The ubiquitin ligase HERC4 suppresses MafA transcriptional activity triggered by GSK3ß in myeloma by atypical K63-linked polyubiquitination.

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 3ß (GSK3ß). 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 GSK3ß 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/GSK3ß/MafA for the treatment of MM.

PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy.

Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 µM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

Induction of zinc finger protein RNF6 auto-ubiquitination for the treatment of myeloma and chronic myeloid leukemia.

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.

Targeting the Otub1/c-Maf axis for the treatment 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 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 is independent of 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 US Food and Drug Administration-approved drugs and natural products, from which the generic cardiac glycoside lanatoside C (LanC) is found to prevent c-Maf deubiquitination 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.

RP58 knockdown contributes to hypoxia-ischemia-induced pineal dysfunction and circadian rhythm disruption in neonatal rats.

Hypoxia-ischemia (HI) of the brain not only impairs neurodevelopment but also causes pineal gland dysfunction, which leads to circadian rhythm disruption. However, the underlying mechanism of circadian rhythm disruption associated with HI-induced pineal dysfunction remains unknown. The zinc finger protein repressor protein with a predicted molecular mass of 58 kDa (RP58) is involved in the development and differentiation of nerve cells. In this study, we established an HI model in neonatal rats to investigate the expression of RP58 and its role in pineal dysfunction and circadian rhythm disruption induced by HI. We demonstrated that RP58 was highly expressed in the pineal gland under normal conditions and significantly downregulated in the pineal gland and primary pinealocytes following HI. Knockdown of RP58 decreased the expression of enzymes in the melatonin (Mel) synthesis pathway (tryptophan hydroxylase 1 [TPH1], acetylserotonin O-methyltransferase [ASMT], and arylalkylamine N-acetyltransferase [AANAT]) and clock genes (circadian locomotor output cycles kaput [CLOCK] and brain and muscle ARNT-like 1 [BMAL1]), and it also reduced the production of Mel, caused pineal cell injury, and disrupted circadian rhythms in vivo and in vitro. Similarly, HI reduced the expression of Mel synthesis enzymes (TPH1, ASMT, and AANAT) and clock genes (CLOCK and BMAL1), and caused pineal injury and circadian rhythm disruption, which were exacerbated by RP58 knockdown. The detrimental effect of RP58 knockdown on pineal dysfunction and circadian rhythm disruption was reversed by the addition of exogenous Mel. Furthermore, exogenous Mel reversed HI-induced pineal dysfunction and circadian rhythm disruption, as reflected by improvements in Mel production, voluntary activity periods, and activity frequency, as well as a diminished decrease in the expression of Mel synthesis enzymes and clock genes. The present study suggests that RP58 is an endogenous source of protection against pineal dysfunction and circadian rhythm disruption after neonatal HI.

Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa. 1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized with Palbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thus identifies USP10 as the first deubiquitinase of CCND3 and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

The deubiquitinase USP10 restores PTEN activity and inhibits non-small cell lung cancer cell proliferation.

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.

TRIM25 activates AKT/mTOR by inhibiting PTEN via K63-linked polyubiquitination in non-small cell lung cancer.

The PTEN/AKT/mTOR signaling pathway is frequently dysregulated in non-small cell lung cancer (NSCLC), but the mechanisms are not well-understood. The present study found that the ubiquitin ligase TRIM25 is highly expressed in NSCLC tissues and promotes NSCLC cell survival and tumor growth. Mechanistic studies revealed that TRIM25 binds to PTEN and mediates its K63-linked ubiquitination at K266. This modification prevents the plasma membrane translocation of PTEN and reduces its phosphatase activity therefore accumulating PI(3,4,5)P3. TRIM25 thus activates the AKT/mTOR signaling. Moreover, we found that the antibacterial nitroxoline can activate PTEN by reducing its K63-linked polyubiquitination and sensitizes NSCLC to cisplatin-induced apoptosis. This study thus identified a novel modulation on the PTEN signaling pathway by TRIM25 and provides a potential target for NSCLC treatment.

The deubiquitinase USP7 stabilizes Maf proteins to promote myeloma cell survival.

