Efficacy Of MLN4924 Research Articles

Gene expression profiling reveals the genomic changes caused by MLN4924 and the sensitizing effects of NAPEPLD knockdown in pancreatic cancer

ABSTRACT MLN4924 inhibits the proteolytic degradation of Cullin-Ring E3 ligase (CRL) substrates and exhibits antitumor activity toward various malignancies, including pancreatic cancer. MLN4924 suppresses tumor growth by altering various key regulator proteins; however, its impact on gene expression in tumors remains unknown. In this study, the genomic changes caused by MLN4924 in pancreatic cancer were examined by gene chip analysis and ingenuity pathway analysis. Eleven pathways were significantly altered (5 activated and 6 inhibited), 45 functions were significantly changed (21 activated and 24 inhibited), and the most activated upstream factor was predicted to be TNF. Of 691 differentially expressed genes, NAPEPLD knockdown showed synergism with MLN4924, as determined by real-time quantitative PCR and high content screening. NAPEPLD knockdown enhanced the effect of MLN4924 on inhibiting proliferation and inducing apoptosis in vitro. In a pancreatic cancer nude mouse model, MLN4924 inhibited tumor growth more significantly in the NAPEPLD knockdown group than in the control group. NAPEPLD expression was higher in pancreatic cancer tissues than in the normal pancreas but was not associated with prognosis. These findings indicate that MLN4924 causes extensive genomic changes in pancreatic cancer cells, and targeting NAPEPLD may increase the efficacy of MLN4924.

EXTH-43. DYNAMIC MULTI-OMICS ANALYSIS OF MLN4924-TREATED GLIOBLASTOMA CELLS REVEALS ABSENCE OF PTEN AS A CANDIDATE DRIVER OF NEDDYLATION INHIBITION RESISTANCE

Abstract Glioblastoma multiforme (GBM) is the most lethal primary brain tumor and calls for novel therapeutic development. The neddylation pathway may be a therapeutic target, as global over activation of neddylation has been found in GBM patients and has correlated with shorter patient survival. Analogous to ubiquitination in reaction scheme and enzyme classes used, neddylation is a post-translational modification that is essential to many protein regulation and biological processes. Although non-cullin NEDD8 substrates have been investigated in recent years, the most well-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), which act as a scaffold for substrates to be ubiquitinated and degraded. The neddylation inhibitor MLN4924 targets NEDD8 Activating Enzyme (NAE), an upstream activator of neddylation, and induces cell cycle arrest, apoptosis and senescence in cancer cells. In this work, we investigated the context of vulnerability to Pevonedistat (MLN4924) in GBM. Efficacy of MLN4924 in glioma cell models was evaluated by measuring cell viability (CellTiterGlo®), colony formation efficiency, and cell cycle progression (flow cytometry with propidium iodide staining). GB1 (IC50= 0.28 μM), LN18 (IC50 = 0.19 μM), and GBM43 (IC50= 0.45 μM) were established as sensitive and M059K (IC50 = 5.5 μM), SNU1105 (IC50 = 20.9 μM), and GBM39 (IC50= 10.3 μM) as non-sensitive cell lines based on IC50 values. To discover genomic and/or proteomic markers of differential response, we collected RNA and protein from sensitive and non-sensitive cell lines after 0, 2, 8 and 24 hour treatment with MLN4924 for RNA sequencing and mass spectroscopy analyses. Multi-OMICS data point towards the absence of functional PTEN as a driver of MLN4924 resistance. Uncovering the mechanism underlying GBM’s vulnerability to MLN4924 will expand the knowledge of neddylation’s role in cancer and may arm physicians with an understanding of whether a GBM patient would respond to MLN4924 treatment or not.

