Targeted therapy against the epidermal growth factor
receptor (EGFR) is used to treat metastatic colorectal cancer (mCRC), which is effective
for 10-20 % of mCRC patients. However, treatment-naive CRC patients with
tumours containing activating KRAS mutations show resistance to EGFR-targeted
therapy, a phenomenon called primary resistance. Additionally, CRC patients
that do respond to EGFR-targeted therapy at first, often stop responding after
three to twelve months of treatment, due to the emergence of  de novo
MAPK pathway activating mutations, a process which is called acquired
resistance. Resistance to EGFR-targeted therapy, both in primary resistant
tumours and tumour cells acquiring de
novo resistance, is often caused by activating KRAS mutations. Currently,
it is not known if activating a KRAS mutation singlehandedly drives resistance
to EGFR targeted therapy, or if this mutation simply gives an advantage in
generating resistance. Furthermore, it is also unknown whether a KRAS mutation
is as potent in primary resistant tumours as it is in tumours with an acquired
resistance. By introducing an oncogenic KRAS mutation in patient-derived CRC
organoids using CRISPR technology, we found that organoids selected on
phenotype are more resistant to targeted therapy than those obtained in a
treatment naïve manner.

With
about 1.2 million patients being diagnosed yearly, colorectal cancer is the
third most common cancer worldwide. It is also the third leading cause of
cancer-related deaths, resulting in 774.000 deaths every year. When diagnosed
with metastatic colorectal cancer, patients have a 5-year survival rate of less
than 5%.1–4The
treatment of metastatic colorectal cancer patients relies mainly on monoclonal
antibodies (mAb) targeting the epidermal growth factor receptor (EGFR). By
binding to the extracellular part of the receptor, activation of the MAPK
pathway will be inhibited, thus suppressing proliferation and cell survival. Two of
the earliest examples of such EGFR targeting mAbs are cetuximab and panitumumab
(Figure 1).5 However, initial response rates
of only 10-20 % in mCRC patients treated with these EGFR inhibitors (EGFRi) was
observed. It soon became clear that this was caused by primary resistance to
EGFR inhibition, which is mainly due to the activation of the RAF-MEK-ERK6 and PI3KCA-PTEN-Akt7 pathways that signal downstream
of the EGFR (Figure 1).Similar
to cetuximab and panitumumab, resistance to small molecule inhibitors targeting
the EGFR and HER28, such as afatinib, is observed
when downstream signalling of the EGFR is altered.9–12

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One of
these downstream targets is the RAS subfamily, which is mutated in about 15% of
all human tumours.3,4,13,14 Constitutive activation of RAS
not only stimulates the RAF-MEK-ERK pathway, but activates the PI3K-Akt pathway
as well. Both KRAS – mutated in 45% of all colorectal cancers – and NRAS –
mutated in 1-6% of all colorectal cancers– were found to play a role in
resistance to EGFRi.14 By influencing several
intracellular pathways, these mutations cause primary (de novo) resistance to EGFRi, which resulted in non-wild
type RAS patients being excluded from EGFR-targeted therapy.6 However, even for patients who
do show a beneficial response to EGFRi treatment at first, disease progression
is often observed after 3-12 months of treatment.15–17 This phenomenon is called
acquired (secondary) resistance,5 in which often similar oncogenic
alterations are found  that also play a
role in primary resistance. Among those, members of the RAS family can be
detected, but also mutations and amplifications in BRAF, HER2 and MET are
frequently detected.6 Currently, it is not known if
mutant KRAS, when present in primary tumours, is able to drive resistance to
EGFR-targeted therapy. It is also not known whether primary mutant KRAS tumours
are as potent in driving EGFRi resistance as acquired mutant KRAS tumours, or if
the primary KRAS mutation merely gives an advantage in obtaining resistance.

Although
the mechanism of resistance in primary and acquired resistant KRAS tumours are
said to be the similar,2,6,10 studies have shown that EGFRi
treatment naïve patients react differently to Afatinib treatment than  patients that have been treated before,18 
suggesting that the underlying mechanisms might not be exactly the same.

In
this work, we introduced an activating KRAS mutation in patient-derived CRC
organoids using CRISPR. By using different selection methods, we obtained
primary and acquired KRAS mutant CRC organoids, which were used to investigate
the difference in sensitivity to EGFRi therapy. We demonstrate that primary
mutant KRAS tumours show increased sensitivity to EGFRi treatment compared
secondary mutant KRAS tumours.