Functional characterisation of EGFR mutations in cancer

Abstract

Large-scale pan-cancer sequencing of tumour samples has identified a plethora of epidermal growth factor (EGFR) mutations along the full length of the gene. However, clinical studies have demonstrated that distinct mutations have different responses to anti-EGFR therapy, leading to a disparity in outcomes for patients. This thesis sought to study EGFR mutations from across all cancer types in order to establish the optimal kinase inhibitors for distinct EGFR mutants. Expression vectors encoding 18 distinct EGFR mutants were generated and expressed in the Ba/F3 model cell line. The sensitivity of these mutants to 9 EGFR inhibitors (EGFRi) and 53 other small molecule inhibitors was examined, confirming previous observations and providing novel insight into the sensitivities of distinct EGFR mutants to different inhibitors. Notably, TAS6417 and poziotinib, which are currently under clinical investigation in non-small cell lung cancer (NSCLC) harbouring exon 20 insertion (Ex20Ins) mutant EGFR, were shown to be active against 6 extracellular domain EGFR mutants (L62R, R108G, A289D/T/V, G598V) for which there are currently no approved anti-EGFR therapies. Furthermore, 6 broad-spectrum kinase inhibitors (dasatinib, saracatinib, bosutinib, cediranib, vandetanib, and ponatinib) were found to selectively inhibit a subset of kinase domain mutants (746_750del, 747_751del, and L858R). This thesis also used N-ethyl-N-nitrosourea (ENU) mutagenesis to investigate resistance mechanisms to poziotinib and TAS6417 in Ba/F3 cells expressing 3 distinct Ex20Ins mutants (A767_769dupASV, S768_dupSVD, and N771_H773dupNPH). T790M and C797S mutations were identified in cells with acquired poziotinib resistance, with T790M occurring more frequently than C797S. Interestingly, the overall frequency of T790M or C797S in poziotinib-resistant cells varied between the distinct Ex20Ins mutants. Only additional C797S mutations were identified in cells with acquired TAS6417 resistance. T790M and C797S mutations were shown to prevent EGFR inhibition following poziotinib treatment and C797S was shown to prevent EGFR inhibition following TAS6417 treatment. Both T790M and C797S mutations conferred similar levels of resistance to poziotinib, but C797S conferred greater resistance to TAS6417 compared to T790M. Finally, the heat shock protein 90 (Hsp90) inhibitor luminespib was shown to overcome T790M- or C797S-mediated resistance to poziotinib or TAS6417.