Molecule Inhibitor Of NEDD8-activating Enzyme Research Articles

Differential dynamics of cullin deneddylation via COP9 signalosome subunit 5 interaction

Cullin-RING E3 ubiquitin ligases (CRLs) spatiotemporally regulate the proteasomal degradation of numerous cellular proteins involved in cell cycle control, DNA replication, and maintenance of genome stability. Activation of CRLs is controlled via neddylation by NEDD8-activating, -conjugating, and -attaching enzymes to the C-terminus of scaffold cullins (CULs), whereas the COP9 signalosome (CSN) inactivates CRLs via deneddylation. Here, we show that the deneddylation rate of each CUL is differentially modulated. Dose- or time-dependent treatment with pevonedistat, a small molecule inhibitor of NEDD8-activating enzyme (NAE), rapidly inhibits neddylation in most CULs, including CUL1, CUL3, CUL4A/B, and CUL5, whereas the deneddylation of CUL2 is slowly increased. We revealed that the different deneddylation speeds of each CUL depend on its binding strength with CSN5, the catalytic core of the CSN complex. Immunoprecipitation analysis revealed that CUL2 has a lower binding affinity for CSN5 than other CULs. Consistently, released cells treated with CSN5 inhibitor showed that CUL2 was slowly converted to the deneddylated form compared to the rapid deneddylation of other CULs. These findings provide mechanistic insights into the different dynamics of CULs in neddylation–deneddylation conversion.

PD-L1 induction via the MEK-JNK-AP1 axis by a neddylation inhibitor promotes cancer-associated immunosuppression

MLN4924 is a first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE), which is currently in several clinical trials for anti-cancer applications. However, MLN4924 also showed some off-target effects with potential to promote the growth of cancer cells which counteracts its anticancer activity. In this study, we found that MLN4924 increases the levels of PD-L1 mRNA and protein in dose- and time-dependent manners. Mechanistic study showed that this MLN4924 effect is largely independent of neddylation inactivation, but is due to activation of both ERK and JNK signals, leading to AP-1 activation, which is blocked by the small molecule inhibitors of MEK and JNK, respectively. Biologically, MLN4924 attenuates T cell killing in a co-culture model due to PD-L1 upregulation, which can be, at least in part, abrogated by either MEK inhibitor or anti-PD-L1 antibody. In an in vivo BALB/c mouse xenograft tumor model, while MLN4924 alone had no effect, combination with either MEK inhibitor or anti-PD-L1 antibody enhanced the suppression of tumor growth. Taken together, our study provides a sound rationale for effective anticancer therapy in combination of anti-PD-L1 antibody or MEK inhibitor with MLN4924 to overcome the side-effect of immunosuppression by MLN4924 via PD-L1 induction.

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.

Pevonedistat, a Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Induces Cell Cycle Deregulation, Anaphase Catastrophe, and Apoptosis in T-Cell Lymphoma Cells

