Expression profiling by high throughput sequencing
Pan-HER TKI neratinib has demonstrated clinical activity in patients with HER2-mutant cancers. However responses are heterogenoeus and not generally prolonged, suggesting de-novo and acquired resistance to neratinib. To study mechanisms of resistance to neratinib we generated neratinib resistant cells by gradual dose escalation until resistance was achieved and performed various analyses including RNAseq.
5637NR and OVCAR8NR cells were maintained under drug-free conditions for 1 week prior to seeding. Parental and neratinib-resistant cells were seeded in triplicate in 10-cm dishes and then treated with or without neratinib (5637 - 600 nM; OVCAR8 - 1 μM); 24h later, cells were harvested and RNA was purified using ReliaPrep RNA Cell Miniprep system (Promega).