Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells

G Wei Xu; Julia I Toth; Sara R da Silva; Stacey-Lynn Paiva; Julie L Lukkarila; Rose Hurren; Neil Maclean; Mahadeo A Sukhai; Rabindra N Bhattacharjee; Carolyn A Goard; Bruno Medeiros; Patrick T Gunning; Sirano Dhe-Paganon; Matthew D Petroski; Aaron D Schimmer

doi:10.1371/journal.pone.0093530

Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells

PLoS One. 2014 Apr 1;9(4):e93530. doi: 10.1371/journal.pone.0093530. eCollection 2014.

Affiliations

¹ Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
² NCI-designated Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
³ Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario, Canada.
⁴ Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario, Canada; HalTech Regional Innovation Centre, Sheridan Institute of Technology and Advanced Learning, Oakville, Ontario, Canada.
⁵ Division of Nephrology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America.

Abstract

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphate / chemistry
Adenosine Triphosphate / metabolism
Antineoplastic Agents / chemistry
Antineoplastic Agents / pharmacology*
Cell Line, Tumor
Cullin Proteins / metabolism
Cyclopentanes / chemistry
Cyclopentanes / pharmacology*
DNA Mutational Analysis
Drug Resistance, Neoplasm / genetics*
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology*
Genotype
Humans
K562 Cells
Leukemia / genetics*
Leukemia / metabolism
Models, Molecular
NEDD8 Protein
Point Mutation
Protein Binding
Protein Conformation
Pyrimidines / chemistry
Pyrimidines / pharmacology*
Structure-Activity Relationship
U937 Cells
Ubiquitin-Activating Enzymes / antagonists & inhibitors*
Ubiquitin-Activating Enzymes / chemistry
Ubiquitin-Activating Enzymes / genetics*
Ubiquitins / genetics
Ubiquitins / metabolism

Substances

Antineoplastic Agents
Cullin Proteins
Cyclopentanes
Enzyme Inhibitors
NEDD8 Protein
NEDD8 protein, human
Pyrimidines
Ubiquitins
Adenosine Triphosphate
Ubiquitin-Activating Enzymes
NAE protein, human
pevonedistat

Grants and funding

This work was supported by the Canadian Cancer Society Research Institute, the Leukemia & Lymphoma Society, the American Cancer Society (M.D.P.), and the V Foundation for Cancer Research (M.D.P.). M.D.P. is an American Cancer Society Research Scholar (RSG-11-224-01-DMC). A.D.S. is a Leukemia and Lymphoma Society Scholar in Clinical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.