Introduction: Despite the significant progress of targeted therapies in B-cell malignancies, T-cell lymphomas remain an area of unmet medical need. Most patients are diagnosed at an advanced stage and have limited treatment options. Moreover, most patients who relapse following initial chemotherapy ultimately succumb to disease. Recent successes of targeting the proteasome (i.e., bortezomib) and E3 ligases (i.e., lenalidomide) identify the ubiquitin-proteasome system (UPS) as a tractable target in lymphoma. Pevonedistat, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE), interferes with activation of NEDD8, a ubiquitin-like modifier. This interference ultimately leads to decreased activity of cullin-RING (E3) ligases and accumulation of their substrates, including inhibitor of NFκB (IκB), the replication licensing protein Cdt1, and p27. We previously demonstrated that targeting NAE affected primary neoplastic B cells via several mechanisms: disruption of NFκB activity as well as induction of Cdt1, DNA damage, and cell cycle arrest. Here, we demonstrate that targeting NAE in T-cell lymphoma cells mediates apoptosis via cell cycle deregulation, accompanied by induction of Cdt1 and p27, and induction of anaphase catastrophe.Methods: Experiments were performed in T-cell lymphoma cell lines (SR, HH, Jurkat, and SUP-T1) as well as circulating primary cells from patients with peripheral T-cell lymphoma and Sezary syndrome. Pevonedistat (TAK-924) was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). Apoptosis was assessed by Annexin V staining.Results: SR (PTCL) cells and primary T-cell lymphoma cells were the most sensitive to pevonedistat (IC50of ~250nM at 24 hours); Jurkat and SUP-T1 cells demonstrated low/intermediate sensitivity, whereas HH (CTCL) cells were resistant. Targeting NAE disrupted cullin neddylation in a dose-dependent manner across all tested cell lines and primary neoplastic T cells, followed by accumulation of phospho-IκBα. Upregulation of phospho-IκBα was notable within 2 hours of pevonedistat treatment across both sensitive and resistant cell lines and primary cells. Concomitantly, we observed induction of p27 and Cdt1. Upregulation of Cdt1 was attenuated in HH cells compared with SR, consistent with the low proliferation rate of the former. Treatment of SR cells with pevonedistat led to DNA damage as evidenced by γH2AX and G2/M arrest.Chromosomal instability is a prominent feature in cancer and poorly studied as a therapeutic target. We have previously shown that cancer cells undergo multipolar anaphase in response to inhibition of cyclin-dependent kinase-2 (CDK2), an interphase CDK, followed by apoptosis and termed this event anaphase catastrophe (Hu et al., 2015; Danilov et al., 2016). As we observed robust accumulation of the endogenous CDK inhibitor p27 in cells treated with pevonedistat, an event presumed to lead to attenuated CDK2 activity, we studied anaphase catastrophe in this setting. We visualized anaphase catastrophe by immunofluorescent staining for nuclear material (DAPI) and γ-tubulin, and scored it in 50 cells per condition. NAE inhibition with pevonedistat induced anaphase catastrophe in SR and Jurkat cells. Upon 24-hour exposure to 250 nM pevonedistat, 9.8±6.0% of SR and 18±4.4% of Jurkat cells demonstrated multipolar anaphases, compared with 1±0.8% and 3.0±2.6% with vehicle control, respectively.Conclusions: Inhibiting NAE with pevonedistat induces apoptosis of T-cell lymphoma cells. We propose deregulation of Cdt1 and p27, followed by anaphase catastrophe, as a key mechanistic event implicated in pevonedistat-induced apoptosis in neoplastic T cells. Our work provides rationale to further investigate neddylation as a therapeutic target in T-cell lymphoma. DisclosuresDanilov:Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy; Genentech: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding.

Phase 3 study of first line pevonedistat (PEV) + azacitidine (AZA) versus single-agent AZA in patients with higher-risk myelodysplastic syndromes (HR MDS), chronic myelomonocytic leukemia (CMML) or low-blast acute myelogenous leukemia (AML).

TPS7077Background: PEV is a first-in-class small molecule inhibitor of NEDD8-activating enzyme, with single-agent clinical activity in patients with relapsed/refractory AML (Swords et al. Br J Haem...

Neddylation inhibitor MLN4924 suppresses growth and migration of human gastric cancer cells.

MLN4924 is a recently discovered small molecule inhibitor of NEDD8-Activating Enzyme (NAE). Because cullin RING ligase (CRL), the largest family of E3 ubiquitin ligase, requires cullin neddylation for its activity, MLN4924, therefore, acts as an indirect inhibitor of CRL by blocking cullin neddylation. Given that CRLs components are up-regulated, whereas neddylation modification is over-activated in a number of human cancers, MLN4924 was found to be effective in growth suppression of cancer cells. Whether MLN4924 is effective against gastric cancer cells, however, remains elusive. Here we showed that in gastric cancer cells, MLN4924 rapidly inhibited cullin 1 neddylation and remarkably suppressed growth and survival as well as migration in a dose-and time-dependent manner. Mechanistic studies in combination with siRNA knockdown-based rescue experiments revealed that MLN4924 induced the accumulation of a number of CRL substrates, including CDT1/ORC1, p21/p27, and PHLPP1 to trigger DNA damage response and induce growth arrest at the G2/M phase, to induce senescence, as well as autophagy, respectively. MLN4924 also significantly suppressed migration by transcriptionally activating E-cadherin and repressing MMP-9. Taken together, our study suggest that neddylation modification and CRL E3 ligase are attractive gastric cancer targets, and MLN4924 might be further developed as a potent therapeutic agent for the treatment of gastric cancer.

