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 EGFRL858R-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.

Lung adenocarcinoma–associated mutations in exons encoding the tyrosine kinase domain (TKD) of EGFR confer sensitivity to first- and second-generation tyrosine kinase inhibitors (TKI) such as erlotinib, gefitinib, afatinib, and dacomitinib (1); however, almost all patients develop acquired resistance to these drugs about 1 year after starting treatment. About 60% of these resistant tumors have a secondary mutation in exon 20 (T790M), which increases the ATP-binding affinity of the mutated protein and renders the tumor resistant to first- and second-generation TKIs (2). Several other mechanisms of acquired resistance to TKIs have been identified, such as mutations in genes encoding proteins that signal downstream of EGFR, histologic transformation, and amplification of other receptor tyrosine kinases (3).

Third-generation inhibitors (e.g., osimertinib) were developed that target mutated EGFR (including the T790M mutation) over wild-type, presumably resulting in decreased toxicity (4). Based on its clinical activity, osimertinib was first approved for the second-line treatment of T790M-positive EGFR-mutant lung cancer (5). However, resistance to second-line osimertinib has also been described in patients—and in approximately 30% of cases, it is due to the emergence of additional mutations in EGFR such as C797S/G, G796S/R, L792F/H, L718Q/V, and G724S (6–16). The C797S mutation removes the cysteine side-chain with which osimertinib reacts covalently, thus preventing drug binding to EGFR. Mutations at L718 and G796 have been predicted to prevent drug binding by sterically altering the drug-binding pocket (7, 17).

Osimertinib has also shown promising activity in the first line (18, 19), leading to its recent approval for use in this setting. This raises important questions concerning likely mechanisms of resistance to first-line osimertinib, about which very little is known. Analysis of circulating tumor DNA (ctDNA) in patients whose tumors progressed on first-line osimertinib (20, 21) has shown that MET amplification was the most common mechanism of resistance (15%), with C797S and L718Q EGFR mutations found in 7% and 2% of cases, respectively. Given this limited information, it remains unclear which therapeutic strategies might be effective at overcoming resistance to first-line osimertinib. One possible strategy is to sequentially apply, or combine, different generations of EGFR TKIs. Several studies have demonstrated that a first-generation TKI is effective in cell line models with C797S (22, 23), and this is supported by case reports of several patients who responded to treatment with a first-generation EGFR TKI (24, 25) or the combination of osimertinib plus a first-generation EGFR TKI (26) after developing resistance to osimertinib mediated by C797S. Indeed, a clinical trial is currently underway to evaluate the combination of osimertinib and gefitinib in TKI-naïve EGFR-mutant lung cancer patients (NCT03122717). Similarly, several in vitro studies and one case report have suggested that second-generation TKIs can be effective in cells with osimertinib resistance through acquisition of an L718 mutation (9, 15, 23, 27).

To investigate mechanisms of resistance to first-line osimertinib more exhaustively, we used a genetically engineered mouse model (GEMM) of EGFR-mutant lung cancer to generate osimertinib-resistant tumors and evaluate the efficacy of sequential or combination EGFR TKIs to overcome or prevent resistance. Further, we analyzed mutational data obtained from a large cohort of patients with lung cancer and uncovered allele specificity of osimertinib resistance EGFR mutations. Finally, we report a case of a patient with a tumor harboring both the L718V and L718Q mutations at resistance to first-line osimertinib who benefited from afatinib treatment.

Mouse husbandry

All animals were kept in pathogen-free microisolator housing under BSL2 guidelines approved by the Yale University's Institutional Animal Care and Use Committee and in agreement with the NIH Guide for the Care and Use of Laboratory Animals. The generation of doxycycline-inducible EGFRL858R, EGFRL858R+T790M, and CCSP-rtTA mice has been previously described (28–30).

Cell culture

HEK 293T cells (purchased from the ATCC) were maintained at 5% CO2 and cultured in DMEM (Gibco) with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) and kept in a 37°C incubator. PC9 and PC9-VanR cells (purchased from ECACC-Sigma-Aldrich) and 11-18 cells (obtained from the global cell bank of AstraZeneca) were maintained at 5% CO2 in RPMI with 10% FCS and 1X GlutaMAX (Thermo Fisher Scientific). H1975 (NCI-H1975) cells (purchased from the ATCC) were cultured at 5% CO2 in RPMI-1640 (Thermo Fisher) supplemented with 10% FBS (Sigma), 1% Glutamax (Thermo Fisher), and 1% penicillin/streptomycin (Thermo Fisher). Cell lines were periodically checked for Mycoplasma contamination and found to be negative, and were authenticated by short tandem repeat profiling.

Patient cell-free DNA analysis

We performed a retrospective review of EGFR mutation–positive cases with advanced non–small cell lung cancer (NSCLC) from the Guardant Health deidentified database of patients who underwent cfDNA sequencing as part of standard clinical care between September 2017 and March 2019 (Guardant Health). Testing was performed in a Clinical Laboratory Improvement Amendments–certified, College of American Pathologists–accredited, New York State Department of Health–approved clinical laboratory at Guardant Health, Inc. Analysis was completed under a Quorum Review Institution Review Board protocol for deidentified and limited datasets and did not require specific written-informed consent. During this study period, the Guardant360 ctDNA assay included complete exon sequencing of critical exons in 73 genes with reporting of single-nucleotide variants in all 73 genes and indels, fusions, and copy-number variation in a subset of genes. Plasma was analyzed per methods previously described (31). From this cohort, we specifically analyzed cases with the common EGFR driver mutations L858R and E746_A750. We looked at unique patient cases and counted the number of reported C797S, L718Q, or L718V mutations co-occurring with each driver mutation. For patients who had serial testing, each resistance mutation was counted only once per patient—the first time it was reported (unless noted in the figure legend). For the analysis of T790M positivity, the status of T790M was considered only within the same blood draw as the osimertinib-resistance mutation. Two cases of T790M-negative/L718Q-positive, noted by a “&” in Fig. 2, later gained a T790M mutation and maintained the L718Q mutations, but were counted as T790M-negative. In cases with “T790M loss” in sequential blood draws, the resistance mutation was present only without T790M, so they were counted as T790M-negative. The complete dataset is available in the Supplementary Materials.

