Anti-cancer drugs commonly target signal transduction proteins activated by mutation. In human melanomas carrying mutated BRAF, small molecule RAF and MEK kinase inhibitors cause dramatic but often transient tumor regression. Drug resistance emerges on multiple time scales. Soon after drug exposure, BRAFV600E melanomas undergo adaptive (reversible) resistance involving disruption of negative feedback on MAPK signaling. By combining computational and experimental modelling, we show here that adaptive resistance involves pulsatile reactivation of the MAPK pathway so that MAPK activity is low on average but high enough in some cells to drive cell division. This is possible due to the co-existence of two MAPK cascades: one driven by BRAFV600E that is drug-sensitive and a second driven by receptor tyrosine kinases that is drug-resistant. The latter is repressed by the former unless RAF and MEK inhibitors are present. MAPK pulsing then occurs in response to factors in the microenvironment, making RAF inhibitors ineffective and reducing the potency of MEK inhibitors. The presence of two MAPK cascades wired differently is likely an Achilles heel with respect to drug resistance in melanoma, but it may explain the high tolerability of RAF/MEK inhibitors in patients.

Targeted quantification of protein abundances and phosphorylation was performed as previously described (Shi et al., 2016). Briefly, cell pellets from A375 cell lines treated with different doses of vemurafenib were lysed in 100 μl of lysis buffer containing 8 M urea in 100 mM NH4HCO3 (pH 7.8). Proteins were reduced by 5 mM dithiothreitol for 1 hour at 37°C and alkylated using 20 mM iodoacetamide for 1 hour at room temperature in the dark. Samples were diluted eightfold with 50 mM NH4HCO3 and digested by sequencing grade modified trypsin at a 1:50 enzyme-to-protein ratio (w/w) at 37°C for 3 hours. Each sample was then desalted by C18 solid phase extraction and concentrated to a volume of ~100 μl. The final peptide concentration was measured using bicinchoninic acid assay with an average of ~4 µg/µL. 10 µg and 100 µg of the peptide mixture per sample were used with the addition of 200 fmol and 50,000 fmol of crude heavy peptides for quantification of protein abundance and protein phosphorylation dynamics, respectively. For protein abundance quantification (Shi et al., 2016)(Yi et al., 2018), crude heavy-isotope labeled synthetic peptides were purchased from Thermo Scientific and two best response peptides were selected to configure final selected reaction monitoring (SRM) assays for each target protein. All samples were measured by regular LC-SRM using the scheduled SRM algorithm (Shi et al., 2017) for simultaneous quantification of the selected target proteins. For targeted quantification of phosphorylation (Yi et al., 2018), phosphopeptides were selected for core component proteins for the EGFR-MAPK pathway. Crude heavy isotope-labeled phosphopeptides were purchased from New England peptides and spiked into the peptide sample prior to phosphopeptide enrichment. Phosphopeptides were enriched by immobilized metal-ion affinity chromatography (IMAC) with Fe3+-NTA agarose beads. Eluted phosphopeptides were dried down and stored at -80°C until further LC-MS/MS analysis. Lyophilized phosphopeptides were reconstituted in 0.1% FA and subjected to LC-SRM analysis immediately. All LC-SRM measurements were performed using the nanoACQUITY UPLC system coupled online to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific). SRM data were analyzed using Skyline software (MacLean et al., 2010) and the best transitions without interferences were used for quantification. The SRM peak area ratios of the endogenous light peptides over heavy peptide standards (i.e., the L/H ratio) were reported for all SRM measurements.

A375 cells were grown for at least 2 passages to 80% confluence in a 10 cm Petri dish. After washing with PBS, cells were incubated with 0.25% trypsin/2.21 mM EDTA 1× (Corning) for 2 minutes for cell detachment. After adding 20 ml complete medium, cells were thoroughly homogenized into a single cell suspension which was confirmed by microscopy. Cell count was determined using a TC20 automated cell counter (Bio-Rad). The cell suspension was serially diluted with complete medium (containing 10% FBS) to a final concentration of 20 cells in 15 ml and 150 μl of that dilution was dispensed in wells of a 96-well plate (0.2 cells per well). After about 14 days, wells that showed clonal growth (15-20 per 96-well plate) were expanded by passaging the cell line into larger dishes in complete medium.