NEDD8-activating Enzyme Inhibitor MLN4924 Research Articles

The NEDD8 activating enzyme inhibitor MLN4924 mitigates doxorubicin-induced cardiotoxicity in mice

Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.

Inhibition of Neddylation Suppresses Osteoclast Differentiation and Function In Vitro and Alleviates Osteoporosis In Vivo.

Neddylation, or the covalent addition of NEDD8 to specific lysine residue of proteins, is a reversible posttranslational modification, which regulates numerous biological functions; however, its involvement and therapeutic significance in osteoporosis remains unknown. Our results revealed that during the soluble receptor activator of nuclear factor-κB ligand (sRANKL)-stimulated osteoclast differentiation, the neddylation and expression of UBA3, the NEDD8-activating enzyme (NAE) catalytic subunit, were dose- and time-dependently upregulated in RAW 264.7 macrophages. UBA3 knockdown for diminishing NAE activity or administering low doses of the NAE inhibitor MLN4924 significantly suppressed sRANKL-stimulated osteoclast differentiation and bone-resorbing activity in the macrophages by inhibiting sRANKL-stimulated neddylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-activated transforming growth factor-β-activated kinase 1 (TAK1) downstream signaling for diminishing nuclear factor-activated T cells c1 (NFATc1) expression. sRANKL enhanced the interaction of TRAF6 with the neddylated proteins and the polyubiquitination of TRAF6’s lysine 63, which activated TAK1 downstream signaling; however, this process was inhibited by MLN4924. MLN4924 significantly reduced osteoporosis in an ovariectomy- and sRANKL-induced osteoporosis mouse model in vivo. Our novel finding was that NAE-mediated neddylation participates in RANKL-activated TRAF6–TAK1–NFATc1 signaling during osteoclast differentiation and osteoporosis, suggesting that neddylation may be a new target for treating osteoporosis.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice

Blood-brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti-ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Nedd8-activating enzyme inhibitor MLN4924 (Pevonedistat), inhibits miR-1303 to suppress human breast cancer cell proliferation via targeting p27Kip1

MLN4924/Pevonedistat, a Nedd8-activating enzyme (NAE, E1) inhibitor, has shown notable anti-cancer effect in pre-clinical trials, but it still faces tolerance resistance risk. Combination target therapy indicates a much better clinical effect than single target, and miRNAs are beneficial for easy detection in bodily fluids and tissues. Up to now, MLN4924 and miRNA-targeting combination approaching to treat breast cancer patients remains largely unknown. Here, microRNA-seq analysis showed that the expression of miR-1303 was significantly decreased after MLN4924 treatment in breast cancer cells. Moreover, miR-1303 was abnormally high in breast cancer tissues, and breast cancer patients with high miR-1303 showed poor prognosis. Functionally, excessive miR-1303 promoted the malignant phenotypes of breast cancer cells. Excessive miR-1303 accelerated cell cycle progression by promoting G2/M arrest. Furthermore, we revealed that miR-1303 targeted p27Kip1 to release G2/M arrest. Notably, excessive miR-1303 partially disturbed the anti-cancer effect of MLN4924. These findings provide potential evidences for combined anti-cancer target therapy of breast cancer patients in the future.

Targeting Protein Neddylation for Cancer Therapy.

Neddylation is a posttranslational modification that conjugates a ubiquitin-like protein NEDD8 to substrate proteins. The best-characterized substrates of neddylation are the cullin subunits of cullin-RING E3 ubiquitin ligase complexes (CRLs). CRLs as the largest family of E3 ubiquitin ligases control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. The process of protein neddylation is overactivated in multiple types of human cancers, providing a sound rationale as an attractive anticancer therapeutic strategy, evidenced by the development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Recently, increasing evidence strongly indicates that neddylation inhibition by MLN4924 exerts anticancer effects mainly by triggering cell apoptosis, senescence, and autophagy and causing angiogenesis suppression, inflammatory responses, and chemo-/radiosensitization in a context-dependent manner. Here, we briefly summarize the latest progresses in this field, focusing on the preclinical studies to validate neddylation modification as a promising anticancer target.

