title: 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.
authors.affiliation: Discovery and Medical Biostatistics, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Blank
authors.forename: Xiaozhen J
authors.initials: XJ
authors.lastname: Liu
authors.forename: Katherine
authors.initials: K
authors.lastname: Cosmopoulos
authors.forename: David C
authors.initials: DC
authors.lastname: Bouck
authors.forename: Khristofer
authors.initials: K
authors.lastname: Garcia
authors.forename: Hugues
authors.initials: H
authors.lastname: Bernard
authors.forename: Olga
authors.initials: O
authors.lastname: Tayber
authors.forename: Greg
authors.initials: G
authors.lastname: Hather
authors.forename: Ray
authors.initials: R
authors.lastname: Liu
authors.forename: Usha
authors.initials: U
authors.lastname: Narayanan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Milhollen
authors.forename: Eric S
authors.initials: ES
authors.lastname: Lightcap
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D003517:Cyclopentanes / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D000075687:NEDD8 Protein; D016133:Polymerase Chain Reaction; D011743:Pyrimidines / Q000494:pharmacology*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D003517:Cyclopentanes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011743:Pyrimidines; D014452:Ubiquitins; C539933:pevonedistat