Quantitative proteomic analysis of cellular protein modulation upon inhibition of the NEDD8-activating enzyme by MLN4924

Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.     

MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants

The conjugation of the ubiquitin-like modifier NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN8/RELATED TO UBIQUITIN1 (NEDD8/RUB1; neddylation) is best known as an important posttranslational modification of the cullin subunits of cullin-RING-type E3 ubiquitin ligases (CRLs). MLN4924 has recently been described as an inhibitor of NEDD8-ACTIVATING ENZYME1 (NAE1) in human. Here, we show that MLN4924 is also an effective and specific inhibitor of NAE1 enzymes from Arabidopsis (Arabidopsis thaliana) and other plant species. We found that MLN4924-treated wild-type seedlings have phenotypes that are highly similar to phenotypes of mutants with a partial defect in neddylation and that such neddylation-defective mutants are hypersensitive to MLN4924 treatment. We further found that MLN4924 efficiently blocks the neddylation of cullins in Arabidopsis and that MLN4924 thereby interferes with the degradation of CRL substrates and their downstream responses. MLN4924 treatments also induce characteristic phenotypes in tomato (Solanum lycopersicum), Cardamine hirsuta, and Brachypodium distachyon. Interestingly, MLN4924 also blocks the neddylation of a number of other NEDD8-modified proteins. In summary, we show that MLN4924 is a versatile and specific neddylation inhibitor that will be a useful tool to examine the role of NEDD8- and CRL-dependent processes in a wide range of plant species.     

A gatekeeper residue for NEDD8-activating enzyme inhibition by MLN4924

Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecule's inhibitory effects, allowing cancer cell survival.     

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.     

Mutations in UBA3 Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924 in Human Leukemic Cells

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562_MLN, R-U937_MLN) were selected. R-K562_MLN and R-U937_MLN cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562_MLN cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.     

Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway

MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme, which showed antitumor effect in several types of malignant tumor types. However, the mechanism of action of MLN4924 in acute myeloid leukemia (AML) requires further investigation. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the mRNA levels of genes. Gene expression was knocked down by short hairpin RNA (shRNA). Moreover, the protein expression was detected by Western blotting (WB) assay. The proliferation and apoptosis of AML cells were measured by Cell Counting Kit-8 (CCK8) assay and flow cytometry (FCM). In the present study, we observed that the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1 in AML patients were up-regulated compared with healthy controls, which were correlated with worse overall survival (OS) of patients. Besides, knockdown of UBA3, UBE2M and RBX1 inhibited the NEDDylation of CULs and increased the protein expression of p53 and p21 in MOLM-13 cell line. In AML cells, MLN4924 inhibited cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G₂/M phase. As revealed by experiments in vivo and in vitro, the NEDDylation of CULs was significantly inhibited and the p53 signaling pathway was activated after MLN4924 treatment. So, we concluded that NEDD8, UBA3, UBE2M and RBX1 may serve as the prognostic biomarkers and novel therapeutic targets for AML. Inhibition of the NEDDylation pathway resulted in an anti-leukemia effect by activating the p53 signaling pathway.     

Targeting cullin-RING ligases for cancer treatment: rationales,advances and therapeutic implications

New therapeutic intervention strategies for the treatment of human malignancies are always desired. Approval of bortezomib as a front-line treatment for multiple myeloma highlighted the significance of ubiquitin–proteasome system (UPS) as a promising therapeutic target. However, due to the broad impact of proteasome inhibition, deleterious side effects have been reported with bortezomib treatment. Cullin RING ligases (CRLs)-mediated ubiquitin conjugation process is responsible for the ubiquitin conjugation of 20 % cellular proteins that are designated for degradation through the UPS, most of them are critical proteins involved in cell cycle progression, signaling transduction and apoptosis. Studies have depicted the upstream NEDDylation pathway that controls the CRL activity by regulating the conjugation of an ubiquitin-like-protein NEDD8 to the cullin protein in the complex. A specific pharmaceutical inhibitor of NEDD8 activating enzyme (NAE; E1) MLN4924 was recently developed and has been promoted to Phase I clinical trials for the treatment of several human malignancies. This article summarizes the most recent understanding about the process of NEDD8 conjugation, its relevance for cancer therapy and molecular mechanisms responsible for the potent anti-tumor activity of MLN4924.     