The NEDD8-activating enzyme inhibitor MLN4924 sensitizes a TNFR1+ subgroup of multiple myeloma cells for TNF-induced cell death

The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase βTrCP. MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment.

The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors

ABSTRACT The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients

Adenovirus oncoprotein E4orf6 triggers Cullin5 neddylation to activate the CLR5 E3 ligase for p53 degradation

The human adenovirus oncoprotein E4orf6 hijacks intracellular Cullin 5-based E3 ubiquitin ligases (CRL5s) to induce the degradation of host proteins, including p53, that impede efficient viral replication. The complex also relies on another viral protein, E1B55K, to recruit substrates for ubiquitination. However, the determinants of adenoviral E4orf6-CRL5 E3 ligase-mediated p53 degradation in the scaffolding protein Cullin5 remain rarely investigated. Here, we demonstrated that the viral protein E4orf6 triggered relocalization of the Cullin5 protein from the cytoplasm to the nucleus and induced activation of the CRL5 E3 ligase via facilitating neddylation. The expression of the deneddylase SENP8/Den1 was significantly downregulated by E4orf6. We then identified SENP8 as a natural restriction factor for E4orf6-induced p53 degradation. Furthermore, our results indicated that the NEDD8-conjugating E2 enzyme UBE2M was essential for E4orf6-mediated p53 degradation and that its dominant negative mutant UBE2M C111S dramatically blocked E4orf6 functions. The Nedd8-activating enzyme inhibitor MLN4924 decreased E4orf6-induced neddylation of the cullin5 protein and subsequently suppressed p53 degradation. Collectively, our findings illuminate the strategy by which this viral oncoprotein specifically utilizes the neddylation pathway to activate host CRL E3 ligases to degrade host restriction factors. Disrupting this post-translational modification is an attractive pharmacological intervention against human adenoviruses.

Abstract 71: NEDD8-activating enzyme inhibitor MLN4924 reduces cell viability and induces apoptosis in chemoresistant cancer stem-like cells of human urothelial carcinomas in vitro and in vivo

Abstract Emerging evidence indicates that cancer stem cells (CSCs) may play a crucial role in cancer tumorigenesis, metastasis, and drug resistance. To develop treatment to inhibit CSCs provides promising target for cancer therapy. In this study, we identified a subpopulation of chemoresistant cancer stem-like cells of UCs (T24/R) after chemotherapeutic drug selection from T24, a bladder UC cell line. The T24/R cells displayed stemness markers (SOX2, Nanog, and Kruppel-like factor 4), high tumorigenicity in vivo, and enhanced invasion ability because of their epithelial-mesenchymal transition (EMT) properties. A neddylation inhibitor, MLN4924, efficiently inhibited viability and migration as well as induced apoptosis in the T24/R cells with concomitant suppression of stemness and EMT markers. Our findings suggest that MLN4924 effeiciently suppressed chemoresistant cancer stem-like UC cells and was a promising agent to conquer drug resistance in human bladder UCs. Citation Format: Shih-Ming Liao, Fu-Shun Hsu, Shao-Ping Yang, Yu-Wei Chang, Po-Ming Chow, Yeong-Shiau Pu, Yu-Chieh Tsai, Kuan-Lin Kuo, Kuo-How Huang. NEDD8-activating enzyme inhibitor MLN4924 reduces cell viability and induces apoptosis in chemoresistant cancer stem-like cells of human urothelial carcinomas in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 71.

Neddylation Inactivation Facilitates FOXO3a Nuclear Export to Suppress Estrogen Receptor Transcription and Improve Fulvestrant Sensitivity.

