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A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
Published on: December 7, 2014
Evolution of resistance to Aurora kinase B inhibitors in leukaemia cells
Timothy W Failes1, Gorjana Mitic, Heba Abdel-Halim
1Childrens Cancer Institute Australia, Lowy Cancer Research Centregh, University of New South Wales, Randwick, Australia.
Abstract:
Aurora kinase inhibitors are new mitosis-targeting drugs currently in clinical trials for the treatment of haematological and solid malignancies. However, knowledge of the molecular factors that influence sensitivity and resistance remains limited. Herein, we developed and characterised an in vitro leukaemia model of resistance to the Aurora B inhibitor ZM447439. Human T-cell acute lymphoblastic leukaemia cells, CCRF-CEM, were selected for resistance in 4 µM ZM447439. CEM/AKB4 cells showed no cross-resistance to tubulin-targeted and DNA-damaging agents, but were hypersensitive to an Aurora kinase A inhibitor. Sequencing revealed a mutation in the Aurora B kinase domain corresponding to a G160E amino acid substitution. Molecular modelling of drug binding in Aurora B containing this mutation suggested that resistance is mediated by the glutamate substitution preventing formation of an active drug-binding motif. Progression of resistance in the more highly selected CEM/AKB8 and CEM/AKB16 cells, derived sequentially from CEM/AKB4 in 8 and 16 µM ZM447439 respectively, was mediated by additional defects. These defects were independent of Aurora B and multi-drug resistance pathways and are associated with reduced apoptosis mostly likely due to reduced inhibition of the catalytic activity of aurora kinase B in the presence of drug. Our findings are important in the context of the use of these new targeted agents in treatment regimes against leukaemia and suggest resistance to therapy may arise through multiple independent mechanisms.
Insights
Developing resistance to Aurora kinase inhibitors in leukemia involves multiple mechanisms. A key mutation in Aurora B kinase causes resistance, with further defects impacting apoptosis and drug inhibition in advanced resistance models.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Aurora kinase inhibitors are novel mitosis-targeting drugs for treating hematological and solid malignancies.
- Understanding molecular factors influencing sensitivity and resistance to these drugs is crucial for effective cancer therapy.
- Limited knowledge exists regarding the mechanisms of resistance to Aurora kinase inhibitors, particularly Aurora B inhibitors.
Purpose of the Study:
- To develop and characterize an in vitro leukemia model of resistance to the Aurora B inhibitor ZM447439.
- To investigate the molecular mechanisms underlying acquired resistance to Aurora B inhibition in leukemia cells.
- To identify potential cross-resistance or hypersensitivity patterns in resistant leukemia models.
Main Methods:
- Development of resistant leukemia cell lines (CCRF-CEM) through sequential selection with increasing concentrations of ZM447439.
- Assessment of cross-resistance to other anti-cancer agents (tubulin inhibitors, DNA-damaging agents) and hypersensitivity to Aurora kinase A inhibitors.
- Genetic sequencing to identify mutations in Aurora B kinase domain and molecular modeling to predict drug-binding alterations.
- Analysis of apoptosis and inhibition of Aurora kinase B activity in resistant cells.
Main Results:
- Acquired resistance to ZM447439 in CEM/AKB4 cells was associated with a G160E mutation in the Aurora B kinase domain, preventing active drug-binding.
- Resistant cells showed no cross-resistance to other drug classes but were hypersensitive to an Aurora kinase A inhibitor.
- Further resistance in CEM/AKB8 and CEM/AKB16 cells involved additional, independent defects reducing apoptosis, likely due to impaired inhibition of Aurora kinase B activity.
Conclusions:
- Leukemia cells can develop resistance to Aurora B inhibitors through distinct molecular mechanisms, including kinase domain mutations and defects affecting apoptosis.
- These findings highlight the potential for multiple, independent resistance pathways to emerge during targeted therapy for leukemia.
- Understanding these mechanisms is critical for optimizing the use of Aurora kinase inhibitors in clinical treatment regimens.
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