Patient and clinical sample collection

The patient underwent treatment with EGFR-targeted therapies based on the treating oncologist's recommendations. The patient's response to therapy was monitored by CT or MRI and clinical examinations as per standard practice. Response assessment was determined by the treating clinician. Formalin-fixed paraffin-embedded sections were prepared from tumor biopsies for histologic assessment and genetic testing at various time points. For the pretreatment biopsy, the sequencing performed was a targeted next-generation sequencing (NGS) Illumina MiSeqDx platform, and the liver biopsy at progression was analyzed using Foundation One CDx sequencing. The case report was included with written-informed consent from the patient, approved by the Institutional Review Board (IRB #2055-13) at Lifespan Cancer Institute at the Rhode Island Hospital and in accordance with the Declaration of Helsinki.

Acquired resistance to first-line osimertinib is mediated by secondary EGFR mutations in vivo

To identify mechanisms of resistance to first-line osimertinib in EGFR-mutant lung adenocarcinoma, we modeled acquired resistance to this TKI using the CCSP-rtTA;TetO-EGFRL858R GEMM (28). In this model, lung-specific expression of the human EGFR transgene is induced by doxycycline administration, and the mice develop multifocal peripheral lung adenocarcinomas that closely mirror the human disease. Mice were treated continuously 5 days/week with osimertinib once a day (QD) and monitored by MRI for the emergence of osimertinib-resistant tumors (Fig. 1A). The 5 mg/kg dose was used initially (4), but in subsequent studies, it was determined that 25 mg/kg QD was approximately equivalent to the 80 mg QD human clinical dose (32). Therefore, once tumors resistant to 5 mg/kg appeared, mice were switched to 25 mg/kg to confirm resistance. We also treated an independent cohort of mice with 25 mg/kg osimertinib QD from the start. MRIs from a representative subset of 16 mice revealed that 15 (94%) exhibited complete responses to 25 mg/kg osimertinib after 1 month of treatment (Fig. 1B), and all (100%) exhibited complete responses by 2 months.

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-EGFRL858R mice were administered doxycycline for the duration of the experiment and developed tumors after approximately 6 weeks on doxycycline. When tumors were detected by MRI (see pretreatment image), osimertinib treatment was initiated (QD M-F), which elicited a response (see representative response MRI), and mice were treated until the emergence of resistant tumors by MRI. Coronal MR images are shown. H, heart; red arrowheads, 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-EGFRL858R 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 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-EGFRL858R mice were administered doxycycline for the duration of the experiment and developed tumors after approximately 6 weeks on doxycycline. When tumors were detected by MRI (see pretreatment image), osimertinib treatment was initiated (QD M-F), which elicited a response (see representative response MRI), and mice were treated until the emergence of resistant tumors by MRI. Coronal MR images are shown. H, heart; red arrowheads, 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-EGFRL858R 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.

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In the 5→25 mg/kg cohort, the first osimertinib-resistant tumor, defined by confirmed growth in subsequent MRIs and a size of at least 30 mm3, emerged after 4.4 months of treatment (average 5.3 months). Twelve of the 14 mice (86%) developed one or more resistant tumors. To ensure availability of sufficient material for analysis, resistant tumors were allowed to grow to 200 to 300 mm3 before mice were sacrificed and tumors collected. To establish the mechanism of resistance to osimertinib in the tumors, we used targeted Sanger sequencing of cDNA extracted from the tumors to identify secondary mutations in the EGFR transgene. A mutation encoding the C797S substitution was seen in 5 of 17 (29.4%) osimertinib-resistant tumors (Supplementary Fig. S1). An additional 5 tumors (29.4%) harbored L718V mutations, and the remaining 7 (41.2%) had no acquired EGFR mutation. None of the tumors had the T790M mutation commonly seen at resistance to first-generation TKIs, consistent with clinical observations (20, 21), and all tumors retained the activating L858R mutation. Sequencing of cDNA from a subset of these tumors confirmed that the resistance mutations emerged in cis with L858R (Supplementary Table S1). All resistance mechanisms found were mutually exclusive within each resistant tumor, and distinct resistance mechanisms were uncovered in different tumors from within the same mouse.

In the cohort of mice treated with 25 mg/kg osimertinib QD only, 26 (93%) developed osimertinib-resistant tumors—on average after 7.8 months. Of the 54 tumors collected, there were 14 (25.9%) C797S, 7 (13%) L718Q, and 3 (5.6%) L718V-mutant tumors, whereas the remaining 30 (55.6%) had no secondary EGFR mutations (Fig. 1C). Therefore, the two dosing regimens resulted in different frequencies of L718V-mutated tumors (P = 0.0162). Histologic analysis of the resistant tumors confirmed that all tumors were lung adenocarcinomas consistent with treatment-naïve tumors from this model. No resistant tumors exhibited cellular state or mesenchymal changes.

To examine the effects of these secondary mutations on EGFR phosphorylation in Western blots, we engineered the C797S, L718V, and L718Q mutations individually into EGFRL858R cDNAs and transiently transfected the resulting plasmids into 293T cells. Whereas osimertinib efficiently suppresses EGFR phosphorylation in EGFRL858R-mutant cells, it fails to block phosphorylation of the EGFRL858R+C797S or EGFRL858R+L718Q variants even at 1 μmol/L (Fig. 1D). The EGFRL858R+L718V variant exhibits an intermediate phenotype. These data suggest that, although all three mutations confer osimertinib resistance, the L718V mutation does so less effectively than the C797S and L718Q mutations. This is consistent with L718V mutations arising more frequently in tumors that originally received lower osimertinib doses in the mice. Interestingly, EGFRL858R+L718Q-mutant cells also exhibit reduced levels of EGFR protein and of basal EGFR phosphorylation, suggesting reduced stability and/or kinase activity of this variant.

Until recently, osimertinib was predominantly used as a second-line EGFR-targeted therapy for T790M-positive tumors. To compare osimertinib resistance in T790M-negative tumors with that observed in T790M-positive tumors directly, we utilized the CCSP-rtTA;TetO-EGFRL858R+T790M GEMM (29) and treated mice continuously with osimertinib (Fig. 2A). Twenty out of the 49 treated mice (41%) developed osimertinib-resistant tumors, on average after 8.0 months. Of the 27 tumors collected, we found 12 (44.4%) C797S, 1 (3.7%) C797G, 2 (7.4%) L718Q, 1 (3.7%) L718V-mutant tumor with concomitant loss of T790M, 1 (3.7%) L792H, and 10 tumors (37.0%) with no acquired EGFR mutation (Fig. 2B). Interestingly, the C797G and L792H mutations were never observed in the EGFRL858R model, only in the context of T790M. These data suggest that C797X mutations may be more frequent in the EGFRL858R+T790M than EGFRL858R setting, although the difference is not significant (P = 0.0787) due to low numbers in the EGFRL858R+T790M model.