Abstract 248: Dynamic multi-OMICS analysis of glioblastoma cells reveals context of vulnerability to neddylation inhibition by pevonedistat

Abstract As the most lethal primary brain tumor, glioblastoma multiforme (GBM) calls for novel therapeutic development. Global over activation of neddylation (a post-translational modification) has recently been found in GBM patients and has correlated with shorter patient survival. Significant accumulation of neddylation in recurrent GBM tissues indicates its importance in tumorigenesis and tumor progression. Analogous to the ubiquitination pathway, neddylation is essential to many protein regulation and biological processes. Although most well-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), non-cullin NEDD8 substrates have been investigated in recent years. Neddylation and subsequent degradation of PARC, p53, MDM2 and EGFR exemplify the broader functional role of neddylation. The neddylation inhibitor MLN4924 targets NEDD8 Activating Enzyme (NAE), an upstream activator of neddylation, and, as a result, induces cell cycle arrest, apoptosis and senescence in cancer cells. In this work, we investigated the context of vulnerability to Pevonedistat (MLN4924) in GBM by comparing the dynamic response of sensitive and non-sensitive cells using transcriptomics and proteomics profiling, using long-established and patient derived glioma cell lines. Efficacy of MLN4924 in glioma cell models was evaluated by measuring cell viability (CellTiterGlo®), colony formation efficiency, and cell cycle progression (flow cytometry with propidium iodide staining). GB1 (IC50= 0.28 μM), LN18 (IC50 = 0.19 μM), and GBM43 (IC50= 0.45 μM) were established as sensitive and M059K (IC50= 5.5 μM), SNU1105 (IC50 = 20.9 μM), and GBM39 (IC50= 10.3 μM) as non-sensitive cell lines based on IC50 values. Western blot analysis of known cell cycle regulatory pathways and DNA damage response pathway did not show significant dynamic differences between sensitive and non-sensitive glioma cell models. To discover genomic and/or proteomic markers of differential response we collected RNA and protein for LN18 (sensitive) and SNU1105 (Non-sensitive) cells after 0, 2, 8 and 24 h treatment with MLN4924 at 100 nM and 500 nM concentration for transcriptomics and proteomics analysis. RNA sequencing was utilized for dynamic transcriptomic analysis. Cell lysates were processed using bottom-up proteomics workflow and the data was acquired on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. Proteins were identified by querying spectral data against canonical and RNA-Seq predicted proteins and differential analysis was carried out to identify candidate determinats of vulnerability. An understanding of determinants of vulnerability to MLN4924 will expand knowledge of neddylation’s role in cancer and may point to signatures of GBM patients most likely to respond to this targeted intervention. Citation Format: Shayesteh R. Ferdosi, Brett Taylor, Nanyun Tang, Rita Bybee, Sen Peng, Victoria David-Dirgo, Krystine Garcia-Mansfield, Ritin Sharma, Patrick Pirrotte, Michael Berens, Harshil Dhruv. Dynamic multi-OMICS analysis of glioblastoma cells reveals context of vulnerability to neddylation inhibition by pevonedistat [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 248.

Abstract 881: Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM)

Abstract Neddylation is a post-translational mechanism that marks proteins for degradation through activity of NEDD8 Activating Enzyme (NAE). NAE activates cullin-RING ligases (CRL), which ubiquitylate selected substrates and mark them for proteosomal degradation. MLN4924, or Pevonedistat, targets NAE and inhibits Neddylation and induces apoptosis in sensitive cells. To assess the preferential sensitivity of cell lines to MLN4924, we performed a 10-point drug dose response (DDR) assay on long-term established GBM cell lines. Efficacy of MLN4924 in glioma cell lines was evaluated by measuring cell viability (CellTiterGlo®) and cell cycle progression (flow cytometry with propidium iodide staining). To identify mechanism of differential response to MLN4924 treatment, cell cycle regulatory pathway and DNA damage were also examined by Western blotting. GB1 (IC50 = 0.28 μM) & LN18 (IC50 = 0.19 μM) were established as sensitive and M059K (IC50 = 5.5 μM) & SNU1105 (IC50 = 20.9 μM) as non-sensitive cell lines based on the IC50 values. Flow cytometry analysis of DNA content revealed significant arrest of cells in G2/M even at low doses of 100 nM of MLN4924 preferentially in GB1 and LN18. This was consistent with an increase in CRL substrates p21, p27 and WEE1 in GB1 and LN18 possibly contributing to the G2/M arrest. While CDT1 accumulation was observed starting at 2h post MLN4924 treatment in sensitive cell lines, it took upto 8h for CDT1 accumulation in the non-sensitive cell lines. Increases in CDT1 induced re-replication causing massive arrest in G2/M phase lead to increased DNA damage, validated by higher expression of γH2AX in the sensitive cell lines. Additionally, we also investigated the efficacy of MLN4924 against orthotopic glioma PDX models in vitro and in vivo to validate our findings. In a cohort of glioblastoma PDX models we discovered that GBM PDX models with lower Neddylation gene set enrichment score (GBM116, GBM59, SF7300) were markedly more vulnerable to MLN4924 than GBM PDX models with higher Neddylation gene set enrichment score (GBM91 and GBM102) in vitro and in vivo. Orthotopic PDX models of selected GBM revealed survival prolongation of GBM116, but minimal survival benefit to GBM102 tumors. Validation of the predictive markers of vulnerability to MLN4924 in additional PDX models will set the stage for prospective clinical trials of MLN4924 in glioblastoma patients. Citation Format: Leena Chaudhuri, Rita L. Bybee, Lauren K. Hartman, Sen Peng, Darren Finlay, Kristiina Vuori, Michael E. Berens, Harshil D. Dhruv. Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 881.