How the neddylation pathway functions in breast tumor and regulation of estrogen receptor (ER) expression is rarely reported. The purpose of this study was to identify the role of neddylation in breast cancer and ER expression, and further explore the underlying mechanisms. Expression patterns of nedd8-activating enzyme (NAE) and nedd8, two key proteins in the neddylation pathway, were examined in human breast specimens. ER-α expression was investigated using animal 18F-FES-PET/CT and immunoblotting upon NAE inhibitor MLN4924 treatment. Chromatin immunoprecipitation assay, luciferase reporter promoter assay, and the CRISPR-Cas9 system were used to elucidate the mechanism of ER-α regulation by MLN4924. The ER-positive breast cancer mouse model was used to determine the synergetic effect of MLN4924 and fulvestrant on tumor growth. All statistical tests were two-sided. Both NAE1 and nedd8 expressions were higher in the ER-positive subgroup. Higher expressions of NAE1 and nedd8 indicated poorer prognosis. Importantly, ER-α expression was significantly downregulated upon MLN4924 treatment in vitro and in vivo. Mechanistically, MLN4924 treatment delayed serum and glucocorticoid-induced protein kinase (SGK) degradation and induced Forkhead box O3a (FOXO3a) nuclear export as well as decreased binding to the ESR1 promoter. Importantly, MLN4924 single or synergized with fulvestrant significantly suppressed the growth of ER-positive breast cancer in vitro and in vivo. Our proof-of-principle study determines the activation of neddylation in breast tumor tissues for the first time and reveals a new ER-α regulatory mechanism, as well as further explores an effective approach to improve fulvestrant sensitivity through a neddylation inactivation combination.

Effects of neddylation modification inhibitor MLN4924 on proliferation and migration of human gastric cancer cells

Objective To explore the effects of NEDD8 activating enzyme (NAE) inhibitor MLN4924 on gastric cancer cell proliferation and migration. Methods Human gastric cancer cell lines NCI-N87 and SGC-7901 were treated with different concentrations of MLN4924, and the inhibitory concentrations were detected by cell counting kit-8 (CCK-8) method. 20% maximal inhibitory concentration (IC20) was used to treat gastric cancer cells. The cell proliferation was measured by CCK-8 and clone formation assay, and cell migration was determined by cell scratch test. Results When human gastric cancer cell lines NCI-N87 and SGC-7901 were treated with 0.528 and 0.375 μmol/L respectively, the inhibitory rate reached to 20%. CCK-8 method showed that MLN4924 inhibited the proliferation of human gastric cells in a dose- and time-dependent manner. In the clone formation assay, the clone number of NCI-N87 cells was 71.00±10.19 in MLN4924 treated group and 215.33±9.56 in the control group. The clone number of SGC-7901 cells was 23.66±18.66 in MLN4924-treated group, and 32.33±34.12 in the control group.In vitro wound scratch assay revealed that the healing rate of MLN4924-treated NCI-N87 cells was (5.30±0.78)%, and (51.05±6.35)% in the control group, and that of MLN4924-treated SGC-7901 cells was (3.12±0.52)%, and (23.05±2.01)% in the control group. Conclusion Neddylation modification inhibitor MLN4924 suppresses the growth and migration of gastric cancer cells. Key words: Gastric cancer; Neddylation modification inhibitor; Proliferation; Migration

NEDD8-conjugated Cullin4 positive regulates antimicrobial peptides expression in Eriocheir sinensis