Endothelial deletion of Sag/Rbx2/Roc2 E3 ubiquitin ligase causes embryonic lethality and blocks tumor angiogenesis

SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin-RING ligase (CRL). Our recent study showed that Sag total knockout caused embryonic lethality at E11.5–12.5 days with associated defects in vasculogenesis. Whether Sag is required for de novo vasculogenesis in embryos and angiogenesis in tumors is totally unknown. Here, we report that Sag endothelial deletion also causes embryonic lethality at E15.5 with poor vasculogenesis. Sag deletion in primary endothelial cells or knockdown in MS-1 endothelial cells inhibits migration, proliferation and tube formation with p27 accumulation being responsible for the suppression of migration and proliferation. Furthermore, Sag deletion significantly inhibits angiogenesis in an in vivo Matrigel plug assay, and tumor angiogenesis and tumorigenesis in a B16F10 melanoma model. Finally, MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that inhibits CRL, suppresses in vitro migration, proliferation, and tube formation, as well as in vivo angiogenesis and tumorigenesis. Taken together, our study, using both genetic and pharmaceutical approaches, demonstrates that Sag is essential for embryonic vasculogenesis and tumor angiogenesis, and provides the proof-of-concept evidence that targeting Sag E3 ubiquitin ligase may have clinical value for anti-angiogenesis therapy of human cancer.

Abstract 1657: Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis in breast cancer cells.

Abstract 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 breast cancer cells. Mechanistically, by inactivating CRLs, MLN4924 causes accumulation of DEPTOR and HIF1α. The siRNA knockdown and gene KO studies showed that DEPTOR and the HIF1α-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. Citation Format: Yongchao Zhao, Xiufang Xiong, Lijun Jia, Yi Sun. Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1657. doi:10.1158/1538-7445.AM2013-1657

MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Abstract 3717The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures:No relevant conflicts of interest to declare.

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.

The NEDD8-Activating Enzyme Inhibitor, MLN4924, Cooperates with TRAIL to Augment Apoptosis through Facilitating c-FLIP Degradation in Head and Neck Cancer Cells

TNF-related apoptosis-inducing ligand (TRAIL) is a tumor-selective cytokine with potential anticancer activity and is currently under clinical testing. Head and neck squamous cell carcinoma (HNSCC), like other cancer types, exhibits varied sensitivity to TRAIL. MLN4924 is a newly developed investigational small molecule inhibitor of NEDD8-activating enzyme with potent anticancer activity. This study reveals a novel function of MLN4924 in synergizing with TRAIL to induce apoptosis in HNSCC cells. MLN4924 alone effectively inhibited the growth of HNSCC cells and induced apoptosis. When combined with TRAIL, synergistic effects on decreasing the survival and inducing apoptosis of HNSCC cells occurred. MLN4924 decreased c-FLIP levels without modulating death receptor 4 and death receptor 5 expression. Enforced expression of c-FLIP substantially attenuated MLN4924/TRAIL-induced apoptosis. Thus c-FLIP reduction plays an important role in mediating MLN4924/TRAIL-induced apoptosis. Moreover, MLN4924 decreased c-FLIP stability, increased c-FLIP ubiquitination, and facilitated c-FLIP degradation, suggesting that MLN4924 decreases c-FLIP levels through promoting its degradation. MLN4924 activated c-jun-NH(2)-kinase (JNK) signaling, evidenced by increased levels of phospho-c-Jun in MLN4924-treated cells. Chemical inhibition of JNK activation not only prevented MLN4924-induced c-FLIP reduction, but also inhibited MLN4924/TRAIL-induced apoptosis, suggesting that JNK activation mediates c-FLIP downregulation and subsequent enhancement of TRAIL-induced apoptosis by MLN4924. Because knockdown of NEDD8 failed to activate JNK signaling and downregulate c-FLIP, it is likely that MLN4924 reduces c-FLIP levels and enhances TRAIL-induced apoptosis independent of NEDD8 inhibition.

