Abstract

Targeted therapies are currently hampered by
the ability of tumours to evolve resistance mechanisms involving genetic
aberrations. NSCLC tumours containing EGFR Ex19_Del/L858R in
combination with T790M mutations are resistant to tyrosine kinase inhibitors
(TKIs) such as gefitinib and are therefore treated with novel TKIs like
osimertinib, which have been generated to target the double mutant. Recently, DNA analysis from relapsed patients
has identified C797S as a novel resistant triple mutant. In addition to the
C797S mutation, other EGFR mutations
such as L718Q/V, L792F/H/Y, G796D/R/S and C797G mutations have also been
reported in recent clinical studies within a small subset of patients. However,
it remains unclear if these mutations are really causative of the tumour resistance
to osimertinib or if they are only passengers. To address this question, we
designed a strategy based on CRISPR-Cas9 technology which enables a quick osimertinib
resistance assessment of any observed clinical EGFR mutations at an endogenous level in a lung cancer cellular
model. Mutations were introduced into cells through CRISPR-Cas9-mediated
homology-directed repair via
single-strand DNA oligonucleotides containing an exact point mutation or a
degenerated codon in the presence or absence of the EGFR secondary mutation T790M. After osimertinib treatment, amplicon-sequencing
analysis was performed on resistant cells to confirm mutations and to compare
enrichment before and after drug treatment.

Thanks to this method, named TEMPR (Targeted
Endogenous Mapping of Pharmacological Resistance), we were able to quickly confirm
that three out of seven mutations found in the clinic can confer resistance at
least in vitro. These new resistant
mutations could then be characterised with the aim of finding new suitable
drugs.