Withdrawal of MEK1/2 inhibitor reverses acquired resistance driven by BRAF amplification but drives EMT and chemoresistance in cells with amplified KRAS

Acquired resistance to MEK1/2 inhibitors can arise through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and selumetinib resistance are fully reversible following drug withdrawal. Resistant cells with BRAFV600E amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling (2-3% of total ERK1/2 active, here quantified by mass spectrometry), that is optimal for cell survival and proliferation. The magnitude of ERK1/2 activation following selumetinib withdrawal (~20% active) drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, which selects against those cells with amplified BRAFV600E. ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of selumetinib resistance. Furthermore, growth of selumetinib-resistant cells with BRAFV600E amplification as tumour xenografts is inhibited in mice that do not receive selumetinib. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, providing a rationale for intermittent dosing to forestall resistance. In striking contrast, selumetinib resistance driven by KRASG13D amplification is not reversible. In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents arguing strongly against the use of ‘drug holidays’ in cases of KRASG13D amplification. Cells were treated with either selumetinib or DMSO and treated for 4,16,or 48 hours before being harvested and assayed