Treatment Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML and DLBCL Models

Abstract 1413MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). MLN4924 is a mechanism-based inhibitor of NAE as demonstrated through the formation of a NEDD8-MLN4924 covalent adduct that is a tight-binding inhibitor of NAE. The antitumor activity of MLN4924 has been linked to two mechanisms in pre-clinical models: (1) the induction of DNA re-replication and cell death through dysregulation of Cdt-1 and (2) the inhibition of NF-κB signaling. Importantly, modulation of both pathways has been demonstrated in pharmacodynamic (PD) studies from patients administered MLN4924. To characterize potential mechanisms of resistance to MLN4924 xenograft models of AML and Diffuse Large-B cell Lymphoma (DLBCL) were used. Mice bearing HL-60 and THP-1 (AML) and OCI-Ly10 (DLBCL) xenografts were treated with MLN4924 twice weekly for 95–110 days and most animals achieved a complete tumor regression. However, four of ten HL-60, six of ten THP-1 and one of ten OCI-Ly10 xenografts became refractory and re-grew during the MLN4924 treatment period. These tumors were harvested and subjected to microarray analysis and DNA sequencing of genes required for NEDD8 activation by NAE. Mutations in NAEβ resulting in amino acid substitutions were detected in several xenografts in the ATP binding pocket (A171T) and NEDD8-binding cleft (E204K) suggesting that mutations in the target enzyme may play a role in emergence of refractory tumors. Tumors containing mutations in NAEβ were passaged into new mice and shown to be completely insensitive to MLN4924 confirming resistance. In addition, cell lines derived from the resistant xenografts were shown to be resistant to MLN4924 in vitro but remained sensitive to other chemotherapeutic agents. Biochemical analysis of NAEβ mutants revealed a slower rate of NEDD8-MLN4924 adduct formation and demonstrated that the adduct was no longer bound tightly by the mutant enzyme. Thus, treatment emergent mutations in NAEβ lead to MLN4924 resistance in pre-clinical models through reduced inhibitory potency. These data serve as the framework for understanding mechanisms of resistance to MLN4924, provide rationale for patient selection approaches and offer a strategy to overcome treatment emergent mutations that may arise in clinical studies. Disclosures:Milhollen:Millennium Pharmaceuticals: Employment. Thomas:Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Riceberg:Millennium Pharmaceuticals: Employment. Sells:Millennium Pharmaceuticals: Employment. Amidon:Millennium Pharmaceuticals: Employment. Manfredi:Millennium Pharmaceuticals: Employment. Bence:Millennium Pharmaceuticals: Employment. Brownell:Millennium Pharmaceuticals: Employment. Dick:Millennium Pharmaceuticals: Employment. Koenig:Millennium Pharmaceuticals, Inc: Employment. Gavin:Millennium Pharmaceuticals: Employment. Smith:Millennium Pharmaceuticals: Employment.

Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models

Abstract 578MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures:Smith:Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

Inactivation of SAG E3 ubiquitin ligase blocks embryonic stem cell differentiation and sensitizes leukemia cells to retinoid acid.

Sensitive to Apoptosis Gene (SAG), also known as RBX2 (RING box protein-2), is the RING component of SCF (SKP1, Cullin, and F-box protein) E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES) to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag−/− mES cells were much more sensitive to all-trans retinoic acid (RA)-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag−/− mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy). We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.

Abstract C28: Treatment emergent mutations in NAEbeta confer resistance to the investigational NEDD8-activating enzyme inhibitor MLN4924.

