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A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants
Published on: October 10, 2022
Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells
G Wei Xu1, Julia I Toth2, Sara R da Silva3
1Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
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.
Insights
Mechanisms of resistance to the anti-cancer drug MLN4924 involve mutations in the NEDD8-activating enzyme (NAE). These mutations reduce drug effectiveness, highlighting the need for new NAE inhibitors to overcome resistance in leukemia.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The NEDD8-activating enzyme (NAE) is crucial for neddylation, a post-translational modification process.
- MLN4924, a selective NAE inhibitor, shows anti-tumor activity and clinical promise in hematologic malignancies.
Purpose of the Study:
- To investigate the mechanisms underlying resistance to the NAE inhibitor MLN4924.
- To identify specific genetic alterations conferring resistance to MLN4924 in leukemia cells.
Main Methods:
- Leukemia cell lines (K562, U937) were cultured with MLN4924 for six months to select for resistant populations.
- Genetic sequencing identified mutations in the UBA3 gene (catalytic subunit of NAE) in resistant cells.
- Biochemical assays assessed the impact of mutations on NAE enzyme kinetics and MLN4924 potency.
Main Results:
- Resistant cell lines (R-K562(MLN), R-U937(MLN)) acquired specific mutations (I310N, Y352H) in the UBA3 gene.
- These UBA3 mutations increased NAE affinity for ATP and decreased affinity for NEDD8.
- Mutations reduced MLN4924 potency in vitro but maintained sufficient NAE activity for cell survival.
- Resistant cells exhibited cross-resistance to other NAE inhibitors but remained sensitive to pan-E1 inhibitors.
Conclusions:
- Acquired mutations in UBA3 are a key mechanism of resistance to MLN4924 in leukemia.
- Understanding these resistance mechanisms is essential for developing next-generation NAE inhibitors.
- Targeting NAE remains a viable strategy, but resistance pathways must be considered for therapeutic success.
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