Molecular dynamics investigation on the poor sensitivity of A171T mutant NEDD8-activating enzyme (NAE) for MLN4924

MLN4924 is an adenosine sulfamate analog that generates the inhibitory NEDD8-MLN4924 covalent complex. A single nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3 reduces the enzyme’s sensitivity for MLN4924. Our molecular dynamics simulation study revealed that A171T transition brought remarkable conformational changes in enzyme structure (open ATP binding pocket), which reduced the interaction between MLN4924 and ATP binding pocket while wild form completely covered the MLN4924. A total difference of ?49.75?kJ/mol was noticed in interaction energy (electrostatic and van der Waals) during simulation between mutant and wild form with MLN4924. Superimposition of final 20?ns mutant structure with reference structure showed significant change in native binding position as compared to wild form. Results were found in coherence with the recently reported in vitro studies which states that A171T transition leads to change in ATP binding pocket structure.     

Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis

MLN4924, a newly discovered small molecule inhibitor of NEDD8-activating enzyme (NAE), inactivates Cullin-RING E3 ubiquitin Ligases (CRLs) by blocking cullin neddylation. As a result, MLN4924 causes accumulation of several key substrates of CRLs and effectively suppresses tumor cell growth by inducing apoptosis and senescence. However, the role of MLN4924 in induction of autophagy and its biological significance are totally unknown. Here we showed that MLN4924 effectively induces autophagy in both time- and dose-dependent manners in multiple human cancer lines, indicating a general phenomenon. Mechanistically, by inactivating CRLs, MLN4924 causes accumulation of DEPTOR and HIF1α. The siRNA knockdown and gene KO studies showed that DEPTOR and the HIF1-REDD1-TSC1 axis are responsible for MLN4924-induced autophagy via inhibiting mTORC1. Biologically, autophagy is a survival signal to tumor cells, and blockage of autophagy via siRNA knockdown, gene KO and small molecule inhibitor remarkably enhanced MLN4924-induced apoptosis. Our study reveals an uncharacterized mechanism of MLN4924 action and provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an autophagy inhibitor for maximal killing of tumor cells via enhancing apoptosis.     

Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells

The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.     

Neddylation inhibitor MLN4924 suppresses cilia formation by modulating AKT1

The primary cilium is a microtubule-based sensory organelle. The molecular mechanism that regulates ciliary dynamics remains elusive. Here, we report an unexpected finding that MLN4924, a small molecule inhibitor of NEDD8-activating enzyme (NAE), blocks primary ciliary formation by inhibiting synthesis/assembly and promoting disassembly. This is mainly mediated by MLN4924-induced phosphorylation of AKT1 at Ser473 under serum-starved, ciliary-promoting conditions. Indeed, pharmaceutical inhibition (by MK2206) or genetic depletion (via siRNA) of AKT1 rescues MLN4924 effect, indicating its causal role. Interestingly, pAKT1-Ser473 activity regulates both ciliary synthesis/assembly and disassembly in a MLN4924 dependent manner, whereas pAKT-Thr308 determines the ciliary length in MLN4924-independent but VHL-dependent manner. Finally, MLN4924 inhibits mouse hair regrowth, a process requires ciliogenesis. Collectively, our study demonstrates an unexpected role of a neddylation inhibitor in regulation of ciliogenesis via AKT1, and provides a proof-of-concept for potential utility of MLN4924 in the treatment of human diseases associated with abnormal ciliogenesis.     

Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells

The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.     

Ubiquitin E3 Ligase CRL4CDT2/DCAF2 as a Potential Chemotherapeutic Target for Ovarian Surface Epithelial Cancer

Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4^CDT2 repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4^CDT2 is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.     

Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.     

Chk1 inhibitor SCH 900776 enhances the antitumor activity of MLN4924 on pancreatic cancer

MLN4924 inhibits the cullin-RING ligases mediated ubiquitin-proteasome system, and has showed antitumor activities in preclinical studies, but its effects and mechanisms on pancreatic cancer (PC) remains elusive. We found that MLN4924 inhibited the proliferation and clonogenicity of PC cells, caused DNA damage, particularly double-strand breaks, and leaded to Chk1 activation and cell-cycle arrest. Chk1 inhibitor SCH 900776 alone exhibited minimal cytotoxicity, and caused no DNA damage on PC cells. But in the combination therapy, SCH 900776 enhanced the cytotoxicity and DNA damage caused by MLN4924, likely by abrogating G2/M arrest and promoting DNA re-replication. In vivo study on a xenograft PC mouse model also showed that SCH 900776 increased the efficacy of MLN4924. We also evaluated the level of NEDD8-activating enzyme (NAE), the direct target of MLN4924, and found that NAE level was elevated in PC tissues compared with normal pancreas, but was irrelevant with prognosis. Our findings provide the preclinical evidence and the rationale of the combination therapy of MLN4924 with SCH 900776 or other Chk1 inhibitors to treat PC.     