The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin-proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.

Glaucocalyxin A suppresses multiple myeloma progression in vitro and in vivo through inhibiting the activation of STAT3 signaling pathway.

BACKGROUND: Multiple myeloma (MM) is the most common malignant hematological disease in the people worldwide. Glaucocalyxin A (GLA) is a bioactive ent-kauranoid diterpenoid, that is derived from Rabdosia japonica var. GLA has been demonstrated that it had various pharmacological activities, such as anti-coagulation, anti-bacterial, anti-tumor, anti-inflammation, antioxidant activities. Although GLA has effective anti-tumor properties, its effects on multiple myeloma remain unclear. The aim of this study was to examine the possible anti-cancer effects of GLA and their molecular mechanisms on MM cells in vitro and in vivo. METHODS: To evaluate the role of GLA on the proliferation of MM cells in vitro and in vivo, we used MTT method to detect the role of GLA on the proliferation of MM cells. Cell apoptosis and cell cycle assay were evaluated by flow cytometry. Protein expressions in GLA-treated and untreated MM cells were evaluated by western blot analyses. MM xenograft nude mice model was used to investigate the role of GLA on the proliferation of MM cells in vivo. IHC assay was used to examine the role of GLA on the MM xenograft model in vivo. RESULTS: In the present study, we firstly reported the potent anti-myeloma activity of GLA on MM cells. We found that GLA could induce apoptosis in vitro and in vivo. GLA could inhibit the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and downregulate interleukin IL-6 induced STAT3 phosphorylation in MM. Overexpression of STAT3 could significantly prevent apoptosis induced by GLA; while knockdown of STAT3 enhanced it. Moreover, GLA could inhibit cell proliferation by inducing the cell cycle arrest. GLA reduced the expression of cell cycle-related proteins CCNB1, CCND1, CCND2, and CCND3 and increased the expression of p21 in MM cell lines. In addition, in the MM xenograft nude mice model, GLA exhibited very good anti-myeloma activity. Administration of GLA almost completely inhibited tumor growth within 19 days without physical toxicity. And the IHC results showed GLA significantly inhibited cell proliferation and interfered STAT3 pathway on MM xenograft model tumor tissues. CONCLUSIONS: Taken together, our present research indicated that GLA inhibits the MM cell proliferation, induces MM cell apoptosis and cell cycle arrest through blocking the activation of STAT3 pathway. Thus, GLA may be a potential therapeutic candidate for MM patients in the future.

Inhibition of the Otub1/c-Maf axis by the herbal acevaltrate induces myeloma cell apoptosis.

BACKGROUND: The oncogenic transcript factor c-Maf is stabilized by the deubiquitinase Otub1 and promotes myeloma cell proliferation and confers to chemoresistance. Inhibition of the Otub1/c-Maf axis is a promising therapeutic target, but there are no inhibitors reported on this specific axis. METHODS: A luciferase assay was applied to screen potential inhibitors of Otub1/c-Maf. Annexin V staining/flow cytometry was applied to evaluate cell apoptosis. Immunoprecipitation was applied to examine protein ubiquitination and interaction. Xenograft models in nude mice were used to evaluate anti-myeloma activity of AVT. RESULTS: Acevaltrate (AVT), isolated from Valeriana glechomifolia, was identified based on a bioactive screen against the Otub1/c-Maf/luciferase system. AVT disrupts the interaction of Otub1/c-Maf thus inhibiting Otub1 activity and leading to c-Maf polyubiquitination and subsequent degradation in proteasomes. Consistently, AVT inhibits c-Maf transcriptional activity and downregulates the expression of its target genes key for myeloma growth and survival. Moreover, AVT displays potent anti-myeloma activity by triggering myeloma cell apoptosis in vitro and impairing myeloma xenograft growth in vivo but presents no marked toxicity. CONCLUSIONS: The natural product AVT inhibits the Otub1/c-Maf axis and displays potent anti-myeloma activity. Given its great safety and efficacy, AVT could be further developed for MM treatment. Video Abstract.