Figure 2.

Allele-specific pattern of osimertinib resistance mutations in mice and patients. A, Schema of the experiment. CCSP-rtTA;TetO-EGFRL858R+T790M mice were administered doxycycline for the duration of the experiment and developed tumors after approximately 3 months on doxycycline. When tumors were detected by MRI (see pretreatment image), mice were put on continuous osimertinib treatment (QD M-F), which elicited a response (see representative response MRI), and were treated until the emergence of resistant tumors. Coronal MR images are shown. H, heart; and red arrowheads, 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 EGFRL858R+T790M mice. #, tumor that lost T790M. C, Estimated growth rates of TKI-naïve tumors for both the EGFRL858R and EGFRL858R+T790M models. D, Estimated growth rates of the osimertinib-resistant tumors treated with 25 mg/kg osimertinib by resistance mutation for both the EGFRL858R and EGFRL858R+T790M models. Growth rates are depicted as the relative change in cell count per day (log-scale). Error bars, SEM. P values were obtained by performing two-sided t tests. E and F, Graphs showing the frequency of the indicated EGFR mutations in cfDNA from patients with tumors harboring the indicated baseline EGFR mutation (E) or L858R-positive cases with or without a T790M mutation (F). ⁁, the mutations in this case were called in two separate blood draws. &, two of the T790M-negative L718Q cases listed here later gained a T790M mutation and maintained L718Q in a later blood draw. Two-sided Fisher exact test; *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

Figure 2.

Allele-specific pattern of osimertinib resistance mutations in mice and patients. A, Schema of the experiment. CCSP-rtTA;TetO-EGFRL858R+T790M mice were administered doxycycline for the duration of the experiment and developed tumors after approximately 3 months on doxycycline. When tumors were detected by MRI (see pretreatment image), mice were put on continuous osimertinib treatment (QD M-F), which elicited a response (see representative response MRI), and were treated until the emergence of resistant tumors. Coronal MR images are shown. H, heart; and red arrowheads, 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 EGFRL858R+T790M mice. #, tumor that lost T790M. C, Estimated growth rates of TKI-naïve tumors for both the EGFRL858R and EGFRL858R+T790M models. D, Estimated growth rates of the osimertinib-resistant tumors treated with 25 mg/kg osimertinib by resistance mutation for both the EGFRL858R and EGFRL858R+T790M models. Growth rates are depicted as the relative change in cell count per day (log-scale). Error bars, SEM. P values were obtained by performing two-sided t tests. E and F, Graphs showing the frequency of the indicated EGFR mutations in cfDNA from patients with tumors harboring the indicated baseline EGFR mutation (E) or L858R-positive cases with or without a T790M mutation (F). ⁁, the mutations in this case were called in two separate blood draws. &, two of the T790M-negative L718Q cases listed here later gained a T790M mutation and maintained L718Q in a later blood draw. Two-sided Fisher exact test; *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

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The reversion to wild-type T790 seen with gain of L718V in one tumor (Supplementary Fig. S2) was surprising and mirrors clinical case reports (9, 33). We subcloned the cDNA extracted from this tumor and confirmed that loss of T790M had occurred on the allele with the L718V mutation. Because the T790M mutation is in the EGFR transgene in the mice, this finding suggests that it has reverted back to wild-type. To confirm this hypothesis, we performed targeted deep sequencing of osimertinib-resistant tumors and untreated control tumors. We did not detect wild-type T790 alleles in any of 8 untreated control tumors. Further, all other osimertinib-resistant tumors retained T790M, indicating that the loss of T790M was unique to the L718V-mutant tumor.

Motivated by previous findings that the T790M mutation confers a growth disadvantage in the absence of TKI selection (34), we next investigated whether the osimertinib resistance mutations alter the growth dynamics of EGFR-mutant tumors. We estimated the growth rates by applying a linear mixed effects model to our MRI tumor volume measurements. The growth rates are estimated as the relative change in number of tumor cells per day (see Supplementary Methods). We found that tumors in the EGFRL858R+T790M model do indeed exhibit a slower growth rate than tumors in the EGFRL858R model (P = 0.007; Fig. 2C). Within the EGFRL858R model, L718V-mutant tumors grew slower than both the C797S- and L718Q-mutant tumors (both P < 0.0001). Within the EGFRL858R+T790M model, L718Q-mutant tumors exhibited faster growth than C797S-mutant tumors (P = 0.0205; Fig. 2D). In addition, C797S-mutant tumors grew faster in the EGFRL858R than EGFRL858R+T790M model (P = 0.0002). These results indicate that the resistance mutations can indeed differentially alter the growth dynamics of resistant tumors, which would affect the spectrum of resistance mutations that emerge.

L718V/Q and C797S mutations are observed at different frequencies in different allelic contexts in patient cfDNA

To explore the prevalence of mutations that confer osimertinib resistance in patients, we investigated a large cohort of EGFR-mutated cases—1,117 L858R and 1,123 E746_A750 (“Del19”)—from the Guardant Health deidentified database of patients with advanced NSCLC undergoing routine clinical genomic testing by plasma next-generation sequencing (31). Within each driver mutation group, we examined the frequency of C797S, L718V, L718Q, and any combination of these specific mutations (Fig. 2E)—without considering the treatment for patients, because this was not available for all cases. Collectively, these mutations were observed in 3.9% of L858R and 5.7% of Del19 tumors (P = 0.0606). The C797S mutation was present in 25 L858R (2.2%) and 61 Del19 cases (5.4%), whereas L718V was found in 9 L858R (0.8%), and L718Q was observed in 8 L858R cases (0.7%). We found that although the C797S mutation was observed more frequently in Del19- than L858R-mutant tumors (P < 0.0001), L718V and L718Q were more frequent in L858R cases (L718V, P = 0.0019; L718Q, P = 0.0038). In fact, the L718Q mutation was only observed with a Del19 mutation when C797S was also present (n = 3), and the L718V mutation was never observed with a Del19 mutation.

We further separated the data into T790M-positive and -negative subsets. The C797S, L718V, and L718Q mutations occurred more frequently with T790M than without it (Fig. 2F and Supplementary Fig. S3). This could potentially be due to the fact that a higher proportion of patients who have received osimertinib to date are within the T790M-positive subset and might be overrepresented in the database. However, within the L858R subset, C797S mutations occurred more frequently together with T790M (28 times more T790M-positive than –negative) than L718V (1.9 times) or L718Q (1.4 times) mutations (L718V, P = 0.0074; L718Q, P = 0.0041). The ratio of C797S T790M-positive to –negative cases was similar in the Del19 subset (Supplementary Fig. S3). These data further indicate that the relative frequency of L718Q/V mutations compared with C797S may be higher in T790M-negative compared with T790M-positive tumors.

