Drug Sensitivity and Allele Specificity of First-Line Osimertinib Resistance EGFR Mutations

Abstract

Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFR^L858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance in vivo. SIGNIFICANCE: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/2017/F1.large.jpg.

Figure 1.. Acquired resistance to first-line osimertinib arises primarily due to the emergence of secondary mutations in EGFR.

A. Schema of the experiment. CCSP-rtTA;TetO-EGFR^L858R mice were administered doxycycline for the duration of the experiment and developed tumors after ~6 weeks on doxycycline. When tumors were detected by MRI (see pre-treatment image), osimertinib treatment was initiated (QD M-F) which elicited a response (see representative response MRI) and treated until the emergence of resistant tumors by MRI. Coronal MR images are shown, in which ‘H’ indicates heart and red arrows indicate tumor. The osimertinib-resistant tumors were then collected and analyzed to determine the resistance mechanisms present. B. Waterfall plot showing tumor volume changes after 1 month of osimertinib treatment in individual CCSP-rtTA;TetO-EGFR^L858R mice, normalized to baseline tumor. C. Pie-charts illustrating the resistance mechanisms found in osimertinib-resistant tumors treated with 25 mg/kg of osimertinib. D. Western blot analysis of 293T cells transiently transfected with pcDNA3.1(–) plasmids encoding the indicated EGFR alleles, treated for 1 hour with varying concentrations of osimertinib. Blots are representative of n=2 biological replicates. EV, empty vector.

Figure 2.. Allele-specific pattern of osimertinib resistance mutations in mice and patients.

A. Schema of the experiment. CCSP-rtTA;TetO-EGFR^L858R+T790M mice were administered doxycycline for the duration of the experiment and developed tumors after ~3 months on doxycycline. When tumors were detected by MRI (see pre-treatment image), mice were put on continuous osimertinib treatment (QD M-F) which elicited a response (see representative response MRI) and treated until the emergence of resistant tumors. Coronal MR images are shown, in which H indicates heart and red arrows indicate tumor. The osimertinib-resistant tumors were then collected and analyzed to identify the resistance mechanism. B. Pie-chart illustrating the resistance mechanisms found in osimertinib-resistant tumors from EGFR^L858R+T790M mice. #, tumor that lost T790M. C. Estimated growth rates of TKI-naïve tumors for both the EGFR^L858R and EGFR^L858R+T790M models. D. Estimated growth rates of the osimertinib-resistant tumors treated with 25 mg/kg osimertinib by resistance mutation for both the EGFR^L858R and EGFR^L858R+T790M models. Growth rates are depicted as the relative change in cell count per day (log-scale). Error bars represent SEM. P-values were obtained by performing two-sided t tests. E and F. Graphs showing the frequency of the indicated osimertinib resistance mutations in cfDNA from E. patients with tumors harboring the indicated baseline EGFR mutation or F. L858R-positive cases with or without a T790M mutation. ^, the mutations in this case were called in 2 separate blood draws. &, 2 of the T790M-negative L718Q cases listed here later gained a T790M mutation and maintained L718Q in a later blood draw. Two-sided Fisher’s exact test; *, p<0.05; **, p<0.005; ****, p<0.0001.

Figure 3.. EGFR L718Q and L718V mutations confer resistance to osimertinib in human lung cancer cells.

A. Schematic workflow of the CRISPR experiment (see Supplementary Methods). B-D. Cell growth of CRISPR-edited-PC9 (blue), PC9-VanR (red), and II-18 (green, lines superimposed) cells during osimertinib treatment as measured by Cell Metric. E. Histograms showing the difference in the proportion of reads for the indicated codon in the corresponding sample before (pre) and after osimertinib selection (post). As a reference, the mean of the top 5 indels found in the control sample is shown. The dashed line is the background threshold. Error bars show SEM for three biological replicates for PC9 and two for PC9-VanR and II-18 for B-D and E. Two-sided t test, * p<0.05.

Figure 4.. Afatinib suppresses phosphorylation of EGFR mutants containing the L718V/Q mutations.

Western blots of 293T cells transiently transfected with pcDNA3.1(–) containing EGFR with the indicated mutations, treated for one hour with varying concentrations of the indicated TKIs. A and B show a direct comparison of EGFR^L858R, EGFR^L858R+C797S, EGFR^L858R+L718V and EGFR^L858R+L718Q treated with 100 nM osimertinib, erlotinib, or afatinib. C and D show dose-dependent changes in EGFR phosphorylation of EGFR^L858R+L718V and EGFR^L858R+L718Q compared with EGFR^L858R+C797S and EGFR^L858R+T790M treated with increasing doses of afatinib. E. Comparison of the effect of 100 nM osimertinib on L858R and exon 19 deletion mutants. Sensitivity of the exon 19 deletion mutants with or without the indicated mutations to varying concentrations of osimertinib (F), erlotinib (G) or afatinib (H). All blots are representative of n=2 biological replicates. EV, empty vector.

Figure 5.. The sensitivity of osimertinib-resistant tumors to erlotinib or afatinib treatment depends on the specific osimertinib resistance mutation present.

A. Schema of the experiment. CCSP-rtTA;TetO-EGFR^L858R mice were treated with 25 mg/kg osimertinib until the emergence of resistant tumors, as in Figure 1. Mice were then switched to either erlotinib, afatinib, or the combination of erlotinib plus osimertinib for 1–3 weeks. B, C, and D. Average tumor volume changes for the osimertinib-resistant tumors switched to 25 mg/kg erlotinib for 3 weeks (B), 25 mg/kg afatinib for 10 days (C), and erlotinib plus osimertinib (D, 25 mg/kg each) as determined by MRI. Tumor volume is normalized to the point of TKI switch. Error bars represent SEM. For B, curves are the average of n=11 total tumors (C797S n=5; L718V n=3; L718Q n=3). For C, n=12 total tumors (C797S n=7; L718Q n=5). For D, n=10 total tumors (C797S n=7; L718Q n=3). E. Graphs of tumor growth rates for the osimertinib-resistant tumors before and after they were switched to erlotinib (left), afatinib (center), or erlotinib plus osimertinib (right). Growth rates are depicted as the relative change in cell count per day (log-scale). F. Schema of the experiment. TKI-naïve CCSP-rtTA;TetO-EGFR^L858R mice were treated with the combination of osimertinib plus erlotinib (25 mg/kg each) until the emergence of resistant tumors. Error bars represent SEM. (* indicates p<0.05). O, osimertinib; E, erlotinib; A, afatinib; C, erlotinib plus osimertinib.

Figure 6.. Benefit from afatinib in a patient who developed resistance to first-line osimertinib.

A. Timeline of the patient’s treatment history and clinical testing results. Osimertinib was given at 80 mg QD and afatinib was given at 40 mg QD. Timeline not to scale. B. CT scans of a lung lesion (indicated with red arrow) that was new prior to starting afatinib, and then shrunk after treatment with afatinib.

Figure 7.. Possible structural consequences of L718V/Q-mutations for EGFR binding to TKIs.

Structural models for the L858R-mutated EGFR kinase domain bound to osimertinib, erlotinib, or afatinib are shown, with (or without) the incorporation of L718V or L718Q mutations as described in the Supplementary Methods. In each case, the bound drug molecule of the energy minimized model has been replaced with that bound to the L858R TKD lacking an L718 mutation in order to show how drug binding is impacted. Interactions with drug are lost in all L718V variants (and drug is reoriented slightly to compensate). Clashes between the Q718 side-chain and drug (denoted by yellow lightning bolt) are seen for the osimertinib and erlotinib complexes (with resulting drug reorientation), but not for afatinib.