Inhibition of the deubiquitinase USP9x induces pre-B cell homeobox 1 (PBX1) degradation and thereby stimulates prostate cancer cell apoptosis.

Chemoresistance is a leading obstacle in effective management of advanced prostate cancer (PCa). A better understanding of the molecular mechanisms involved in PCa chemoresistance could improve treatment of patients with PCa. In the present study, using immune histochemical, chemistry, and precipitation assays with cells from individuals with benign or malignant prostate cancer or established PCa cell lines, we found that the oncogenic transcription factor pre-B cell leukemia homeobox-1 (PBX1) promotes PCa cell proliferation and confers to resistance against common anti-cancer drugs such as doxorubicin and cisplatin. We observed that genetic PBX1 knockdown abrogates this resistance, and further experiments revealed that PBX1 stability was modulated by the ubiquitin-proteasomal pathway. To directly probe the impact of this pathway on PBX1 activity, we screened for PBX1-specific deubiquitinases (Dubs) and found that ubiquitin-specific peptidase 9 X-linked (USP9x) interacted with and stabilized the PBX1 protein by attenuating its Lys-48-linked polyubiquitination. Moreover, the USP9x inhibitor WP1130 markedly induced PBX1 degradation and promoted PCa cell apoptosis. The results in this study indicate that PBX1 confers to PCa chemoresistance and identify USP9x as a Dub of PBX1. We concluded that targeting the USP9x/PBX1 axis could be a potential therapeutic strategy for managing advanced prostate cancer.

RNF6 promotes myeloma cell proliferation and survival by inducing glucocorticoid receptor polyubiquitination.

RNF6, a RING-type ubiquitin ligase, has been identified as an oncogene in various cancers but its role in multiple myeloma (MM) remains elusive. In the present study we first showed that the expression levels of RNF6 in MM were significantly elevated compared with the bone marrow cells of healthy donors. Overexpression of RNF6 in LP1 and PRMI-8266 MM cell lines promoted cell proliferation, whereas knockdown of RNF6 led to apoptosis of MM cells. Furthermore, we revealed that RNF6, as a ubiquitin ligase, interacted with glucocorticoid receptor (GR) and induced its K63-linked polyubiquitination. Different from current knowledge, RNF6 increased GR stability at both endogenous and exogenous contexts. Such an action greatly promoted GR transcriptional activity, which was confirmed by luciferase assays and by the increased expression levels of prosurvival genes including Bcl-xL and Mcl-1, two typical downstream genes of the GR pathway. Consistent with these findings, ectopic expression of RNF6 in MM cells conferred resistance to dexamethasone, a typical anti-myeloma agent. In conclusion, we demonstrate that RNF6 promotes MM cell proliferation and survival by inducing atypical polyubiquitination to GR, and RNF6 could be a promising therapeutic target for the treatment of MM.

The transmembrane protein TMEPAI induces myeloma cell apoptosis by promoting degradation of the c-Maf transcription factor.

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.

Mebendazole elicits potent antimyeloma activity by inhibiting the USP5/c-Maf axis.

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.

The ubiquitin ligase HERC4 mediates c-Maf ubiquitination and delays the growth of multiple myeloma xenografts in nude mice.

The transcription factor c-Maf is extensively involved in the pathophysiology of multiple myeloma (MM), a fatal malignancy of plasma cells. In the present study, affinity chromatography and mass spectrometry were used to identify c-Maf ubiquitination-associated proteins, from which the E3 ligase HERC4 was found to interact with c-Maf and catalyzed its polyubiquitination and subsequent proteasome-mediated degradation. HERC4 mediated polyubiquitination at K85 and K297 in c-Maf, and this polyubiquitination could be prevented by the isopeptidase USP5. Further analysis on the NCI-60 cell line collection revealed that RPMI 8226, a MM-derived cell line, expressed the lowest level of HERC4. Primary bone marrow analysis revealed HERC4 expression was high in normal bone marrow, but was steadily decreased during myelomagenesis. These findings suggested HERC4 played an important role in MM progression. Moreover, ectopic HERC4 expression decreased MM proliferation in vitro, and delayed xenograft tumor growth in vivo. Therefore, modulation of c-Maf ubiquitination by targeting HERC4 may represent a new therapeutic modality for MM.

