Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma

Abstract

Purpose: Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to the substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance.

Experimental design: Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then done to characterize the biological properties of a novel mutant EGFR allele.

Results: A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R-mutant lung adenocarcinoma after long-term treatment with tyrosine kinase inhibitors. The T854A mutation was not detected in a pretreatment tumor sample. The crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays show that the EGFR T854A mutation abrogates the inhibition of tyrosine phosphorylation by erlotinib. Such resistance seems to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992.

Conclusions: The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket.

Figure 1. Identification of a novel EGFR mutation in a patient with acquired resistance to EGFR inhibitors

Sequencing chromatograms showing presence of the EGFR T854A mutation along with the L858R mutation in pleural fluid cells collected from the index patient after prolonged treatment with gefitinib and erlotinib. Only the L858R mutation was detected in a pretreatment metastatic brain tumor specimen.

Figure 2. T854A mutation reduces inhibition of autophosphorylation of EGFR by erlotinib

Lysates from 293T cells transiently transfected with cDNAs encoding either EGFR WT, T854A, L858R, or L858R plus T854A and treated with different concentrations of erlotinib were immunoblotted for phospho-EGFR (Y¹⁰⁹²), total EGFR, and Actin. T854A reduced the degree of inhibition of phospho-EGFR by erlotinib relative to WT or L858R EGFR. Actin is shown as a loading control.

Figure 3. T854A mutation is not analogous to other known kinase mutations and is located at a drug contact site

A) Alignment of the kinase domain of EGFR with other tyrosine kinases adapted from the Mutagrator Tool reveals no other identified mutations (highlighted in orange) at analogous residues. Overall the EGFR T854 position is not highly conserved among other kinases. B) Crystal structure of the L858R EGFR mutant bound to gefitinib (adapted from (18)) reveals the T854 residue is at the “bottom” of the ATP-binding site, on the C-lobe. Residues with known mutations associated with acquired resistance in patients are shown in red.

Figure 4. BIBW 2992 more potently inhibits both sensitive and resistant EGFR mutants, including T854A

A) Chemical structure of BIBW 2992. Adapted from (21). B) Cell growth inhibition assays of three EGFR mutant cell lines treated with various does of erlotinib (dashed line) or BIBW 2992 (solid line). PC-9 cells have a drug-sensitive exon 19 deletion in EGFR, H3255 cells have the drug-sensitive L858R mutation, and H1975 cells have both L858R and the T790M resistance mutation. All plots are relative to a DMSO-only control and error bars indicate one standard deviation from six replicates. All assays were performed three independent times and representative plots are shown. IC₅₀ values were calculated using BioDataFit 1.02. C) Lysates from 293T cells transiently transfected with cDNAs encoding either EGFR WT, L858R, L858R plus T790M, or L858R plus T854A and treated with different concentrations of BIBW 2992 were immunoblotted for phospho-EGFR, total EGFR, and actin. Actin is shown as a loading control. D) Lysates from 293T cells transiently transfected with L858R plus T854A EGFR cDNA and treated with different concentrations of erlotinib or BIBW 2992 were immunoblotted for phospho-EGFR, total EGFR, and actin.