CRISPR-edited human lung adenocarcinoma cell lines harboring L718Q/V mutations are selected for in the presence of osimertinib

To test whether the L718Q and L718V substitutions in EGFR confer resistance to osimertinib in human lung cancer cells, we utilized CRISPR/Cas9 technology to model the presence of subclonal cell populations containing these mutations in an endogenous gene context. We designed a specific sgRNA targeting EGFR proximal to L718 as well as donor single-strand DNA (ssDNA) containing either the L718Q or L718V mutations (see Supplementary Methods and Table S2). Three human lung adenocarcinoma cell lines, PC9 (EGFRDel19), PC9-VanR (EGFRDel19+T790M; vandetanib-resistant), and 11-18 (EGFRL858R), were transfected with ribonucleoprotein complexes and the specific ssDNA and seeded into two wells (Fig. 3A). Three days after transfection, cells from one well were collected as the “pre-osimertinib” sample. In parallel, the other well was treated with osimertinib for 3 to 4 weeks then collected (“post-osimertinib”). This timepoint was chosen to ensure that drug-resistant growth would be due to the specific CRISPR-edited mutations, and not to other resistance mechanisms that might emerge in the cells under long-term osimertinib treatment.

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 11-18 (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 11-18 for B–D and E. Two-sided t test: *, P < 0.05.

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 11-18 (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 11-18 for B–D and E. Two-sided t test: *, P < 0.05.

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PC9, PC9-VanR, and 11-18 populations that contained cells with the L718Q or L718V mutations showed more proliferation in the presence of osimertinib than control cells (Fig. 3BD and Supplementary Fig. S4). Amplicon-sequencing analysis confirmed that the L718Q/V mutations were enriched in all cell lines (Fig. 3E; Supplementary Fig. S5A). The T790M mutation was absent from PC9-VanR cells that grew out with the L718Q/V mutations (Supplementary Fig. S5B)—resembling the GEMM tumor and patient case described above that both gained L718V and lost T790M, further suggesting that L718 mutations may be more likely to confer resistance in the absence of T790M.

Erlotinib is effective in cells with C797S but not L718Q/V mutations, whereas afatinib is effective in both

Having developed models of resistance to first-line osimertinib treatment, we next sought to investigate therapeutic strategies to treat these tumors. Previous studies have suggested that a first-generation TKI is effective against lung cancer cells harboring C797S (22, 24, 25, 27). In transiently-transfected cells, erlotinib inhibited pEGFR in both EGFRL858R+C797S- and EGFRL858R+L718V-mutated variants but did not decrease phosphorylation in the EGFRL858R+L718V mutant to the same extent as in EGFRL858R cells (Fig. 4A; Supplementary Fig. S6A). The EGFRL858R+L718Q mutant was not inhibited substantially by erlotinib, although the low basal level of pEGFR makes it difficult to interpret any differences (Fig. 4A; Supplementary Fig. S6A). Conversely, afatinib caused complete inhibition of EGFRL858R+L718V/Q phosphorylation (Fig. 4B; Supplementary Fig. S6B). Afatinib also inhibited the EGFRL858R+C797S variant, but required 10-fold more drug for complete inhibition of C797S- than L718Q/V-mutant cells—consistent with previous data (23). Most importantly, complete inhibition of the EGFRL858R+L718V/Q variants by afatinib was achieved at concentrations of approximately 10-fold lower than those needed to inhibit EGFRT790M (Fig. 4C and D). This is highly significant because it is well-established that, although afatinib can suppress EGFRL858R+T790M activity in vitro, it cannot do this at clinically achievable doses. We extended these studies to the Del19 setting and saw similar changes in EGFR phosphorylation (Fig. 4EH). These data are consistent with a report from Nishino and colleagues, in which they performed cell inhibition assays with Ba/F3 cells stably expressing either L858R or Del19 with or without the osimertinib-resistance mutations. They demonstrated that L718Q and L718V confer an >70-fold increase in the IC50 for osimertinib compared with the L858R parental cells. They also showed that the IC50 for erlotinib on L718Q- and L718V-mutant cells was >20-fold than that for the parental cells. In contrast, cells harboring the L718Q and L718V mutations had only slightly different IC50s for afatinib (5.5-fold and 1.1-fold, respectively; ref. 27).

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 1 hour with varying concentrations of the indicated TKIs. A and B show a direct comparison of EGFRL858R, EGFRL858R+C797S, EGFRL858R+L718V, and EGFRL858R+L718Q treated with 100 nmol/L osimertinib, erlotinib, or afatinib. C and D, Dose-dependent changes in EGFR phosphorylation of EGFRL858R+L718V and EGFRL858R+L718Q compared with EGFRL858R+C797S and EGFRL858R+T790M treated with increasing doses of afatinib. E, Comparison of the effect of 100 nmol/L 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 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 1 hour with varying concentrations of the indicated TKIs. A and B show a direct comparison of EGFRL858R, EGFRL858R+C797S, EGFRL858R+L718V, and EGFRL858R+L718Q treated with 100 nmol/L osimertinib, erlotinib, or afatinib. C and D, Dose-dependent changes in EGFR phosphorylation of EGFRL858R+L718V and EGFRL858R+L718Q compared with EGFRL858R+C797S and EGFRL858R+T790M treated with increasing doses of afatinib. E, Comparison of the effect of 100 nmol/L 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.

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To determine whether the observed effects of the TKIs on the different osimertinib-resistant mutations in vitro correlated with in vivo drug sensitivity, we treated mice with EGFRL858R osimertinib-resistant tumors with erlotinib or afatinib for 3 weeks (Fig. 5A). Tumors were considered “resistant” when their volume had increased by ≥20% from the point at which the TKI treatment was switched, and “sensitive” when their volume had decreased by ≥30%. We followed 24 osimertinib-resistant tumors that were switched to erlotinib and found that 7 (29.2%) responded to the TKI. Five of these harbored the C797S mutation (Fig. 5B; Supplementary Fig. S7A), whereas no acquired EGFR mutation could be identified in the other two. By contrast, 16 tumors (66.7%) became resistant, and 1 (4.2%) remained stable. Six of the erlotinib-resistant tumors harbored mutations at L718 (3 L718V and 3 L718Q). Although L718Q-mutant tumors never decreased in size following erlotinib treatment and grew rapidly, tumors with the L718V mutation exhibited a transient response to erlotinib before becoming truly resistant (Fig. 5B; Supplementary Fig. S7A). No acquired EGFR mutations could be detected in the remaining 10 resistant tumors or the 1 stable tumor.

