Melanoma subpopulations that rapidly escape MAPK pathway inhibition incur DNA damage and rely on stress signalling

Despite the increasing number of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance. While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug. Here we track the responses of thousands of single melanoma cells to BRAF inhibitors and show that a subset of cells escapes drug via non-genetic mechanisms within the first three days of treatment. Cells that escape drug rely on ATF4 stress signalling to cycle periodically in drug, experience DNA replication defects leading to DNA damage, and yet out-proliferate other cells over extended treatment. Together, our work reveals just how rapidly melanoma cells can adapt to drug treatment, generating a mutagenesis-prone subpopulation that expands over time.

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April 2020
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Sample size was not predetermined. For single cell analysis (live-cell imaging, immunofluorescence, FISH), the number of cells in each replicates were analyzed in this study. For each condition, there are usually more than 500 cells, which is sufficient to make clear conclusion.
Additionally, for imaging experiments, 96-well plate formats were used, where at least 6 wells, 4 sites/well were used as technical replicates per condition tested.
Data exclusions In scRNA-seq section, we excluded cells having proliferation probability greater or equal than exp(-40) but strictly less than 1 from the analysis since a reliable classification might not be possible.

Replication
All attempts at replication were successful. At least two independent repeats were preformed for all experiments in this paper.
Randomization Not relevant to our study since we seed cells into a 96-well plate and image the whole plate with automated microscopy.
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