Abstract 1177: Targeting NEDD8 to uncover an exceptional responder molecular subtype in glioblastoma

Abstract Neddylation is a post-translational mechanism that marks proteins for degradation through activity of NEDD8 Activating Enzyme (NAE). NAE blocks cullin-RING ligases from initiating proteosomal degradation of select substrates including cell cycle regulators and apoptosis modulators. MLN4924, or Pevonedistat, targets NAE and inhibits Neddylation and induces apoptosis in sensitive cells. We have discovered, in a cohort of glioblastoma PDX models, an exceptional responder to MLN4924 (GBM102). Most pertinently the effects we observe are in a PDX cultured as 3D neurospheres that more closely resemble the true tumor architecture, heterogeneity, and “stem-like” phenotype characteristic of tumor growth. We have leveraged RNAseq expression data from Cancer Cell Line Encyclopedia (CCLE) and Pevonedistat response data from The Cancer Therapeutics Response Portal (CTRP) to apply a network-based analysis to identify pathways enriched with differential dependencies between cell lines sensitive and non-sensitive to MLN4924. The analysis also identifies potential mediating genes that appear to play critical roles in such differential dependency networks. Identified differential networks and mediators provide insight for cellular mechanisms underlying drug response. Additionally, we also investigated the efficacy of MLN4924 against orthotopic glioma PDX models (GBM102 and GBM116) in vivo to validate our findings in vitro. Thus genomic characterization of patient samples may lead to the identification of a molecular signature which is associated with a subset of GBMs vulnerable to MLN4924. As the treatment options for GBM are extremely limited, this may highlight a novel alternative opportunity to treat a select fraction of patients with this aggressive disease. Citation Format: Lauren K. Hartman, Darren Finlay, Peiwen Pan, Seungchan Kim, Gil Speyer, Jeff Kiefer, Harshil Dhruv, Kristiina Vuori, Michael Berens. Targeting NEDD8 to uncover an exceptional responder molecular subtype in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1177. doi:10.1158/1538-7445.AM2017-1177