A Metal-Based Inhibitor of NEDD8-Activating Enzyme

A cyclometallated rhodium(III) complex [Rh(ppy)₂(dppz)]⁺ (1) (where ppy = 2-phenylpyridine and dppz = dipyrido[3,2-a:2′,3′-c]phenazine dipyridophenazine) has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE). The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme.     

NEDDylation regulates RAD18 ubiquitination and localization in response to oxidative DNA damage

Genome integrity is important for cell growth, development and proliferation. The E3 ligase RAD18 plays a vital role in the DNA damage response (DDR) to maintain genome integrity. Recent studies reveal that RAD18 has non-ubiquitinated and mono-ubiquitinated form in normal cells. However, whether RAD18 undergoes other post-translational modification remains to be investigated. Here we show that RAD18 is a target of NEDD8, an ubiquitin-like protein. In response to hydrogen peroxide (H₂O₂)-induced oxidative stress, RAD18 NEDDylation increases significantly, while its ubiquitination decreases. Moreover, NEDD8 overexpression or deNEDDylase NEDP1 deletion further antagonizes RAD18 ubiquitination. In addition, treatment with MLN4924, an inhibitor of NEDD8-activating Enzyme, reduces the interaction between PCNA and RAD18, which blocks the localization of RAD18 to form foci, and thus inhibiting polymerase η recruitment after oxidative stress. Together, our study demonstrates that RAD18 NEDDylation regulates its localization and involves in the DDR pathway by modulating RAD18 ubiquitination.     

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.     

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97^UFD1/NPL4), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.     

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.The ubiquitin–proteasome system ensures timely destruction of intracellular proteins. In the past decade, protein degradation has become a pharmacologic target: proteasome inhibitors (e.g., bortezomib) are currently being used in therapy of plasma and B-cell neoplasms. Inhibiting the ubiquitination process upstream of the proteasome represents a promising alternative approach. In this regard, ubiquitin-like modifiers (Ubl) such as NEDD8, ISG15 (interferon-stimulated gene 15), and SUMO (small ubiquitin-like modifier) regulate diverse cellular processes, depending on the exact Ubl and substrate involved. One such Ubl, NEDD8, modulates Cullin-RING E3 ubiquitin ligase (CRL) activity through covalent modification, neddylation.¹Chronic lymphocytic leukemia (CLL) B cells are highly dependent on cell–cell interactions in the lymph node and bone marrow microenvironment.² Stromal-mediated upregulation of B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in these niches ensures apoptosis evasion and promotes proliferation and clonal expansion.³ We recently reported that MLN4924 (pevonedistat), an investigational inhibitor of the NEDD8-activating enzyme (NAE), successfully abrogates NF-κB pathway activity, CLL cell survival and chemoresistance in an in vitro co-culture model that mimics the lymph node microenvironment.⁴ NAE adenylates NEDD8 at its C-terminus and allows its transfer to a specific cysteine within NAE, thus initiating a process of neddylation. Active NEDD8 is then transferred to the cysteine of the ubiquitin-conjugating enzyme (E2) specific for the pathway (Ubc12), and is finally conjugated to the CRLs.⁵ CRLs are responsible for ubiquitination and degradation of their substrate proteins. NAE–NEDD8 interaction is disrupted when a covalent adduct is formed between NEDD8 and MLN4924.⁶ Ultimately, this prevents ubiqitination of CRL target proteins, extending their half-life, thereby increasing levels of inhibitor of NF-κB (IκB), a negative pathway modulator.^{6, 7, 8} However, CRLs process a variety of proteins that, in addition to signal transduction (IκBα, DEPTOR, β-catenin, hypoxia-inducible factor-1α) and apoptosis (NOXA, Bim_EL), are important regulators of cell cycle and DNA replication (e.g., p21^Cip1, p27^Kip1, Wee1, Cyclin D1 and Cdt1).^{9, 10, 11, 12, 13, 14} Because of the diversity of CRL target substrates, the biological consequences of their inhibition are tissue dependent. In adherent solid tumor cell lines, inhibition of neddylation resulted in characteristic deregulation of cell cycle with DNA re-replication, checkpoint activation and cell cycle arrest, thought to be secondary to stabilization of the replication-licensing protein Cdt1 (chromatin licensing and DNA replication factor 1) and cyclin-dependent kinase (CDK) inhibitor p21^Cip1.^{11, 15, 16, 17} However, the importance of this mechanism in primary neoplastic B cells has not been studied. Here we determined that, under the conditions promoting cell replication and growth, MLN4924 induces checkpoint activation and cell cycle arrest in primary CLL B -cells. This mechanism complements abrogation of NF-κB pathway activity to induce apoptosis in CLL.