The ubiquitin-proteasome system is involved in numerous cellular processes, such as signal transduction, autophagy, cell cycle control, embryogenesis, and regulation of immune response. Neural precursor cell expressed developmentally downregulated 8 (NEDD8) is a ubiquitin-like protein that activates Cullin-RING ligases and modifies substrates via neddylation. However, there is limited information on how neddylation regulates innate immunity in crustaceans. In the present study, we identified the evolutionarily conserved NEDD8 with the ubiquitin homologue domain in the Chinese mitten crab (Eriocheir sinensis), named it EsNEDD8. Then, we analyzed the expression patterns and cellular location of its substrate, EsCullin4. qRT-PCR showed that both EsNEDD8 and EsCullin4 were widely expressed in all the selected tissues, and EsCullin4 was significantly upregulated in hemocytes after bacterial stimulation. Moreover, silencing of EsCullin4 significantly suppressed the expression of antimicrobial peptides (AMPs) in the hemocytes after bacterial stimulation, and inhibition of EsCullin4 neddylation by treatment with the NEDD8-activating enzyme inhibitor MLN4924 significantly inhibited the expression of the AMPs. Thus, the results show that EsNEDD8-modified EsCullin4 could control antimicrobial activities via regulation of AMPs expression in the Chinese mitten crab.

Neddylation inhibitor MLN4924 induces G2 cell cycle arrest, DNA damage and sensitizes esophageal squamous cell carcinoma cells to cisplatin.

Inhibiting the protein neddylation pathway using the NEDD8-activating enzyme inhibitor MLN4924 represents an attractive anticancer strategy having been demonstrated to exhibit promising anticancer efficacy and with tolerable levels of toxicity; however, the mechanism by which MLN4924 inhibits cell proliferation in human esophageal squamous cell carcinoma (ESCC) cells requires further investigation. The present study revealed that MLN4924 treatment led to G2 cell cycle arrest and enhanced the protein stability of Wee1-like protein kinase and cyclin dependent protein kinase inhibitor 1A and B and p27. Furthermore, MLN4924 induced DNA damage and sensitized esophageal cancer cells to cisplatin by enhancing apoptosis. These findings extend the understanding of the function and mechanism of MLN4924 in ESCC and provide further evidence for the future development of neddylation inhibitors in clinical trials of esophageal cancer therapy, either alone or in combination.

Effects of the NEDD8-Activating Enzyme Inhibitor MLN4924 on Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

ABSTRACTThe switch of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is a key event for viral dissemination and pathogenesis. MLN4924, a novel neddylation inhibitor, reportedly causes the onset of KSHV reactivation but impairs later phases of the viral lytic program in infected cells. Thus far, the molecular mechanism involved in the modulation of the KSHV lytic cycle by MLN4924 is not yet fully understood. Here, we confirmed that treatment of different KSHV-infected primary effusion lymphoma (PEL) cell lines with MLN4924 substantially induces viral lytic protein expression. Due to the key role of the virally encoded ORF50 protein in the latent-to-lytic switch, we investigated its transcriptional regulation by MLN4924. We found that MLN4924 activates the ORF50 promoter (ORF50p) in KSHV-positive cells (but not in KSHV-negative cells), and the RBP-Jκ-binding elements within the promoter are critically required for MLN4924 responsiveness. In KSHV-negative cells, reactivation of the ORF50 promoter by MLN4924 requires the presence of the latency-associated nuclear antigen (LANA). Under such a condition, LANA acts as a repressor to block the ORF50p activity, whereas MLN4924 treatment relieves LANA-mediated repression. Importantly, we showed that LANA is a neddylated protein and can be deneddylated by MLN4924. On the other hand, we revealed that MLN4924 exhibits concentration-dependent biphasic effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)- or sodium butyrate (SB)-induced viral reactivation in PEL cell lines. In other words, low concentrations of MLN4924 promote activation of TPA- or SB-mediated viral reactivation, whereas high concentrations of MLN4924, conversely, inhibit the progression of TPA- or SB-mediated viral lytic program.IMPORTANCE MLN4924 is a neddylation (NEDD8 modification) inhibitor, which currently acts as an anti-cancer drug in clinical trials. Although MLN4924 has been reported to trigger KSHV reactivation, many aspects regarding the action of MLN4924 in regulating the KSHV lytic cycle are not fully understood. Since the KSHV ORF50 protein is the key regulator of viral lytic reactivation, we focus on its transcriptional regulation by MLN4924. We here show that activation of the ORF50 gene by MLN4924 involves the relief of LANA-mediated transcriptional repression. Importantly, we find that LANA is a neddylated protein. To our knowledge, this is the first report showing that neddylation occurs in viral proteins. Additionally, we provide evidence that different concentrations of MLN4924 have opposite effects on TPA-mediated or SB-mediated KSHV lytic cycle activation. Therefore, in clinical application, we propose that MLN4924 needs to be used with caution in combination therapy to treat KSHV-positive subjects.

