MA16.07 Drug Repurposing to Overcome De Novo Resistance of Non-Traditional EGFR Mutations

Abstract

Approximately 10% of EGFR mutant NSCLCs have an in-frame insertion in exon 20 of EGFR resulting in innate resistance to 1st generation TKIs. The reported response rate of patients with EGFR exon 20 insertions to gefitinib and erlotinib is 5% with a median progression free survival of 1.5 months. It has been shown that exon 20 insertions stabilize the active conformation of EGFR and increase affinity to ATP over TKIs. We hypothesize that exon 20 insertions induce conformational changes to the drug binding pocket resulting in EGFR TKI resistance which can be overcome by covalently binding TKIs. Ba/F3 cells expressing 7 different clinically observed EGFR exon 20 insertion mutations spanning the helix (residues 763-766) and loop (residues 767-773) regions were generated and screened against 1st, 2nd, and 3rd generation EGFR inhibitors including erlotinib, gefitinib, afatinib, dacomitinib, neratinib, poziotinib, ibrutinib rocilentinib, EGF816, and osimertinib. Computational modeling was conducted to analyze the conformational changes and drug binding affinity. In Ba/F3 cells with EGFR exon 20 insertions, most 1st and 3rd generation TKIs failed to inhibit growth of EGFR exon 20 insertions after residue 767 with IC50 values above 100nM. However, poziotinib significantly inhibited cell growth of all EGFR exon 20 insertions tested across the helix and loop regions with an average IC50 value of 2.9nM, as compared to osimertinib and rocilentinib (IC50 values =103nM and 850nM, respectively). Further characterization using three-dimensional modeling revealed that exon 20 insertions induce conformational changes which cause a decreased affinity for 1st generation TKIs and steric hindrance of C797 reducing the ability of 3rd generation TKIs to covalently bind. A significant shift of the c-helix and p-loop result in a sterically hindered binding pocket. Therefore, the smaller, more flexible 1,2-dichloro-3-fluorobenzene terminal group of poziotinib can overcome the structural changes induced by the exon 20 insertions, whereas the ridged 1-methylindole terminal group of osimertinib cannot. EGFR exon 20 insertions induce a shift of the p-loop and c-helix resulting in steric hindrance of the binding pocket thereby preventing binding of 1st generation and 3rd generation EGFR inhibitors including rocilentinib and osimertinib. A smaller, more flexible inhibitor such as poziotinib can overcome the steric hindrance of the drug binding pocket. Currently, in vivo studies of the EGFR D770insNPG GEMM, and EGFR H773insNPH PDX model with poziotinib are underway, and a clinical trial testing poziotinib in EGFR exon 20 mutant NSCLC patients will begin enrollment this year.