Silvia La Monica1, Roberta Minari2, Daniele Cretella1, Mara Bonelli1, Claudia Fumarola1, Andrea Cavazzoni1, Maricla Galetti3,4, Graziana Digiacomo1, Federica Riccardi5, Pier Giorgio Petronini1, Marcello Tiseo6,7, Roberta Alfieri1. 1. Department of Medicine and Surgery, University of Parma, Parma, Italy. 2. Medical Oncology Unit, University Hospital of Parma, Parma, Italy. 3. Italian Workers' Compensation Authority (INAIL) Research Center, Parma, Italy. 4. Center of Excellence for Toxicological Research (CERT), University of Parma, Parma, Italy. 5. Regional Reference Centre for Inherited Bleeding Disorders, University Hospital of Parma, Parma, Italy. 6. Department of Medicine and Surgery, University of Parma, Parma, Italy. marcello.tiseo@unipr.it. 7. Medical Oncology Unit, University Hospital of Parma, Parma, Italy. marcello.tiseo@unipr.it.
Abstract
BACKGROUND: Osimertinib is a new third-generation, epidermal growth factor receptor-tyrosine kinase inhibitor highly selective for the epidermal growth factor receptor with both activating and T790M mutations. A recent phase III trial showed a statistically significant progression-free survival benefit with osimertinib vs. gefitinib or erlotinib as first-line treatment for EGFR-mutated non-small cell lung cancer, and preliminary data are available on resistance mechanisms to first-line osimertinib therapy. OBJECTIVE: The objective of this study was to examine potential in vitro mechanisms of acquired resistance to osimertinib in a cell model carrying an EGFR exon 19 deletion. METHODS: PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. Three clones resistant to osimertinib (half maximal inhibitory concentration > 1 μM) were isolated, genotyped by next-generation sequencing and tested for drug sensitivity. Cell proliferation and migration, cell death, and signaling transduction pathways were analyzed. RESULTS: Our study revealed that all the three resistant clones developed acquired resistance via the BRAF G469A mutation maintaining a constitutive activation of the ERK pathway. Stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib. Treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation. CONCLUSIONS: Our in vitro studies revealed the BRAF G469A-activating mutation as a potential mechanism of acquired resistance to first-line osimertinib treatment, and provide a strategy of intervention to overcome this mechanism of resistance.
BACKGROUND: Osimertinib is a new third-generation, epidermal growth factor receptor-tyrosine kinase inhibitor highly selective for the epidermal growth factor receptor with both activating and T790M mutations. A recent phase III trial showed a statistically significant progression-free survival benefit with osimertinib vs. gefitinib or erlotinib as first-line treatment for EGFR-mutated non-small cell lung cancer, and preliminary data are available on resistance mechanisms to first-line osimertinib therapy. OBJECTIVE: The objective of this study was to examine potential in vitro mechanisms of acquired resistance to osimertinib in a cell model carrying an EGFR exon 19 deletion. METHODS: PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. Three clones resistant to osimertinib (half maximal inhibitory concentration > 1 μM) were isolated, genotyped by next-generation sequencing and tested for drug sensitivity. Cell proliferation and migration, cell death, and signaling transduction pathways were analyzed. RESULTS: Our study revealed that all the three resistant clones developed acquired resistance via the BRAF G469A mutation maintaining a constitutive activation of the ERK pathway. Stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib. Treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation. CONCLUSIONS: Our in vitro studies revealed the BRAF G469A-activating mutation as a potential mechanism of acquired resistance to first-line osimertinib treatment, and provide a strategy of intervention to overcome this mechanism of resistance.
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Authors: Silvia La Monica; Cristina Caffarra; Francesca Saccani; Elena Galvani; Maricla Galetti; Claudia Fumarola; Mara Bonelli; Andrea Cavazzoni; Daniele Cretella; Rita Sirangelo; Rita Gatti; Marcello Tiseo; Andrea Ardizzoni; Elisa Giovannetti; Pier Giorgio Petronini; Roberta R Alfieri Journal: PLoS One Date: 2013-10-22 Impact factor: 3.240
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