Targeting neddylation pathway with MLN4924 (Pevonedistat) induces NOXA-dependent apoptosis in renal cell carcinoma

Inhibition of protein neddylation pathway has emerged an attractive anticancer strategy in preclinical studies by using Nedd8-activating enzyme (NAE) inhibitor MLN4924 (Pevonedistat). Previous studies have reported the antitumor activity of MLN4924 mediated by its efficacy on apoptosis, autophagy and senescence. However, whether MLN4924 has any effect on renal carcinoma cells (RCC) remains unexplored. Here we reported that MLN4924 specifically inhibited protein neddylation pathway, leading to statistically significantly suppress the proliferation, survival and migration of RCC cells by inducing G2 cell-cycle arrest, followed by apoptosis in a MLN4924 dose-dependent manner. Further mechanistic study revealed that MLN4924-induced apoptosis was mediated by substantial up-regulation of pro-apoptotic NOXA. These findings highlighted the anticancer effects of the neddylation inhibitors (e.g. MLN4924) for the treatment of RCC.

Radiosensitization by the investigational NEDD8-activating enzyme inhibitor MLN4924 (pevonedistat) in hormone-resistant prostate cancer cells.

Salvage radiotherapy (SRT) is the first-line treatment for prostate cancer patients with biochemical recurrence following radical prostatectomy, and new specific radiosensitizers are in urgent need to enhance SRT effect. MLN4924 (also known as Pevonedistat), a specific inhibitor of NEDD8-activating enzyme, has recently entered phase I/II clinical trials in several malignancies. By inhibiting cullin neddylation, MLN4924 inactivates Cullin-RING ligases (CRL), which have been validated as an attractive radiosensitizing target. In our study, we demonstrate that MLN4924 can be used as a potent radiosensitizer in hormone-resistant prostate cancer cells. We found that MLN4924 inhibited cullin neddylation and sensitized prostate cancer cells to irradiation (IR). Mechanistically, MLN4924 enhanced IR-induced G2 cell-cycle arrest, by inducing accumulation of WEE1/p21/p27, three well-known CRL substrates. Importantly, siRNA knockdown of WEE1/p21/p27 partially abrogated MLN4924-induced G2 cell-cycle arrest, indicating a causal role of WEE1/p21/p27 in MLN4924-induced radiosensitization. Further mechanistic studies revealed that induction of DNA damage and apoptosis also contributed to MLN4924 radiosensitization in hormone-resistant prostate cancer cells. Our findings lay the foundation for future application of MLN4924 as a potential radiosensitizer in hormone refractory prostate cancer (HRPC).

The NEDD8-activating enzyme inhibitor MLN4924 induces G2 arrest and apoptosis in T-cell acute lymphoblastic leukemia.

The first-in-class compound MLN4924 is a small molecule inhibitor that selectively inactivates NEDD8-activating enzyme (NAE). The anticancer effects of MLN4924 have been attributed to impaired neddylation of Cullin proteins. Here, we show that treatment of T-cell acute lymphoblastic leukemia (T-ALL) cells with MLN4924 potently suppressed the neddylation of Cullins and the oncogenic growth of T-ALL cells in-vitro. Moreover, MLN4924 induced disease regression in an in vivo xenograft model. MLN4924 also induced cell cycle arrest at G2 phase and apoptosis in T-ALL cells. However, inhibition of the neddylation of Cullins alone could not explain the effects of MLN4924 in T-ALL cells. Gene expression profiling indicated ribosome function, steroid biosynthesis, and hematopoietic cell lineage pathways were affected by MLN4924 treatment. MLN4924 also induced nucleolar disruption, suggesting nucleolar stress signaling might contribute to the anticancer effects of MLN4924 in T-ALL cells. In addition, MLN4924 treatment reduced 14-3-3ξ\\δ protein levels in T-ALL cells. Thus, MLN4924 may inhibit T-ALL cell proliferation via several pathways.

