LAUSR

Spontaneous genetic mutations allow an initially drug-sensitive population of cancer cells to acquire a drug-resistant phenotype. However, little is known about how drug-sensitive cells first evade drug action and survive in the presence of drug, referred to as "drug tolerance," a crucial step on the road to resistance. In this study, we combined single-cell time-lapse microscopy and MATLAB-based automated cell tracking to identify the molecular basis of drug tolerance. By simultaneously monitoring CDK2 and ERK activity in single cells, we found that melanoma cells harboring a BRAF-V600E mutation have high ERK activity and consecutively cycled with an inter-mitotic time of around 14 hr in full growth media. Upon treatment with dabrafenib, an ATP competitive B-Raf inhibitor, all cells entered a CDK2-low quiescence with reduced ERK activity. Strikingly, after 72 hr of dabrafenib treatment, a fraction of cells escaped drug action, re-entered the cell cycle, and divided once in presence of drug before re-entering a transient quiescence. Additional cells continued to escape and divide in the presence of drug, with a steady-state level of about 10% of the population engaged in the cell cycle at any point during the week-long observation period. We refer to cells that re-entered the cell cycle as "escapees" and cells that stayed in quiescence during the observation period as "non-escapees." Remarkably, we found that escapees showed significantly higher ERK activity than non-escapees after a few days of treatment, even though their initial ERK activities were indistinguishable. To test whether reactivation of the MAPK pathway is the cause of cell cycle re-entry, we co-treated cells with dabrafenib and the MEK inhibitor trametinib. This co-treatment resulted in even lower ERK activity and elimination of the escapee population. This result implies that dabrafenib, even at high doses, does not fully turn off the MAPK pathway, thereby enabling eventual escape from drug. By monitoring escaping cells at the molecular level in real time, our work thus suggests a possible mechanism for cells to develop drug tolerance in less than three days, long before acquiring genetic mutations. Citation Format: Chen Yang, Chengzhe Tian, Sabrina L. Spencer. Single-cell imaging reveals MAPK pathway reactivation in drug-tolerant melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2833.