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-EGFRL858R mice were treated with 25 mg/kg osimertinib until the emergence of resistant tumors, as in Fig. 1. Mice were then switched to either erlotinib, afatinib, or the combination of erlotinib plus osimertinib for 1 to 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 for 3 weeks (25 mg/kg each; D), as determined by MRI. Tumor volume is normalized to the point of TKI switch. Error bars, 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 (middle), 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-EGFRL858R mice were treated with the combination of osimertinib plus erlotinib (25 mg/kg each) until the emergence of resistant tumors. Error bars, SEM. *, P < 0.05. O, osimertinib; E, erlotinib; A, afatinib; C, erlotinib plus osimertinib.

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-EGFRL858R mice were treated with 25 mg/kg osimertinib until the emergence of resistant tumors, as in Fig. 1. Mice were then switched to either erlotinib, afatinib, or the combination of erlotinib plus osimertinib for 1 to 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 for 3 weeks (25 mg/kg each; D), as determined by MRI. Tumor volume is normalized to the point of TKI switch. Error bars, 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 (middle), 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-EGFRL858R mice were treated with the combination of osimertinib plus erlotinib (25 mg/kg each) until the emergence of resistant tumors. Error bars, SEM. *, P < 0.05. O, osimertinib; E, erlotinib; A, afatinib; C, erlotinib plus osimertinib.

Close modal

We next assessed the effect of afatinib treatment on 24 osimertinib-resistant EGFRL858R tumors (Fig. 5A). Fourteen tumors (58.3%) regressed after this switch, whereas 7 (29.2%) grew by ≥20% on afatinib within 1 to 3 weeks of treatment, and 3 (12.5%) remained stable. The disease control rate (sensitive and stabilized tumors) on afatinib (70.8%) was greater than on erlotinib (33.3%, P = 0.0199). Mice were treated with afatinib for 10 days because in the first set of mice, 2 responding tumors responded completely, leaving no tumor cells for analysis of the resistance mechanisms (Supplementary Fig. S7B). Of the 12 tumors responding to afatinib that could be sequenced, all but one had secondary mutations in EGFR: 7 C797S and 4 L718Q. An additional stable tumor also harbored the L718Q mutation (Fig. 5C; Supplementary Fig. S7C). This demonstrates in vivo that tumors with these mutations respond to a second-generation EGFR TKI. It is important to note that the dose of afatinib used in these experiments is not effective against tumors with the T790M mutation in vivo (35).

We treated a cohort of 7 osimertinib-resistant EGFRL858R tumors with 7.5 mg/kg QD afatinib and monitored their growth for 3 weeks. One tumor (14%) responded, 1 (14%) was stable, and 5 (71%) were resistant. Three of these tumors harbored the C797S mutations and exhibited mixed responses, whereas two tumors harboring the L718Q mutation were resistant to this dose of afatinib (Supplementary Fig. S7D and S7E). Two additional resistant tumors did not have a secondary mutation in EGFR. This result demonstrates that the response to afatinib of C797S- and L718Q-mutant tumors is dose-dependent.

We also treated a set of osimertinib-resistant EGFRL858R tumors with the combination of 25 mg/kg erlotinib plus 25 mg/kg osimertinib to assess whether using these TKIs together could overcome resistance to first-line osimertinib. Intriguingly, this strategy has been shown to be effective on cells harboring the L718Q mutation (23), despite the mutation conferring resistance to each TKI individually. Eight out of 16 tumors (50%) responded, 2 (13%) were stable, and 6 (38%) were resistant. Seven sensitive tumors harbored the C797S mutation (Fig. 5D; Supplementary Fig. S7F). The remaining responding tumor—as well as the two stable tumors—harbored L718Q mutations (Fig. 5D; Supplementary Fig. S7F). None of the six resistant tumors had secondary EGFR mutations.

We compared the growth rates of tumors with specific mutations before and after they were switched to erlotinib, afatinib, or the combination of erlotinib plus osimertinib (Fig. 5E). The positive or negative growth rates correlated with resistance or sensitivity to the TKI, respectively. We observed that only the C797S-mutant tumors exhibited a negative growth rate on erlotinib treatment compared with positive growth on osimertinib (P = 0.0115, Fig. 5E, left). Both the C797S- and L718Q-mutant tumors exhibited negative growth rates on afatinib treatment (Fig. 5E, center). Both C797S- and L718Q-mutant tumors exhibited negative growth rates under the combination of erlotinib plus osimertinib, though this was lower for C797S (P = 0.0392; Fig. 5E, right). These data suggest that L718Q-mutant tumors may respond (or at least stabilize) when treated with the combination of first- and third-generation EGFR TKIs.

Combination first-line erlotinib plus osimertinib prevents secondary mutations in EGFR

Given that L718V/Q mutations are not observed at resistance to first-line erlotinib, and the combination of erlotinib and osimertinib was effective against L718Q-mutant tumors, we wondered whether this combination as initial therapy could prevent the emergence of L718 mutations. To test this hypothesis, we treated 17 tumor-bearing CCSP-rtTA;TetO-EGFRL858R mice with a combination of osimertinib and erlotinib (25 mg/kg QD continuously M-F each; Fig. 5F). Six out of 17 mice (35.3%) developed resistant tumors, on average after 6.9 months. This time to resistance is slightly shorter than it was for the EGFRL858R mice treated with 25 mg/kg osimertinib only, although the difference is not significant (6.9 vs. 7.8 months, P = 0.2767). Of 10 resistant tumors collected, none harbored secondary mutations in EGFR. This result suggests that the combination of first- and third-generation TKIs may indeed be effective at preventing secondary EGFR mutations from arising as a resistance mechanism, although it did not necessarily delay the emergence of resistance overall. Ten mice (58.8%) were sacrificed before the end of the study without developing resistant tumors due to declining health, possibly due to kidney toxicity, as morphologic abnormalities were found in the kidneys including fibrosis and some atrophy. Therefore, it is possible that dosing will need to be optimized when using this combination of TKIs.

