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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 369

incorporation. This G1 cell cycle arrest was maintained for at least 12 days following selumetinib withdrawal,
and a subpopulation of cells exhibited features of senescence, including senescence-associated β-galactosidase
activity and elevated secretion of cytokines that form part of the senescence-associated secretory phenotype .
[11]
Both proliferative arrest and senescence were ERK1/2-dependent as they could be prevented using the ERK1/2
inhibitor SCH772984 . Co-culture of COLO205 and C6244-R cells confirmed that C6244-R cells exhibited
[11]
a fitness disadvantage in the absence of MEKi, with a ~35-fold enrichment of COLO205 parental cells vs.
resistant C6244-R cells after 7 days . This is consistent with the proliferative arrest in C6244-R being the
[11]
selection pressure that drives reversal of resistance to selumetinib. As the concentration of selumetinib was
increased the fitness of C6244-R increased and at concentrations > 0.1 µmol/L selumetinib C6244-R dominated
the culture .
[11]
When selumetinib was withdrawn, C6244-R cells exhibited a rapid and sustained hyperactivation of ERK1/2
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far beyond the level seen in parental COLO205 cells [9,11] . This resulted in the anticipated induction of p21 .
However, p21 expression was transient, peaking 4-8 h post MEKi-withdrawal before subsiding back to
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basal levels . Thus, p21 expression did not correlate with the sustained G1 cell cycle arrest observed
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[11]
following MEKi withdrawal; indeed, siRNA-mediated knock-down of p21 confirmed that the proliferative
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deficit was p21 -independent. p21 expression was also low relative to other CRC cell lines, such as
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HCT116, possibly because p53, an important transcriptional activator of CDKN1A/p21 , is mutated in
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COLO205 cells . However, expression of the related CDKI p57 KIP2 correlated well with cell cycle arrest, loss
[11]
of cyclin A and p-RB following selumetinib withdrawal. Moreover, knock-out of p57 KIP2 by CRISPR/Cas9
gene editing demonstrated that the G1 cell cycle arrest following MEKi removal was wholly dependent on
p57 KIP2 . Importantly, knock-out of p57 KIP2 also prevented or delayed reversal of resistance, demonstrating
that the p57 KIP2 -dependent proliferative arrest was a key selection pressure that drove reversion to MEKi
sensitivity [Figure 2] .
[11]
BRAF V600E -mutant HT29 cells also acquire resistance to selumetinib through BRAF amplification [Figure 1].
[11]
Again 12 clonal populations of these selumetinib-resistant HT29 (HT6244-R) cells exhibited very similar
BRAF upregulation and, in the presence of selumetinib, near-identical p-ERK1/2 levels to parental HT29
cells . Withdrawal of MEKi resulted in equivalent hyperactivation of ERK1/2 and RSK in all clones.
[11]
Reversion of resistance was near-complete in some populations (clonal and non-clonal) after just 5 weeks
selumetinib withdrawal and complete in all populations by 10 weeks [Figure 3] . BRAF expression and
[11]
p-ERK1/2 reverted to parental MEKi-naïve levels, and BRAF amplification was lost in these revertant
cells. HT644-R clonal and non-clonal populations had 4 copies of chromosome 7, with one harbouring an
intrachromosomal BRAF amplification, resulting in a BRAF copy number of 12 vs. 4 in parental HT29 cells .
[11]
Both clonal and non-clonal resistant cells lost BRAF amplification following 10 weeks MEKi withdrawal,
with 5 copies of BRAF remaining and the chromosome 7 count maintained at 4 . Given that in these
[11]
revertant cells 3 chromosomes had one copy of BRAF and one chromosome had 2 copies, this suggests
that the amplicon was all-but lost from the chromosome with 2 copies of BRAF remaining. That resistance
and BRAF amplification were reversible from clonal populations of both C6244-R and HT6244-R, and that
entire chromosomes (C6244-R) or BRAF amplicons (C6244-R and HT644-R) were lost during reversal of
resistance, supports the conclusion that loss of BRAF copy number was from individual cells harbouring
BRAF amplification, rather than the result of outgrowth of rare dormant parental-like cells that persisted
in the population and were selected for when MEKi was withdrawn. The cytogenetic mechanisms that
underpin intrachromosomal BRAF amplification, and its subsequent loss, are unclear but given these data
reversal of resistance must presumably involve at least slow division of cells with BRAF amplification.

Short-term MEKi withdrawal from HT6244-R cells caused a pronounced but transient G1 cell cycle arrest that
peaked at 16 hours and returned to a near basal cell cycle profile after 72 hours . This short-lived cell cycle
[11]
disruption correlated with induction of p21 expression. However, from 6 days MEKi withdrawal onwards
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