ABSTRACT
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, Gadd45alpha, 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.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Cell Death/drug effects , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Pyrazines/pharmacology , RNA, Small Interfering/genetics , Bortezomib , Cell Death/genetics , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA Damage , Gene Knockdown Techniques , HCT116 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Ribosomes/drug effects , TOR Serine-Threonine Kinases , TransfectionABSTRACT
The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.
