MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G 2 -M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G 2 phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924. Cancer Res; 73(1); 225-34. Ó2012 AACR.

Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45α, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death. Cancer Res; 70(11); 4318-26. ©2010 AACR.

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. Thirtyeight 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

MLN4924 is an investigational small-molecule inhibitor of the Nedd8-activating enzyme currently in phase I clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standard-of-care agents and may affect the clinical development of MLN4924. As such, we studied its interaction with other DNA-damaging agents. Mitomycin C, cisplatin, cytarabine, UV radiation, SN-38, and gemcitabine demonstrated synergy in combination with MLN4924 in vitro. The combination of mitomycin C and MLN4924 was shown to be synergistic in a mouse xenograft model. Importantly, depletion of genes within the ataxia telangiectasia and Rad3 related (ATR) and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. In addition, comet assay demonstrated increased DNA strand breaks with the combination of MLN4924 and mitomycin C. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924 results in frequent replication fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may provide useful information for the clinical development of these combinations. Mol Cancer Ther; 13(6); 1625-35. Ó2014 AACR.

Platinum-based therapies, both cisplatin and carboplatin, are utilized as part of first-line standard of care regimens for advanced non-small cell lung cancer (NSCLC), but overall response rate and overall survival remain limited, with a 5-year survival rate of 18%. Therefore, agents that can improve responses and survival are needed. Pevonedistat (MLN4924) is an investigational small molecule inhibitor of the Nedd8-activating enzyme (NAE) currently in Phase Ib clinical trials. Nedd8 is a small ubiquitin-like protein whose activation of the cullin family of E3 ubiquitin ligases is dependent on NAE activity. Importantly, pevonedistat synergized with carboplatin in a cell viability assay for 6 of 20 NSCLC cell lines tested in vitro, and synergy was also detected in the triple combination of carboplatin, pevonedistat, and paclitaxel. Order of addition experiments in the A549 NSCLC cell line demonstrated a benefit of simultaneous treatment or addition of pevonedistat prior to platinum treatment, while a reduced combination benefit was observed when cisplatin was added 24-48 hrs before pevonedistat. Levels of NAE pathway inhibition were similar in pevonedistat and pevonedistat+cisplatin treated cells, indicating that a drug-drug interaction was not likely the cause of the synergy. Using patient-derived xenograft (PDX) models a combination benefit of carboplatin and pevonedistat was demonstrated in a carboplatin-insensitive model but a similar improvement in response was not observed in a carboplatin-sensitive model, adding to evidence that pevonedistat might target platinum resistance mechanisms. To evaluate the mechanism of synergy between pevonedistat and platinum, in vitro experiments with RNAi were performed in 4 cell lines. These studies identified 2 mechanisms of synergy between pevonedistat and platinums in vitro. Depletion of genes within the TC-NER (transcription-coupled nucleotide excision repair) and ICR (interstrand crosslink repair) pathways reduced the synergy between pevonedistat and platinum, with the contribution of each pathway varying by cell line. Because these pathways are also implicated in the response to single agent platinums, the results suggest pevonedistat could delay completion of platinum-induced DNA repair and therefore result in enhanced cell death. Inhibition of neddylation of the E3 ubiquitin ligase CUL4-RBX1-DDB1-ERCC8 by pevonedistat may delay the TC-NER pathway, providing a direct mechanism of resistance reversal by pevonedistat. The combination of pevonedistat with carboplatin and paclitaxel is under evaluation as part of a phase 1b trial in adult patients with solid tumors (NCT01862328). Citation Format: David C. Bouck, Khristofer Garcia, Jonathan L. Blank, Xiaozhen J. Liu, Hugues Bernard, Allison J. Berger, Mike Kuranda, Erik Koenig, Eric S. Lightcap. Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C55.

<p>PDF file - 337K, Analysis of flow cytometry results of the effect of vehicle (DMSO) or 3 muM MLN4924 for 24 h on multiple cell lines</p>

<p>PDF file - 355K, Effect of Gene Knock-down on Cell Cycle Distribution of A375 Melanoma Cells in Combination with Vehicle</p>

<p>PDF file - 1.6MB, Full Length Western Blots in Support of Figures 4 and 5</p>