Broad and potent antiviral activity of the NAE inhibitor MLN4924.

In terms of infected human individuals, herpesviruses range among the most successful virus families. Subclinical herpesviral infections in healthy individuals contrast with life-threatening syndromes under immunocompromising and immunoimmature conditions. Based on our finding that cytomegaloviruses interact with Cullin Roc ubiquitin ligases (CRLs) in the context of interferon antagonism, we systematically assessed viral dependency on CRLs by utilizing the drug MLN4924. CRL activity is regulated through the conjugation of Cullins with the ubiquitin-like molecule Nedd8. By inhibiting the Nedd8-activating Enzyme (NAE), MLN4924 interferes with Nedd8 conjugation and CRL activity. MLN4924 exhibited pronounced antiviral activity against mouse and human cytomegalovirus, herpes simplex virus (HSV)- 1 (including multi-drug resistant clinical isolates), HSV-2, adeno and influenza viruses. Human cytomegalovirus genome amplification was blocked at nanomolar MLN4924 concentrations. Global proteome analyses revealed that MLN4924 blocks cytomegaloviral replication despite increased IE1 amounts. Expression of dominant negative Cullins assigned this IE regulation to defined Cullin molecules and phenocopied the antiviral effect of MLN4924.

MLN4924 Synergistically Enhances Cisplatin-induced Cytotoxicity via JNK and Bcl-xL Pathways in Human Urothelial Carcinoma.

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma. However, the response rate is only 40–65%. This study investigated the anti-tumor effect and underlying mechanisms of the combination of cisplatin and the NEDD8-activating enzyme inhibitor MLN4924 in human bladder urothelial carcinoma. The combination of cisplatin and MLN4924 exerted synergistic cytotoxicity on two high-grade bladder urothelial carcinoma cell lines, NTUB1 and T24 (combination index <1). MLN4924 also potentiated the cisplatin-induced apoptosis and activation of caspase-3 and -7, phospho-histone H2A.X and PARP. c-Jun N-terminal kinase (JNK) activation and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) were also observed during cisplatin and MLN4924 treatment. Inhibition of JNK activation partially restored cell viability and Bcl-xL expression. Bcl-xL overexpression also rescued cell viability. MLN4924 significantly potentiated cisplatin-induced tumor suppression in urothelial carcinoma xenograft mice. In summary, MLN4924 synergistically enhanced the anti-tumor effect of cisplatin via an increase in DNA damage, JNK activation and down-regulation of Bcl-xL in urothelial carcinoma cells. These findings provide a new therapeutic strategy for the treatment of bladder cancer.

Neddylation Pathway as a Novel Anti-cancer Target: Mechanistic Investigation and Therapeutic Implication.

Protein neddylation, a newly characterized posttranslational modification that adds the ubiquitin-like molecule NEDD8 to substrates, modulates important biological processes, whereas dysfunction of neddylation may cause several serious diseases, such as cancer. Inhibition of neddylation pathway has emerged as a promising anticancer strategy, as evidenced by development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924. Due to its potent anti-cancer efficacy and well-tolerated toxicity, MLN4924 has been evaluated in multiple Phase I clinical trials for solid tumors and hematologic malignancies. Recently, accumulating evidences indicate that neddylation pathway also plays a pivotal role in the regulation of multiple processes of tumor microenvironment (TME), such as tumor angiogenesis and the function of immune cells. In this review, we briefly summarize the latest progresses in this field and highlight neddylation pathway as an attractive therapeutic target against human cancer.