Mutations in Kras confer resistance to osimertinib in vivo

In efforts to identify mechanisms of osimertinib resistance in tumors without EGFR secondary mutations, we first surveyed Kras. Alterations in this gene have been implicated in resistance to osimertinib and previously found at resistance to other EGFR-directed therapies in these models (10–12, 20, 36, 37). In the EGFRL858R tumors with acquired resistance to first-line osimertinib described in Fig. 1C, 28 of 54 tumors (51.9%) resistant to 25 mg/kg osimertinib harbored Kras mutations at codon 12, 13, or 61, as did 4 of 17 (23.5%) tumors resistant to the 5→25 mg/kg osimertinib regimen and 7 out of 27 (25.9%) osimertinib-resistant EGFRL858R+T790M tumors (Supplementary Fig. S8). Finally, 9 of 10 tumors (90%) resistant to combination first-line erlotinib plus osimertinib harbored mutations in Kras (Supplementary Fig. S8). The Kras mutations (Supplementary Table S3) were all mutually exclusive with acquired EGFR mutations, yet the engineered EGFR L858R or L858R+T790M mutations were always maintained in the Kras-mutant tumors.

We also did not observe any significant amplification of Met—an established EGFR TKI resistance mechanism (1–3, 10, 11, 13, 20, 21)—in the tumors that did not acquire either an EGFR or Kras mutation (Supplementary Fig. S9). Therefore, acquired EGFR and Kras mutations account for almost all cases of osimertinib-resistant tumors in these GEMMs (Supplementary Fig. S8).

Benefit from afatinib treatment in a patient with an L858R-mutant tumor harboring L718Q and L718V mutations

To investigate the potential benefit of second-generation EGFR TKIs in overcoming resistance due to L718 mutations in patients, we studied a patient who had developed resistance to first-line osimertinib and was subsequently treated with afatinib. A 62-year-old woman with a 4 pack-year smoking history presented with back and flank pain. CT and MRI scans showed a 3.7 cm mass in the right upper lobe of the lung, multiple subcentimeter lung nodules, 3 liver lesions, an adrenal lesion, multiple lesions in the thoracic spine, a compression fracture at T9 with evidence of spinal cord compression, and punctate lesions in the left parietal lobe and cerebellum. Biopsy of a liver lesion demonstrated adenocarcinoma positive for TTF-1 and Napsin-A by IHC. An EGFRL858R mutation was detected in the tumor with targeted NGS using the Illumina MiSeqDx platform (Fig. 6A; Supplementary Table S4). The patient underwent a T9 corpectomy and spine fusion, followed by consolidative radiation, after which, she was started on 80 mg osimertinib QD. She had an excellent response, with shrinkage of all sites of disease and resolution of brain metastases. Imaging performed every approximately 2 months showed a continued response, until 8.5 months after initiation of osimertinib when the patient was found to have growth of a liver lesion. A repeat biopsy was performed on the growing lesion and showed adenocarcinoma consistent with the original pathology. This tissue was sent for NGS using Foundation One CDx testing. The original EGFRL858R mutation was detected (allelic frequency of 84%), along with EGFRL718Q (73%) and EGFRL718V (3%) mutations. The patient underwent thermal ablation to the enlarging liver lesion; however 2 months later, she was found to have progression in the liver and lungs. At that time, she was started on 40 mg afatinib QD given the acquired L718V and L718Q mutations. Imaging performed approximately 2 months later showed shrinkage in a previously growing lung lesion (Fig. 6B)—although this site may not necessarily harbor the L718Q/V mutations—and stable disease at all other sites. RECIST1.1 measurements confirmed that afatinib led to a partial response (Supplementary Table S5).

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 arrowhead) that was new prior to starting afatinib, and then shrunk after treatment with afatinib.

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 arrowhead) that was new prior to starting afatinib, and then shrunk after treatment with afatinib.

Close modal

After approximately 4.5 months on afatinib treatment, the patient developed disease progression. Another biopsy was performed on a progressing liver lesion, and the tissue was sent for NGS using Foundation One CDx testing. This biopsy revealed the emergence of the EGFRT790M mutation (allelic frequency of 59%). Interestingly, this was accompanied by an increase in the allelic frequency of the L718V mutation (80%), and a decrease in the frequency of the L718Q mutation (3%). This report suggests that although L718Q/V-mutant tumors could benefit from transient responses to afatinib treatment, they can acquire the T790M mutation and eventually become resistant to all available EGFR TKIs.

The mean progression-free survival for osimertinib in the first line is 19 months, with an overall survival of 39 months, which is superior to that observed with other TKIs (18, 19). Patients are not cured, however, and a better understanding of acquired resistance to osimertinib is needed. Here, using GEMMs of EGFR-mutant lung adenocarcinoma, we show that resistance to first-line osimertinib can be due to acquired C797S, L718Q, and L718V mutations in EGFR. This is the first report of an in vivo first-line osimertinib resistance model that acquires EGFR mutations. Data emerging from the FLAURA trial of front-line osimertinib indicate that, at acquired resistance, the C797S and L718Q mutations are detected in 7% and 2% of cases from ctDNA (21). The higher frequency of C797S mutations versus L718Q mutations observed in these patient samples (with a variety of driver mutations) compared with the more equal distribution of C797S and L718Q mutations in our mouse model may reflect the allele specificity of the resistance mutations (14). Indeed, our analysis of cfDNA from patients with EGFR-mutant lung cancer clearly shows that C797S mutations predominate in the Del19 subset of tumors, and that L718Q and L718V mutations generally only arise in the context of L858R, consistent with the few published reports of L718Q/V mutations (8–10, 15, 16, 33). Why L718Q/V mutations are found more readily in the context of L858R mutations in patients remains to be determined. It is possible that it is due to differential stability of the different mutation combinations or differences in other biochemical properties such as their kinase activity or dimerization properties. Interestingly, in vitro data in our study and others’ (23) show that Del19 cells harboring a L718Q/V mutation can exist and are resistant to osimertinib, suggesting the intriguing possibility that resistance mechanisms that emerge spontaneously in vivo may be somewhat different than those observed in vitro.