Abstract NTOC-102: TARGETING NEDDYLATION TO OVERCOME CISPLATIN RESISTANCE IN OVARIAN CANCER

Abstract PURPOSE: Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major problem that limits overall survival and novel therapeutic strategies are urgently needed. The inappropriate loss of several NEDD8-cullin-RING ubiquitin ligase (CRL) targets has been linked to cancer progression and drug resistance, suggesting that this pathway may be a promising new therapeutic target. MLN4924 is a first-in-class NEDD8 activating enzyme (NAE) inhibitor currently under investigation in multiple clinical studies. We investigated its anticancer activity and pharmacodynamic effects in cisplatin-sensitive and cisplatin-resistant ovarian cancer models. EXPERIMENTAL PROCEDURES: Cellular sensitivity to MLN4924/cisplatin was determined by measuring viability, senescence, and apoptosis. The effects of drug treatment on global protein expression, DNA damage, and reactive oxygen species generation were determined. RNA interference approaches were used to identify and validate regulators of therapeutic sensitivity. The in vivo effects of MLN4924/cisplatin on tumor burden and key pharmacodynamic endpoints were assessed in cisplatin-sensitive and -resistant xenograft models. RESULTS: Intrinsic resistance to cisplatin did not significantly impact the efficacy of MLN4924 against ovarian cancer cells, indicating that this agent may be useful for patients with platinum-resistant disease. Detailed pharmacodynamic analyses showed that treatment with MLN4924 induced the stabilization of key NEDD8 substrates and regulators of cellular redox status. Notably, cisplatin-resistant cells displayed significantly higher basal levels of NEDDylated cullins and NEDDylation activity than cisplatin-sensitive cells. Further investigation demonstrated that MLN4924 significantly augmented the activity of cisplatin against cisplatin-resistant cells. This suggests that aberrant NEDDylation may contribute to drug resistance and that targeting this pathway may be a promising strategy to restore chemosensitivity. MLN4924 and cisplatin cooperated to induce DNA damage, oxidative stress, and increased expression of the BH3-only protein NBK/BIK. Targeted NBK/BIK knockdown diminished the pro-apoptotic effects of the MLN4924/cisplatin combination. In addition, administration of MLN4924 to mice bearing ovarian tumor xenografts significantly increased the efficacy of cisplatin against both cisplatin-sensitive and cisplatin-resistant tumors. CONCLUSIONS: Our collective data provide a rationale for the clinical investigation of NAE inhibition as a novel strategy to augment cisplatin efficacy in patients with ovarian cancer and other malignancies where platinum-based chemotherapy is utilized. Citation Format: Steffan T. Nawrocki, Kevin R. Kelly, Claudia M. Espitia, and Jennifer S. Carew. TARGETING NEDDYLATION TO OVERCOME CISPLATIN RESISTANCE IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-102.

Abstract 1780: Targeted inactivation of CRL4-CDT2 E3 ubiquitin ligase as a novel therapeutic strategy for malignant melanoma

Abstract The CRL4CDT2 E3 ubiquitin ligase is emerging as a critical regulator cell cycle progression, primarily through the targeted ubiquitylation and degradation of critical cell cycle regulators including CDT1, p21 and SET8 via the 26S proteasome. CDT2, the substrate receptor of the CRL4CDT2 E3 ligase is overexpressed or amplified in several human malignancies, but its role in the development or progression of these cancers remains elusive. We show that CDT2 is overexpressed in primary and metastatic melanoma. Targeted depletion of CDT2 by si-RNA or its pharmacological inhibition by MLN4924, a selective NEDD8-activating enzyme (NAE) inhibitor, suppressed the proliferation of melanoma cancer cells in vitro and inhibited tumor growth of human melanoma xenografts in mice, irrespective of the BRAF mutation status. Inhibition of CRL4CDT2 in melanoma cells with various genetic backgrounds, but not in immortalized human melanocytes, resulted in robust p21- and SET8-dependent rereplication. These results identify the CRL4CDT2 E3 ubiquitin ligase as a promising new therapeutic target for cutaneous melanoma, and demonstrate the efficacy of MLN4924 as a promising anti-melanoma agent. Citation Format: Mouadh Benamar, Fadila Guessous, Patrick Corbett, Craig L. Slingluff, Tarek Abbas. Targeted inactivation of CRL4-CDT2 E3 ubiquitin ligase as a novel therapeutic strategy for malignant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1780. doi:10.1158/1538-7445.AM2015-1780

589 N-myc downstream regulated gene 1 (NDRG1) as a novel anti-angiogenic and therapeutic target for VEGF/VEGF receptor signaling by vascular endothelial cells

