Targeted therapy against the epidermal growth factorreceptor (EGFR) is used to treat metastatic colorectal cancer (mCRC), which is effectivefor 10-20 % of mCRC patients. However, treatment-naive CRC patients withtumours containing activating KRAS mutations show resistance to EGFR-targetedtherapy, a phenomenon called primary resistance. Additionally, CRC patientsthat do respond to EGFR-targeted therapy at first, often stop responding afterthree to twelve months of treatment, due to the emergence of de novoMAPK pathway activating mutations, a process which is called acquiredresistance. Resistance to EGFR-targeted therapy, both in primary resistanttumours and tumour cells acquiring denovo resistance, is often caused by activating KRAS mutations. Currently,it is not known if activating a KRAS mutation singlehandedly drives resistanceto EGFR targeted therapy, or if this mutation simply gives an advantage ingenerating resistance. Furthermore, it is also unknown whether a KRAS mutationis as potent in primary resistant tumours as it is in tumours with an acquiredresistance. By introducing an oncogenic KRAS mutation in patient-derived CRCorganoids using CRISPR technology, we found that organoids selected onphenotype are more resistant to targeted therapy than those obtained in atreatment naïve manner.
Withabout 1.2 million patients being diagnosed yearly, colorectal cancer is thethird most common cancer worldwide. It is also the third leading cause ofcancer-related deaths, resulting in 774.000 deaths every year. When diagnosedwith metastatic colorectal cancer, patients have a 5-year survival rate of lessthan 5%.1–4Thetreatment of metastatic colorectal cancer patients relies mainly on monoclonalantibodies (mAb) targeting the epidermal growth factor receptor (EGFR).
Bybinding to the extracellular part of the receptor, activation of the MAPKpathway will be inhibited, thus suppressing proliferation and cell survival. Two ofthe earliest examples of such EGFR targeting mAbs are cetuximab and panitumumab(Figure 1).5 However, initial response ratesof only 10-20 % in mCRC patients treated with these EGFR inhibitors (EGFRi) wasobserved.
It soon became clear that this was caused by primary resistance toEGFR inhibition, which is mainly due to the activation of the RAF-MEK-ERK6 and PI3KCA-PTEN-Akt7 pathways that signal downstreamof the EGFR (Figure 1).Similarto cetuximab and panitumumab, resistance to small molecule inhibitors targetingthe EGFR and HER28, such as afatinib, is observedwhen downstream signalling of the EGFR is altered.9–12 One ofthese downstream targets is the RAS subfamily, which is mutated in about 15% ofall human tumours.3,4,13,14 Constitutive activation of RASnot only stimulates the RAF-MEK-ERK pathway, but activates the PI3K-Akt pathwayas 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 inresistance to EGFRi.14 By influencing severalintracellular pathways, these mutations cause primary (de novo) resistance to EGFRi, which resulted in non-wildtype RAS patients being excluded from EGFR-targeted therapy.
6 However, even for patients whodo show a beneficial response to EGFRi treatment at first, disease progressionis often observed after 3-12 months of treatment.15–17 This phenomenon is calledacquired (secondary) resistance,5 in which often similar oncogenicalterations are found that also play arole in primary resistance. Among those, members of the RAS family can bedetected, but also mutations and amplifications in BRAF, HER2 and MET arefrequently detected.
6 Currently, it is not known ifmutant KRAS, when present in primary tumours, is able to drive resistance toEGFR-targeted therapy. It is also not known whether primary mutant KRAS tumoursare as potent in driving EGFRi resistance as acquired mutant KRAS tumours, or ifthe primary KRAS mutation merely gives an advantage in obtaining resistance. Althoughthe mechanism of resistance in primary and acquired resistant KRAS tumours aresaid to be the similar,2,6,10 studies have shown that EGFRitreatment 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.Inthis work, we introduced an activating KRAS mutation in patient-derived CRCorganoids using CRISPR. By using different selection methods, we obtainedprimary and acquired KRAS mutant CRC organoids, which were used to investigatethe difference in sensitivity to EGFRi therapy.
We demonstrate that primarymutant KRAS tumours show increased sensitivity to EGFRi treatment comparedsecondary mutant KRAS tumours.