Abstract MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that has shown clinical activity in Phase I clinical trials. MLN4924 is a mechanism-based inhibitor of NAE as demonstrated through the formation of a NEDD8-MLN4924 covalent adduct that is a tight-binding inhibitor of NAE. The antitumor activity of MLN4924 has been linked to two mechanisms in pre-clinical models: (1) the induction of DNA re-replication and cell death through dysregulation of Cdt-1 and (2) the inhibition of NF- B signaling. Importantly, modulation of both pathways has been demonstrated in pharmacodynamic (PD) studies from patients administered MLN4924. To characterize potential mechanisms of resistance to MLN4924, cell line and xenograft models of solid tumors were used. HCT-116 (colon), NCI-H460 (lung) and Calu-6 (lung) cells were treated with high concentrations of MLN4924 for four days after which the remaining cells were isolated and cultured in drug free media. Six HCT-116, two NCI-H460 and one Calu-6 clone were isolated and found to be at least 10-fold less sensitive to MLN4924, in most cases the isolated clones were completely insensitive indicating they were now resistant to MLN4924. However, the MLN4924 resistant clones were still sensitive to other chemotherapies including bortezomib (proteasome inhibitor), SN-38 (topoisomerase I inhibitor) and doxorubicin (anthracycline) suggesting a resistance mechanism unique to MLN4924. Co-incubation with Pgp, BCRP or MRP2 transporter inhibitors did not re-sensitize cells to MLN4924 indicating that the resistance mechanism was not related to increased drug efflux. DNA sequencing of NAE pathway genes identified heterozygous mutations in NAEbeta resulting in amino acid substitutions in the ATP binding pocket or NEDD8-binding cleft. Nude rats bearing HCT-116 xenografts were treated with the maximum tolerated dose of MLN4924 on a clinically relevant dosing regimen of Days 1, 4, 8 and 11 of a 21 day schedule. In the first cycle of therapy tumor regressions were observed but ultimately tumors regrew over subsequent cycles and were found to contain heterozygous mutations in NAEbeta. Tumors containing mutations in NAEbeta were transplanted into nude rats and shown to be insensitive to MLN4924 confirming resistance. In addition, cell lines derived from the resistant xenografts were shown to be resistant to MLN4924 in vitro but remained sensitive to other chemotherapeutic agents. Biochemical analysis of NAEbeta mutants revealed a slower rate of NEDD8-MLN4924 adduct formation and demonstrated that the adduct was no longer bound tightly by the mutant enzyme. Thus, in pre-clinical models of solid tumors treatment emergent mutations in NAEbeta lead to MLN4924 resistance. DNA sequencing of samples obtained from MLN4924 clinical trials is ongoing. These data may provide rationales for patient selection approaches with MLN4924 and the development of next generation NAE inhibitors that can be designed to overcome treatment emergent mutations found in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C28.

Abstract A196: Development, validation, and clinical implementation of a peripheral blood RT-PCR pharmacodynamic assay for MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE).

Abstract The ubiquitin proteasome system regulates the degradation of intracellular proteins that are involved in many cellular processes. These processes, such as cell cycle, DNA replication, and response to oxidative stress play a key role in tumor growth and survival. The ubiquitination and proteasomal degradation of a subset of proteins involved in these processes is controlled by Cullin-RING ubiquitin ligases (CRLs). NEDD8-activating enzyme (NAE) activates the ubiquitin-like protein NEDD8 for conjugation to CRLs and therefore regulates the proteasomal destruction of CRL substrate proteins. NAE inhibition prevents degradation of CRL substrates (e.g. NRF2, CDT1), leading to their accumulation, subsequent apoptosis and cell death. MLN4924 is an investigational small molecule NAE inhibitor with antitumor activity in preclinical models of several tumor types that is currently in Phase I clinical development. MLN4924 is the first NAE inhibitor to enter clinical development, and evidence of target inhibition and/or downstream pathway modulation were key objectives of the Phase I studies. Here we describe the development, validation and clinical implementation of a pharmacodynamic (PD) RT-PCR assay that quantifies levels of gene transcripts regulated in response to NAE inhibition in human whole blood. To develop a prototype PD assay, Affymetrix gene expression profiling studies on HCT116 cells treated in-vitro with an NAE inhibitor were performed. Genes regulated greater than two-fold by NAE inhibition were identified. The list of genes was observed to be enriched for NRF2 regulated transcripts, consistent with the mechanism of action for MLN4924. Twenty-seven of the most robustly regulated genes were tested by RT-PCR (TaqMan) using RNA samples extracted from MLN4924 treated HCT-116 cells and human PBMCs treated ex-vivo with MLN4924 in order to validate the microarray results and develop a RT-PCR based PD assay in human blood. Transcripts showing a robust response to NAE inhibition in both HCT116 cells and PBMCs were identified for further evaluation in whole blood as a preferred sample matrix for clinical studies. To explore the relationship of MLN4924 regulated transcripts with time and drug concentration, blood samples from multiple healthy volunteers were treated ex-vivo with a range of MLN4924 concentrations for three durations (4, 8, 24 hours). Eight genes (NQO1, SLC7A11, TXNRD1, SRXN1, GSR, GCLM, ATF3, and MAG1) that displayed a robust induction (>3-fold) in whole blood were selected for the PD biomarker panel. Inter- and intra-assay variability was established via testing two pools of cDNA control samples (RNA from MLN4924 ex-vivo treated and untreated blood) in replicates on the same plate, and on multiple plates run separately. All genes had inter- and intra-assay CV values of <5%. This RT-PCR panel has been implemented in four Phase I clinical trials with MLN4924 in both solid tumor and hematological malignancies. Each study demonstrates induction of the NAE-regulated transcripts following MLN4924 administration starting at drug doses below the maximum tolerated dose. These results demonstrate the successful development and implementation of a RT-PCR assay that detects downstream effects of NAE inhibition in human whole blood. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A196.