Results: There was variable efficacy of MLN4924 in pediatric cancer cell lines with IC50 values ranging from 35 nM in leukemia, to 1500 nM in an ATRT. MLN4924 resulted in decreased neddylation of CRLs and the stabilization of CRL substrates p21 and p-IuBa and led to DNA rereplication and delay in S phase and G2/M phase progression. MLN4924induced cell death was partially mediated by the production of reactive oxygen species (ROS), as ROS scavenger N-acetylcysteine protected MLN4924-treated leukemia cells. In drug combination assays MLN4924 did not synergy with many conventional chemotherapeutics including etoposide, methotrexate, and doxorubicin. Interestingly, additive effect to weak synergy was observed with the bromodomain inhibitor JQ1 (CI: 0.7−1.0). Conclusion: We demonstrated that MLN4924 has variable but excellent in vitro efficacy towards pediatric cancer cell lines. It induced cell death and cycle arrest by the inhibition of cullin activity and the production of ROS. MLN4924 displayed combinatorial effects with BET inhibition. These preliminary observations provide the rationale for additional studies and to explore the use of MLN4924 in early phase clinical trials in children with refractory malignancies.

Targeting Neddylation pathways to inactivate cullin-RING ligases for anticancer therapy.

Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach. MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy. In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations. In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s.

Overcoming Platinum Resistance in Preclinical Models of Ovarian Cancer Using the Neddylation Inhibitor MLN4924

The nearly ubiquitous development of chemoresistant disease remains a major obstacle against improving outcomes for patients with ovarian cancer. In this investigation, we evaluated the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, in ovarian cancer cells. Efficacy of MLN4924 both alone and in combination with platinum was assessed. Overall, single-agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. However, the combination of MLN4924 with cisplatin or carboplatin produced synergistic effects in SKOV3 and ES2 cells, as well as in primary ovarian cancer cell lines established from high-grade serous, clear cell, and serous borderline ovarian tumors. The efficacy of cisplatin plus MLN4924 was also evident in several in vitro models of platinum-resistant ovarian cancer. Mechanistically, the combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, abrogation of FANCD2 monoubiquitination, or CHK1 phosphorylation. An siRNA screen was used to investigate the contribution of each member of the cullin RING ligase (CRL) family of E3 ubiquitin ligases, the best-characterized downstream mediators of MLN4924's biologic effects. Cisplatin-induced cytotoxicity was augmented by depletion of CUL3, and antagonized by siCUL1 in both ES2 and SKOV3 ovarian cancer cells. This investigation identifies inhibition of neddylation as a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer.

Abstract 3380: Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924.

Abstract Purpose: The objective of this investigation was to evaluate the preclinical activity of MLN4924, an investigational inhibitor of the NEDD8-activating enzyme, against chemoresistant and chemosensitive ovarian cancer cells. ExperimentalDesign: Efficacy of MLN4924 both alone and in combination with cisplatin was assessed using short-term tetrazolium based and longer term focus formation assays. Mechanistic studies included cell cycle analysis, evaluation of DNA-platinum adduct formation (using an ELISA), and evaluation of DNA damage repair pathways. In addition, we employed an siRNA screen to assess the contribution of each member of the Cullin RING-Ligase (CRL) family of E3 ubiquitin ligases, the best characterized downstream mediators of MLN4924’s biological effects. Results: Single agent MLN4924 exhibited moderate activity in ovarian cancer cell lines. The combination of MLN4924 with cisplatin or carboplatin resulted in significant platinum sensitization in SKOV3 and ES2 cells, as well as, in several primary ovarian cancer cell lines established from high grade serous, clear cell, and serous borderline ovarian tumors. Furthermore, the platinum sensitizing effect of MLN4924 was also observed in several in vitro models of platinum resistant ovarian cancer (using both established and primary chemoresistant ovarian cancer cells). Mechanistically, we show that depletion of CUL3 and CUL5 augment, while knockdown of CUL1 and CUL4A antagonize cisplatin-induced cytotoxicity in ovarian cancer cells. The combination of cisplatin and MLN4924 was not associated with DNA re-replication, altered platinum-DNA adduct formation, or abrogation of FANCD2 monoubiquitination. Conclusions: Our investigation reveals a novel mechanism for overcoming platinum resistance in vitro, and provides a strong rationale for clinical investigations of platinum and MLN4924 combinations in ovarian cancer. This work also highlights that different CRL ubiquitination pathways can have distinct and opposing effects on platinum sensitivity and resistance in ovarian cancer cells. Citation Format: Amir A. Jazaeri, Etsuko Shibata, Jonghoon Park, Jennifer Bryant, Mark R. Conaway, Susan C. Modesitt, Peter G. Smith, Michael Milhollen, Allison J. Berger, Anindya Dutta. Overcoming platinum resistance in ovarian cancer using the novel compound MLN4924. [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 3380. doi:10.1158/1538-7445.AM2013-3380

