A single-step, high-dose selection scheme reveals distinct mechanisms of acquired resistance to oncogenic kinase inhibition in cancer cells

Despite the remarkable clinical efficacy demonstrated by molecularly-targeted cancer therapeutics, the benefits are typically temporary due to the emergence of acquired drug resistance. This has spurred a massive effort by the cancer research community to identify mechanisms used by cancer cells to evade treatment. Among the various methodologies developed and employed to identify such mechanisms, the most commonly used approach has been to model acquired resistance by exposing cancer cells in culture to gradually increasing concentrations of drug over an extended period of time. Here, we employed a less commonly used variation on this approach wherein resistant cells are selected by immediately exposing cancer cells to a continuous, high concentration of drug. Using this approach, we isolated clones representing three distinct mechanisms of resistance to inhibition of MET kinase activity from a single clonally-derived cancer cell line. The emergent clones had acquired resistance through engagement of alternative receptor tyrosine kinases, either through upregulation of FGF3 or HBEGF, or increased MAPK signaling through an activating V600E mutation in BRAF. Importantly, these mechanisms were not identified using the conventional 9ramp-up9 approach in previous studies that employed the same cell line. These results suggest that the particular nature of the selection scheme employed in cell culture modeling studies can determine which potential resistance mechanisms are identified, and which ones may be missed, highlighting the need for careful consideration of the specific approach used to model resistance in cultured cells.