Abstract B178: Molecular determinants of response to the investigational small molecule inhibitor of Nedd8-activating enzyme (NAE) MLN4924 in melanoma cell lines and patient-derived tumor explant models.

Abstract Background: The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to proper functioning of the cell. Recent studies have shown that the UPS is more complex than previously thought, and its role in cell cycle regulation, signal transduction, DNA replication, and thus the evolution of the malignant phenotype of many cancers, is now well appreciated. The proteasome inhibitor bortezomib has provided validation of interfering with the UPS for clinical benefit in cancer patients and has provided the impetus for the development of new drugs that target the UPS more specifically. MLN4924 (Millennium Pharmaceuticals, Inc.) is an investigational first-in-class small molecule inhibitor of the key enzyme in the Nedd8 conjugation pathway, NAE. Inhibition of NAE results in in S-phase arrest, DNA re-replication and apoptosis. In this study, we used both in vitro and in vivo models of melanoma to assess the preclinical efficacy of MLN4924 in conjunction with gene array and gene set enrichment analysis (GSEA) to identify differentially expressed genes and pathways as potential molecular determinants of response to this novel compound. Methods & Results: A panel of thirty-six melanoma cell lines was exposed to MLN4924 and IC50s were determined using the CellTiter Glo method. The cell lines were then classified as sensitive (S) or resistant (R) based on an IC50<0.03μM or >2.59μM, respectively. The most extreme S and R lines were also grown as subcutaneous flank xenograft tumors in nude mice to confirm in vitro responses to MLN4924. Additionally, twelve patient-derived melanoma tumors were implanted in mice and both the cell line and explant xenograft mice were treated s.c. with MLN4924 at 90mg/kg. The compound was well-tolerated and no significant weight loss or lethality was observed. Tumors were measured three times weekly by caliper and tumor growth inhibition (TGI) was analyzed. Of the explant models, five responded favorably to MLN4924, with TGI>50%, with one model exhibiting tumor regression. Pharmacodynamic studies were also performed on melanoma explant tumors from mice exposed to a single dose of MLN4924 which were then harvested over a seven point time course ranging from 30 min to 24 hr post-drug. Immunoblotting of these tumors revealed rapid increases in the levels of caspase 3 as well as phospho-Chk-1 at 1 hr and decreases in neddylated cullin levels beginning at 8 hr, consistent with the known mode of action of MLN4924. Affymetrix 1.0 ST gene array analysis was performed on 6 S and 6 R cell lines in order to identify differentially expressed genes that could be used as predictive biomarkers. GSEA and KEGG pathway analysis of this data identified several DNA repair pathways as being enriched in the S lines while ABC drug transporter pathways were enriched in the R lines. Conclusion: MLN4924 demonstrated strong antitumor activity in a subset of melanoma cell lines and patient-derived explants. Based on these preclinical results, MLN4924 demonstrates promising data supportive of further patient-selective clinical development in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B178.