Ubiquitin-proteasome system, a new anti-tumor target

Ubiquitin-proteasome system (UPS) is a major pathway for intracellular protein degradation. Its target proteins include a broad array of regulatory proteins that play important roles in cell cycle progression, cell development and differentiation, DNA damage responses, and tumorgenesis. Dysfunction of UPS gives rise to various kinds of diseases including infectious diseases, autoimmune diseases and cancers. The ubiquitylation process carries out by three classes of enzymes, E1 (activating enzyme), E2 (conjugating enzyme) and E3 (ubiquitin ligase). The ubiquitin is activated by E1 in an ATP-dependent manner, and then transferred to E2, and finally E3 adds ubiquitin on a target protein covalently as a tag, which can be recognized by proteasome for degradation. The regulation of UPS is rather complicated. Human genome encodes hundreds of E3 ligases super family, which are responsible for specificity of protein degradation. Cullin-RING ligases (CRLs) are the largest family of E3 ubiquitin ligases, which have essential role in controlling numerous cancer-relevant proteins ubiquitylation and proteasomal degradation1. Therefore, UPS becomes potential drug target for ant-tumor medication. The proteasome inhibitor, bortezomib, has been used to treat multiple myeloma and mantle cell lymphoma with great success both clinically and commercially for 5 years. However, as bortezomib targets all ubiquitin-tagged proteins, lacking of substrate specificity may cause many side effects. A series of recently published articles have reported a promising anti-tumor candidate, called MLN4924, which is a small-molecule selective inhibitor of NEDD8-activating enzyme (NAE)2. NAE, a heterodimer of NAEα and NAEβ, adenylates a small ubiquitin-like protein NEDD8 at its C-terminus in an ATP-dependent manner as the first step in the neddylation cascade, and transfers NEDD8 to UBC12. Then, NEDD8 is conjugated to cullin protein on CRLs. This covalent modification actives cullin complex to recruit an ubiquitin-charged E2 and leads to downstream target protein polyubiquitination and proteasomal degradation. MLN4924, which can form a covalent adduct with NEDD8, and block NAE at the first step in this pathway, has shown anti-tumor activity in many kinds of cancer cell lines and xenograft models, including lung cancer, colon cancer, melanoma and lymphoma (Figure 1). The anti-tumor activity of MLN4924 results from inactivation of CLRs, which causes DNA re-replication and inhibition of nuclear factor (NF)-κB signaling, thus leading to cancer cell death3. Figure 1 MLN4924 blocks NAE pathway leading to inactivation of CRLs. MLN4924 is currently in clinical trials, and its safety for human and clinical activity has been confirmed in both solid tumor and hematologic malignancy. However, the resistance to the agent was also reported in some studies. Early in 2012, researchers found that treatment-emergent heterozygous mutations in the ATP binding pocket and NEDD8-binding cleft of NAEβ were responsible for the resistance to MLN4924 in cell line and xenograft models of cancer4. However, the genetic determinants responsible for the sensitivity to MLN4924 are not clear. Further study showed that more than 100 genes contributed to the sensitivity to MLN4924, and approximately a half of them interfered with components of cell cycle, apoptotic machinery, UPS and DNA damage responses. Among them, the genes involved in DNA replication, P53, BRCA1/BRCA2, transcription-coupled repair and base excision repair appeared to be important for the lethality of MLN4924. In colon cancer cells, the genes of G2/M checkpoint affected the sensitivity to MLN4924. Surprisingly, the p21-dependent intra-S-phase checkpoint and extensive re-replication play an essential role in this process5. These investigations on the efficacy of MLN4924 and the genes related to its resistance give us a clearer picture of this potential anti-tumor agent, and fill the gap from bedside to bench work.

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