A novel hedgehog inhibitor for the treatment of hematological malignancies.

The hedgehog-smoothened (HH/SMO) pathway has been proposed as a potential therapeutic target for hematological malignancies. Our previous studies designed a series of HH inhibitors with novel scaffolds distinctive from vismodegib, the first Food and Drug Administration-approved HH inhibitor for the treatment of basal-cell carcinoma and medulloblastoma. In the present study, we evaluated these HH inhibitors against blood cancers and found that HH78 displayed potent activity in suppressing the HH signaling pathway. HH78 competitively bound to SMO and suppressed the transcriptional activity of GLI by the luciferase reporter gene assay and the measurement of HH/SMO-downregulated genes, including cyclin D2, cyclin E, PTCH1, PTCH2, and GLI. HH78 at low micromolar concentrations induced significant cancer cell apoptosis showed by increased caspase-3 activation, annexin V-staining and downregulated prosurvival proteins, including c-Myc, Bcl-2, Mcl-1, and Bcl-xL. In contrast, vismodegib did not show any effects on these apoptotic events. HH78 also suppressed the activation of the AKT/mTOR pathway, which cross-talks with the HH/SMO pathway. Finally, HH78 inhibited the growth of human leukemia K562 in nude mice xenografts with no overt toxicity. Collectively, the present study identified a novel HH inhibitor with great potential for the treatment of hematological malignancies.

The Ring Finger Protein RNF6 Induces Leukemia Cell Proliferation as a Direct Target of Pre-B-cell Leukemia Homeobox 1.

RNF6 is a little-studied ring finger protein. In the present study, we found that RNF6 was overexpressed in various leukemia cells and that it accelerated leukemia cell proliferation, whereas knockdown of RNF6 delayed tumor growth in xenografts. To find out the mechanism of RNF6 overexpression in leukemia, we designed a series of truncated constructs of RNF6 regulatory regions in the luciferase reporter system. The results revealed that the region between -144 and -99 upstream of the RNF6 transcription start site was critical and that this region contained a PBX1 recognition element (PRE). PBX1 modulated RNF6 expression by binding to the specific PRE. When PRE was mutated, RNF6 transcription was completely abolished. Further studies showed that PBX1 collaborated with PREP1 but not MEIS1 to modulate RNF6 expression. Moreover, RNF6 expression could be suppressed by doxorubicin, a major anti-leukemia agent, via down-regulating PBX1. This study thus suggests that RNF6 overexpression in leukemia is under the direction of PBX1 and that the PBX1/RNF6 axis can be developed as a novel therapeutic target of leukemia.

Nitroxoline shows antimyeloma activity by targeting the TRIM25/p53 axle.

The aim of this study was to identify the most potent quinoline-based anti-infectives for the treatment of multiple myeloma (MM) and to understand the molecular mechanisms. A small-scale screen against a panel of marketed quinoline-based drugs was performed in MM cell lines. Cell apoptosis was examined by flow cytometry. Anti-MM activity was also evaluated in nude mice. Western blotting was performed to investigate mechanisms. Nitroxoline (NXQ) was the most effective in suppressing MM cell proliferation. NXQ induced more than 40% MM cell apoptosis within 24 h and potentiated anti-MM activities of current major drugs including doxorubicin and lenalidomide. This finding was shown by activation of caspase-3, a major executive apoptotic enzyme, and by inactivation of PARP, a major enzyme in DNA damage repair. NXQ also suppressed prosurvival proteins Bcl-xL and Mcl-1. Moreover, NXQ suppressed the growth of myeloma xenografts in nude mice models. In the mechanistic study, NXQ was found to downregulate TRIM25, a highly expressed ubiquitin ligase in MM. Notably, NXQ upregulated tumor suppressor p53, but not PTEN. Furthermore, overexpression of TRIM25 decreased p53 protein. This study indicated that the long-term use of anti-infective NXQ has potential for MM treatment by targeting the TRIM25/p53 axle.

The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex.

S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissociated the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two critical targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment.