August 1, 2016
At 24 hours post-seeding, 100 Âľl of serum-free medium with or without cetuximab (5 Îźg/ml) was manually added to the cells. All other drugs were added directly on the plate by TECAN D300e digital dispenser (HP). After 72 hoursâ treatment cell viability was assessed by ATP content using CellTiter-Glo Luminescent Assay (Promega). Viability was normalized as a percentage of control untreated cells. Data from growth-inhibition assays were plotted using the nonlinear regression curve fit modelling from GraphPad Prism-5 (GraphPad Software)
Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. Š2016 AACR. Š2016 American Association for Cancer Research.
Oddo, D; Sennott, EM; Barault, L; Valtorta, E; Arena, S; Cassingena, A; Filiciotto, G; Marzolla, G; Elez, E; van Geel, RM; Bartolini, A; Crisafulli, G; Boscaro, V; Godfrey, JT; Buscarino, M; Cancelliere, C; Linnebacher, M; Corti, G; Truini, M; Siravegna, G; Grasselli, J; Gallicchio, M; Bernards, R; Schellens, JH; Tabernero, J; Engelman, JA; Sartore-Bianchi, A; Bardelli, A; Siena, S; Corcoran, RB; Di Nicolantonio, F;
Journal: Cancer Res. Pages: 4504-4515