The mechanism by which the C797S substitution causes osimertinib resistance is straightforward as it removes the side-chain thiol with which osimertinib reacts covalently. Models obtained using molecular dynamics–based energy minimization provide some insight into how L718V/Q mutations might affect drug-binding. The L718 side-chain contacts the phenyl ring of the drug in a crystal structure of osimertinib-bound wild-type EGFR TKD (PDB ID 4ZAU; ref. 38). Substituting L718 with V or Q is therefore highly likely to alter the mode or orientation of osimertinib binding, as others have noted (8, 17, 23). We confirmed this by modeling noncovalent complexes of L858R-mutated EGFR TKD with osimertinib, erlotinib, and afatinib (Fig. 7). Although the pose of osimertinib in the energy-minimized L858R TKD complex (with leucine at position 718) is maintained, loss of van der Waals interactions is seen for the L718V mutation, and a steric clash with the glutamine side-chain is seen for the L718Q mutation (upper panels, Fig. 7). These effects are compensated for in the models by reorientation of osimertinib in the drug-binding site, which may make its orientation suboptimal for covalent reaction with C797—as argued previously for L718Q/T790M EGFR (17). There is also some reduction in osimertinib-binding energy, suggesting that the affinity of the “encounter complex” prior to covalent reaction may be reduced—which would also reduce efficacy. The same mutations are predicted to have similar effects on erlotinib-binding energy and orientation (middle panel, Fig. 7). In this case, the lack of any covalent aspect suggests that observed resistance must arise from weakened binding. Indeed, L718 contacts erlotinib directly in EGFR/erlotinib crystal structures (39). Finally, the effects of the L718V/Q mutations on afatinib-binding energy are only 80% of those calculated for osimertinib, suggesting that they will have less severe effects on residence time of afatinib for its covalent reaction. Moreover, the orientation of afatinib appears less affected by the mutations—in Fig. 7, there is no clash between afatinib in its L858R-bound orientation and the Q718 side-chain, although interactions with L718 are lost in the L718V variant.

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 was 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.

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 was 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.

Close modal

Through experiments in CRISPR-edited human EGFR-mutant lung adenocarcinoma cell lines—to mimic the acquisition of resistance when low frequency subclonal mutations are present—we show that subclonal L718V/Q mutations confer resistance to osimertinib. However, the L718V mutation retained some sensitivity to osimertinib at higher doses, and its frequency was reduced in vivo with higher osimertinib doses. The only three reports of patient tumor biopsies revealing an L718V mutation also coincided with loss of T790M (9, 10, 33), which was replicated in one of our mice and the CRISPR-edited PC9-VanR cells that gained L718V/Q mutations. It is possible that destabilization of EGFR by L718 mutations prevents them from occurring in the presence of T790M.

With osimertinib now as the preferred first-line therapy for advanced EGFR-mutant tumors, it is critical to develop strategies to overcome resistance. Our findings confirm the erlotinib-sensitivity of C797S-mutant tumors in vivo, suggested by several in vitro studies and case reports (22, 24, 25), but L718V/Q-mutant tumors were not sensitive to erlotinib. Afatinib caused tumor regression in all tumors harboring C797S or L718Q mutations, as seen in previous in vitro studies (9, 23). These data are consistent with our report of a patient who developed resistance to first-line osimertinib through the acquisition of both L718Q and L718V mutations in the tumor and whose disease was stabilized by afatinib treatment, similarly to two recent second-line osimertinib case reports with L858R+L718Q- (15) and L858R+L718V-mutant tumors (33). Afatinib is currently FDA-approved for the treatment of tumors harboring G719X mutations based on its clinical efficacy (40), suggesting a similar mechanism could underlie the sensitivity of L718 and G719 mutants to afatinib.

It is unclear why L718 mutations have never been observed in patient tumors resistant to first-generation EGFR TKIs, given that they confer resistance to these inhibitors in vitro and in vivo based on our work and others’ (9, 23). Whether a mutation emerges may depend on several factors including the ease with which a mutation occurs (i.e., base change requirements), the strength of TKI-binding, and the extent of the selective pressure exerted by the TKI. Thus, a combination of EGFR TKIs may be able to prevent secondary EGFR mutations from emerging or delay the emergence of resistance. We tested the approach of combining a first- and third-generation TKI in vivo and, importantly, none of the ten resistant tumors that developed had acquired mutations in EGFR. However, resistance did emerge. Therefore, it is possible that by effectively suppressing EGFR-dependent mechanisms of resistance with the combination therapy, EGFR-independent mechanisms will become more prominent. How this will affect clinical outcomes for patients remains to be determined. Indeed, clinical studies to evaluate the safety and efficacy of combination EGFR TKIs are ongoing (e.g., NCT03122717). Given that we observed toxicities of the drug combination in vivo, future experiments with optimized doses of the agents will be important to conclusively determine whether this approach can be used to prevent EGFR-dependent resistance mechanisms. This also highlights the importance of dose optimization to minimize toxicities and maximize efficacy in clinical trials. Nevertheless, our data present an encouraging new therapeutic approach to treat EGFR-mutant tumors upfront when tumors are most likely to be maximally addicted to mutant EGFR signaling.

Not all EGFR TKI–resistant tumors acquire secondary mutations in EGFR, and we identified Kras mutations in osimertinib-resistant tumors. This is not surprising given that we previously observed these mutations arising in TKI-naïve tumors and at resistance to other EGFR inhibitors in these models (36, 37, 41), and activation of the MAPK/ERK pathway has been described as a mechanism of resistance to third-generation EGFR TKIs (42, 43). Whether the relatively high abundance of Kras mutations in our mouse model is due to spontaneous tumor development in aging mice (44) or whether these are enriched with osimertinib requires additional investigation. Nevertheless, KRAS mutations are certainly of clinical relevance given that they are being observed at osimertinib resistance in patients (10–12, 20, 45).

Acquired EGFR and Kras mutations account for almost all cases of osimertinib-resistant GEMM tumors described here. However, other mechanisms of resistance have been observed in patients (3, 10, 21, 46) and overall, secondary mutations in EGFR are found more frequently in our mouse models than in osimertinib-resistant human tumors. This could reflect the reduced genomic complexity of the GEMM tumors (41), and the fact that expression of the mutant EGFR transgene is constantly being driven by the presence of doxycycline, which may confer a greater propensity for on-target resistance. Therefore, other models should be used for further discovery of EGFR-independent resistance mechanisms and evaluation of therapeutic approaches to overcome them (43, 47, 48). Nonetheless, our study provides important insight into EGFR-dependent (and proximal) resistance mechanisms to osimertinib and highlights the complexity and heterogeneity of osimertinib resistance. Two key avenues for clinical investigation suggested by our findings are: (i) evaluation of sequential TKI treatment that includes osimertinib, followed by a second-generation TKI such as afatinib or dacomitinib, and (ii) evaluation of TKI combinations in the first line.

