title: Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.
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.
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: G Wei
authors.initials: GW
authors.lastname: Xu
authors.affiliation: NCI-designated Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Julia I
authors.initials: JI
authors.lastname: Toth
authors.affiliation: Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario, Canada.
authors.forename: Sara R
authors.initials: SR
authors.lastname: da Silva
authors.affiliation: Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario, Canada.
authors.forename: Stacey-Lynn
authors.initials: SL
authors.lastname: Paiva
authors.affiliation: Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario, Canada; HalTech Regional Innovation Centre, Sheridan Institute of Technology and Advanced Learning, Oakville, Ontario, Canada.
authors.forename: Julie L
authors.initials: JL
authors.lastname: Lukkarila
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: Rose
authors.initials: R
authors.lastname: Hurren
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: Neil
authors.initials: N
authors.lastname: Maclean
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: Mahadeo A
authors.initials: MA
authors.lastname: Sukhai
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: Rabindra N
authors.initials: RN
authors.lastname: Bhattacharjee
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Goard
authors.forename: Bruno
authors.initials: B
authors.lastname: Medeiros
authors.affiliation: Department of Chemistry, University of Toronto Mississauga, Mississauga, Ontario, Canada.
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Gunning
authors.affiliation: Division of Nephrology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Sirano
authors.initials: S
authors.lastname: Dhe-Paganon
authors.affiliation: NCI-designated Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Petroski
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Schimmer
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D044842:Cullin Proteins / Q000378:metabolism; D003517:Cyclopentanes / Q000737:chemistry / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005838:Genotype; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D000075687:NEDD8 Protein; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D020298:U937 Cells; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D044842:Cullin Proteins; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011743:Pyrimidines; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human; C539933:pevonedistat