Novel DNA Damage Checkpoints Mediating Cell Death Induced by the NEDD8-Activating Enzyme Inhibitor MLN4924

Abstract

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.

Neddylation Inhibition Activates the Extrinsic Apoptosis Pathway through ATF4–CHOP–DR5 Axis in Human Esophageal Cancer Cells

“…Death receptor-mediated (extrinsic) apoptosis represents one of the most important cytotoxic pathways activated by anticancer agents (11,27,(41)(42)(43)(44). In this study, we demonstrated, to our knowledge for the first time, that neddylation inhibition with MLN4924 induces DR5-mediated apoptosis in ESCC cells.…”

“…Targeting neddylation pathway has been recently demonstrated as an attractive anticancer strategy, evidenced by the efficacy of the NAE inhibitor MLN4924, a first-in-class anticancer agent, in a multitude of preclinical studies (1)(2)(3)(11)(12)(13)(14)(15)(16)(17)(18)(19). Currently, MLN4924, used as a single agent or in combination with traditional chemotherapeutics, is under investigation in quite a few of phase I/II clinical trials (http://www.clinicaltrials.gov) and has exhibited promising clinical activity in both advanced solid tumors and relapsed/refractory multiple myeloma or lymphoma (18,19).…”

MLN4924 induces Noxa upregulation in acute myelogenous leukemia and synergizes with Bcl-2 inhibitors

“…Levels of other Bcl-2 family proteins did not appear to change in response to MLN4924 (Figure 2a). Cdt1, implicated in DNA re-replication to trigger apoptosis, did accumulate in response to MLN4924 treatment, although not to the same degree previously documented in solid tumors 1,11,33 even though the drug was clearly active, as verified by the classic disappearance of the Nedd8-modified, active form of Cullin1 (Figure 2a, top panel). Addition of N-acetylcysteine (NAC) during MLN4924 exposure did not alter Noxa accumulation (Supplementary Figure S2a), ruling out a contribution of ROS to the cytotoxic mechanism.…”

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway

“…U2OS cells labeled with arginine and lysine containing either light (R0K0), medium (R6K4), or heavy (R10K8) stable isotopes were either untreated 14 , or treated with ActD (medium), or with MLN4924 (heavy), for 15hrs. Equal numbers of light, medium and heavy cells were mixed, fractionated and nucleoli isolated (Fig.…”

“…Recent studies showed that inhibition of NEDDylation by MLN4924 activates the p53 4 tumour suppressor and that the p53 pathway is relevant for the biological response to MLN4924 treatment 13,14 . Interestingly, MLN4924-induced apoptosis is increased upon knockdown of p53 in tumour cell lines containing wild type p53, suggesting that p53 activation may protect cells against MLN4924 13 .…”