Initial testing of the investigational NEDD8‐activating enzyme inhibitor MLN4924 by the pediatric preclinical testing program

MLN4924 is an investigational first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an essential component of the NEDD8 conjugation pathway, controlling the activity of a subset of ubiquitin-proteasome system (UPS) E3 ligases, multiprotein complexes that transfer ubiquitin molecules to substrate proteins. MLN4924 was tested against the PPTP in vitro panel using 96-hour exposure time at concentrations ranging from 1.0 nM to 10 µM. It was tested in vivo at a dose of 100 mg/kg [66 mg/kg for the acute lymphoblastic leukemia (ALL) xenografts] administered orally twice daily × 5 days. Treatment duration was 3 weeks. The median relative IC(50) for MLN4924 against the PPTP cell lines was 143 nM, (range: 15-678 nM) with that for the Ewing panel being significantly lower (31 nM). MLN4924 induced significant differences in EFS distribution compared to control in 20 of 34 (59%) evaluable solid tumor xenografts. MLN4924 induced intermediate activity (EFS T/C values >2) in 9 of the 33 evaluable xenografts (27%), including 4 of 4 glioblastoma xenografts, 2 of 3 Wilm's tumor xenografts, 2 of 5 rhabdomyosarcoma xenografts, and 1 of 4 neuroblastoma xenografts. For the ALL panel, 5 of 8 evaluable xenografts showed intermediate activity for the EFS T/C measure. MLN4924 did not induce objective responses in the PPTP solid tumor or ALL panels. MLN4924 showed potent activity in vitro and in vivo showed tumor growth inhibitory activity against a subset of the PPTP solid tumor and ALL xenografts.

Antitumor activity of MLN4924, an investigational inhibitor of NEDD8-activating enzyme (NAE), in preclinical models of melanoma.

8594 Background: MLN4924 is a novel, small molecule inhibitor of Nedd8-activating enzyme (NAE) currently in Phase I clinical trials. Conjugation of NEDD8 to cullin scaffold proteins is required for...

Abstract 4782: The NEDD8-activating enzyme inhibitor, MLN4924, cooperates with TRAIL to augment apoptosis through JNK-dependent downregulation of c-FLIP in head and neck cancer cells

Abstract The death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a tumor-selective cytokines with potential anticancer activity and is currently being tested in clinical trials. Like other types of cancers, head and neck squamous cell carcinoma (HNSCC) cells posses varied sensitivity to TRAIL. MLN4924 is a newly developed small molecule inhibitor of NEDD8-activating enzyme with potent anticancer activity. Elevated NEDD8 conjugation has been reported in HNSCC cells. However, the effects of MLN4924 on the growth of HNSCC cells and on TRAIL-induced apoptosis in HNSCC cells have not been evaluated. In this study, we showed that MLN4924 effectively inhibited the growth of HNSCC cells (IC50s < 0.4 μM) with potent apoptosis-inducing activity. Moreover, MLN4924 synergized with TRAIL to decrease the survival and to induce apoptosis of HNSCC cells. Interestingly, MLN4924 decreased c-FLIP levels with minimal effects on DR4 and DR5 expression. Enforced expression of c-FLIP substantially attenuated MLN4924/TRAIL-induced apoptosis. These data indicate that c-FLIP reduction plays an important role in mediating cooperative induction of apoptosis by the MLN4924 and TRAIL combination. Furthermore we found that MLN4924 decreased c-FLIP stability, increased c-FLIP ubiqutination and facilitated c-FLIP degradation, suggesting that MLN4924 decreases c-FLIP levels through promoting its degradation via an ubiqutination and proteasome-dependent mechanism. Additionally, MLN4924 potently activated JNK signaling evidenced by increased levels of phospho-c-Jun in MLN4924-treated cells. Blockage of the JNK activation with the JNK inhibitor SP600125 not only prevented MLN4924-induced c-FLIP reduction, but also inhibited MLN4924/TRAIL-induced apoptosis. These results suggest that JNK activation mediates c-FLIP downregulation and subsequent enhancement of TRAIL-induced apoptosis induced by MLN4924. Our findings thus warrant further evaluation of the efficacy of MLN4924 or its combination with TRAIL against the HNSCC in vivo and highlight a novel mechanism by which MLN4924 modulates apoptosis. (This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award, Department of Defense VITAL W81XWH-04-1-0142 and NIH/NCI Head and Neck Cancer SPORE CA128613 awards. FR Khuri and SY Sun are Georgia Cancer Coalition Distinguished Cancer Scholars) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4782. doi:10.1158/1538-7445.AM2011-4782