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Sale et al. Cancer Drug Resist 2019;2:365-80 I http://dx.doi.org/10.20517/cdr.2019.14 Page 371

activation arising from BRAF V600E mutation, sufficient ERK1/2-responsive tumour suppressive mechanisms
remain intact to drive proliferative arrest or cell death following the hyperactivation of ERK1/2 that occurs
upon MEKi withdrawal.

BRAF V600E -MUTANT CRC CELLS EVOLVE TO REINSTATE THE SAME OPTIMAL LEVEL OF ERK1/2
ACTIVITY REGARDLESS OF WHETHER ERK1/2 ARE INHIBITED OR HYPERACTIVATED
Cell cycle phase profile, EdU incorporation and C6244-R fitness vs. COLO205 were all optimal and/or
maximal when C6244-R cells were maintained in 1 µmol/L selumetinib, the concentration in which they
were selected and at which ERK1/2 phosphorylation matched that in parental cells [9,11] . However, these
observations were not unique to selumetinib. C6244-R cells were cross-resistant to the clinically approved
MEKis cobimetinib and trametinib, and to the ERK1/2 inhibitor SCH772984, and in each case proliferated
optimally at inhibitor concentrations that imposed ERK1/2 or RSK phosphorylation at close to parental
levels . These effects were recapitulated in vivo: C6244-R tumours grew better in mice dosed with 10 mg/kg
[11]
selumetinib compared to those dosed with vehicle only or 25 mg/kg selumetinib . Thus, although achieving
[11]
a steady-state concentration of selumetinib in mice akin to that in vitro is not possible, C6244-R cells were
addicted to a tight window or “sweet-spot” of ERK1/2 pathway output optimal for proliferation both in vitro
and in vivo.

This evolutionary pressure to restore ERK1/2 activity to an optimal “sweet-spot” was strikingly exemplified
in an experiment in which separate COLO205 cell lines with resistance to a range of distinct selumetinib
concentrations were established . The higher the concentration of selumetinib, the longer the cells took to
[11]
evolve resistance and proliferate normally. Remarkably, however, all resistant cells proliferated optimally in
the presence of the selumetinib concentration to which they had adapted, and at this concentration exhibited
equivalent p-ERK1/2 levels as parental COLO205 cells . This was enabled by a progressive increase in
[11]
BRAF expression: cells adapted to higher concentrations of selumetinib through higher BRAF expression
that restored parental ERK1/2 activity and a normal cell cycle profile in the respective drug concentration .
[11]
However, in the absence of selumetinib ERK1/2 were hyperactivated in proportion to the degree of BRAF
expression. Consequently COLO205 cells with resistance to higher concentrations of selumetinib exhibited
greater ERK1/2 activation in the absence of selumetinib and underwent G1 cell cycle arrest .
[11]
Thus regardless of whether ERK1/2 were inhibited in parental COLO205 cells, or ERK1/2 were hyperactivated
following MEKi withdrawal from C6244-R cells, cells evolved accordingly to increase or decrease BRAF copy
number and BRAF expression to a level that restored ERK1/2 activity and pathway output back to parental
levels. Mass spectrometry was used to define this optimal “sweet-spot” of ERK1/2 activation; quantifying
ERK1/2 activation loop dual pT-E-pY phosphorylation revealed that COLO205 cells, and C6244-R cells
maintained in selumetinib, proliferated with just 2%-3% of the total ERK1/2 pool active, and cellular p-ERK1
and p-ERK2 concentrations of ~2 nmol/L and 3 nmol/L, respectively . MEKi withdrawal increased the
[11]
stoichiometry of phosphorylated ERK1/2 to ~20%-30%, and cellular p-ERK1 and p-ERK2 concentrations to
~10 and 20 nmol/L, respectively . HT29 cells, and HT6244-R cells in selumetinib, also exhibited a ~2%-5%
[11]
stoichiometry of ERK1/2 phosphorylation and cellular p-ERK1 and p-ERK2 concentrations of < 2 nmol/L and
< 5 nmol/L, respectively . This suggests, even in tumour cells with BRAF V600E mutation, there is substantial
[11]
spare capacity within the ERK1/2 pathway under basal conditions.

MEK1/2 INHIBITOR WITHDRAWAL FROM KRAS-MUTANT CRC CELLS WITH ACQUIRED MEK1/2
INHIBITOR RESISTANCE PROMOTES EMT AND CHEMORESISTANCE
HCT116 CRC cells harbour a KRAS G13D mutation and acquired resistance to selumetinib through KRAS G38A
gene amplification and striking upregulation of KRAS protein [Figure 4]. As with the BRAF V600E -amplified
[9]