcriptional activity and downregulated the expression of STAT3-regulated genes. Further studies showed that SC09 selectively inhibited JAK2 activation but not other kinases including c-Src, ERK, p38 and mTOR that are all associated with STAT3 activation. Moreover, SC09 obviously induced MM cell death in vitro and delayed MM tumor growth in vivo. SC09-induced MM cell death was dependent on the endogenous STAT3 status, and this effect could be attenuated by enforced expression of STAT3. All the results collectively indicated that SC09 blocks the JAK2-STAT3 signaling thus displaying anti-MM activity. Given its well tolerance and anti-MM potency, SC09 is credited for further investigation as a promising drug for MM treatment.

UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown.

Clioquinol is an anti-microbial drug, and it was recently found to induce cancer cell death. In the present study, clioquinol was found to trigger autophagy by inducing LC3 lipidation and autophagosome formation which was abolished by an autophagy inhibitor 3-methyladenine. Further study showed clioquinol displayed no effects on PI3KC3 or Beclin 1 expression but downregulated the expression and the enzymatic activity of mammalian target of Rapamycin (mTOR), a critical modulator of autophagy. Moreover, clioquinol inhibited the catalytic activity of the mTOR complex 1, thus suppressing phosphorylation of P70S6K and 4E-BP1, two major proteins associated with autophagy in the mTORC1 signaling pathway. Clioquinol induced leukemia and myeloma cell apoptosis, however, addition of autophagy inhibitor 3-methyladenine attenuated this kind of cell death. Therefore, this study demonstrated that clioquinol induces autophagy in associated with apoptosis in leukemia and myeloma cells by disrupting mTOR signaling pathway.

As a deubiqutinase Otub1 stabilizes and promotes the oncogenic activity of the transcription factor c-Maf in multiple myeloma (MM), a malignancy of plasma cells. In the screen for bioactive inhibitors of the Otub1/c-Maf axis for MM treatment, nanchangmycin (Nam), a polyketide antibiotic, was identified to suppress c-Maf activity in the presence of Otub1. By suppressing Otub1, Nam induces c-Maf polyubiquitination and subsequent degradation in proteasomes but does not alter its mRNA level. Consistently, Nam downregulates the expression of CCND2, ARK5, and ITGB7, the downstream genes regulated by c-Maf, and promotes MM cell apoptosis as evidenced by PARP and Caspase-3 cleavage, as well as Annexin V staining. In line with the hypothesis, overexpression of Otub1 partly rescues Nam-induced MM cell apoptosis, and interestingly, when Otub1 is knocked down, Nam-decreased MM cell survival is also partly ablated, suggesting Otub1 is essential for Nam anti-MM activity. Nam also displays potent anti-MM activity synergistically with Doxorubicin or lenalidomide. In the in vivo assays, Nam almost completely suppresses the growth of MM xenografts in nude mice at low dosages but it shows no toxicity. Given its safety and efficacy, Nam has a potential for MM treatment by targeting the Otub1/c-Maf axis.

Dihydrorotenone (DHR) is a natural pesticide widely used in farming industry, such as organic produces. DHR is a potent mitochondrial inhibitor and probably induces Parkinsonian syndrome, however, it is not known whether DHR is toxic to other systems. In the present study, we evaluated the cytotoxicity of DHR on human plasma cells. As predicted, DHR impaired mitochondrial function by decreasing mitochondrial membrane potential in plasma cells. Because mito-dysfunction leads to unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, we examined the signature proteins in ER stress, including GRP78, ATF4, and CHOP. After DHR treatment, these proteins were significantly upregulated. It is reported that activation of the mitogen-activated protein kinases p38 and JNK are involved in endoplasmic reticulum stress. However, in the subsequent study, DHR was found to activate p38 but not the JNK signaling. When pre-treated with p38 inhibitor SB203580, activation of p38 and cell apoptosis induced by DHR was partially blocked. Thus, we found that DHR induced human plasma cell death by activating the p38 but not the JNK signaling pathway. Because plasma cells are very important in the immune system, this study provided a new insight in the safety evaluation of DHR application.

The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent.

Recent clinical trials have demonstrated targeting PI3K pathway is a promising strategy for the treatment of blood cancers. To identify novel PI3K inhibitors, we performed a high throughput virtual screen and identified several novel small molecule compounds, including PIK-C98 (C98). The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino acid residues. The cellular assays demonstrated that C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, PTEN, and STAT3. Inhibition of PI3K by C98 led to myeloma cell apoptosis. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analyses showed that C98 was well penetrated into myeloma tumors. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development.