M.E. Cuomo is Associate Director at AstraZeneca. K.S. Price is Medical Science Liaison at, and has an ownership interest (including patents) in, Guardant Health. S.M. Kaech is Academic Editor at the Journal of Experimental Medicine and has an unpaid consultant/advisory board relationship with Celsius Therapeutics. F. Michor is a consultant at Flagship Pioneering. S.B. Goldberg is an advisory board member at AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Amgen, Spectrum, and Eli Lilly, and reports receiving commercial research grant from AstraZeneca and Boehringer Ingelheim. P.D. Smith is Senior Principal Scientist at AstraZeneca. D.A.E. Cross is Medical Affairs Leader at AstraZeneca. K. Politi is a consultant at AstraZeneca, Dynamo Therapeutics, and Maverick Therapeutics; reports receiving commercial research grant from AstraZeneca, Roche/Genentech, and Symphogen; has an ownership interest (including patents) in MSKCC/Molecular MD; and has an unpaid consultant/advisory board relationship with Lung Cancer Research Foundation. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.H. Starrett, M.E. Cuomo, S.M. Kaech, S.B. Goldberg, M.A. Lemmon, P.D. Smith, K. Politi

Development of methodology: J.H. Starrett, A.A. Guernet, M.E. Cuomo, A.M. Unni, F. Michor, K. Politi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.H. Starrett, A.A. Guernet, I.K. van Alderwerelt van Rosenburgh, A. Nagelberg, D. Farnsworth, H. Khan, M. Gaefele, T.F. Stewart, R. Homer, W.W. Lockwood

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.H. Starrett, A.A. Guernet, M.E. Cuomo, K.E. Poels, I.K. van Alderwerelt van Rosenburgh, A. Nagelberg, K.S. Price, H. Khan, K.D. Ashtekar, S.M. Kaech, A.M. Unni, R. Homer, W.W. Lockwood, F. Michor, S.B. Goldberg, D.A.E. Cross, K. Politi

Writing, review, and/or revision of the manuscript: J.H. Starrett, A.A. Guernet, K.E. Poels, I.K. van Alderwerelt van Rosenburgh, K.S. Price, H. Khan, D. Ayeni, T.F. Stewart, S.B. Goldberg, M.A. Lemmon, P.D. Smith, D.A.E. Cross, K. Politi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.S. Price, K. Politi

Study supervision: F. Michor, M.A. Lemmon, K. Politi

Other (performed computational analysis of the drug bound form of EGFR mutant): K.D. Ashtekar

Other (I was working on this model to profile the immune infiltrate in collaboration with the Politi lab during the conception of this project. As such, I was present at and provided feedback of the data as it was gathered by the first author for the first three years of its creation): A. Kuhlmann

The authors would like to thank all members of the Politi Laboratory for helpful discussions and support. We also thank Emily Forrest for critical reading and insights into the article. We thank Kaya Bilguvar, Christopher Castaldi, James Knight, and The Yale Center for Genome Analysis for assistance with the NGS and analysis of mouse tumors. We also thank Damla Etal, Julia Lindgren, Ben Taylor, and Mike Firth for the CRISPR NGS run and analysis. We thank Fangyong Li and The Yale Center for Analytical Sciences for assistance with statistical analysis. We also thank Daniel O’Neill for assistance with data analysis.

This work was supported by Yale's Specialized Program of Research Excellence in Lung Cancer grant (to K. Politi, S.B. Goldberg, and M.A. Lemmon, P50 CA196530) and funding from AstraZeneca (to K. Politi). Additional support came from the NIH/NCI-funded Yale Cancer Biology Training Program T32 CA193200-01A1 and F31 CA228268-01A1 (to J.H. Starrett), R01 CA198164 (to M.A. Lemmon), the Ginny and Kenneth Grunley Fund for Lung Cancer Research, and the Canadian Institutes of Health Research Project Grant PJT-148725 (to W.W. Lockwood). W.W. Lockwood is supported by a Michael Smith Foundation for Health Research Scholar and NIHR New Investigator Awards, A. Guernet is a fellow funded by the IMED AstraZeneca postdoc program, A. Nagelberg is supported by a scholarship from the CIHR, and K.D. Ashtekar is an Arnold and Mabel Beckman Foundation Postdoctoral Fellow. Yale Cancer Center Shared Resources used in this article were in part supported by NIH/NCI Cancer Center Support Grant P30 CA016359.

1.
Camidge
DR
,
Pao
W
,
Sequist
LV
. 
Acquired resistance to TKIs in solid tumours: learning from lung cancer
.
Nat Rev Clin Oncol
2014
;
11
:
473
81
.
2.
Pao
W
,
Chmielecki
J
. 
Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer
.
Nat Rev Cancer
2010
;
10
:
760
74
.
3.
Ohashi
K
,
Maruvka
YE
,
Michor
F
,
Pao
W
. 
Epidermal growth factor receptor tyrosine kinase inhibitor-resistant disease
.
J Clin Oncol
2013
;
31
:
1070
80
.
4.
Cross
DA
,
Ashton
SE
,
Ghiorghiu
S
,
Eberlein
C
,
Nebhan
CA
,
Spitzler
PJ
, et al
AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer
.
Cancer Discov
2014
;
4
:
1046
61
.
5.
Janne
PA
,
Yang
JC
,
Kim
DW
,
Planchard
D
,
Ohe
Y
,
Ramalingam
SS
, et al
AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer
.
N Engl J Med
2015
;
372
:
1689
99
.
6.
Thress
KS
,
Paweletz
CP
,
Felip
E
,
Cho
BC
,
Stetson
D
,
Dougherty
B
, et al
Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M
.
Nat Med
2015
;
21
:
560
2
.
7.
Ou
SI
,
Cui
J
,
Schrock
AB
,
Goldberg
ME
,
Zhu
VW
,
Albacker
L
, et al
Emergence of novel and dominant acquired EGFR solvent-front mutations at Gly796 (G796S/R) together with C797S/R and L792F/H mutations in one EGFR (L858R/T790M) NSCLC patient who progressed on osimertinib
.
Lung Cancer
2017
;
108
:
228
31
.
8.
Bersanelli
M
,
Minari
R
,
Bordi
P
,
Gnetti
L
,
Bozzetti
C
,
Squadrilli
A
, et al
L718Q mutation as new mechanism of acquired resistance to AZD9291 in EGFR-mutated NSCLC
.
J Thorac Oncol
2016
;
11
:
e121
3
.
9.
Liu
Y
,
Li
Y
,
Ou
Q
,
Wu
X
,
Wang
X
,
Shao
YW
, et al
Acquired EGFR L718V mutation mediates resistance to osimertinib in non-small cell lung cancer but retains sensitivity to afatinib
.
Lung Cancer
2018
;
118
:
1
5
.
10.
Yang
Z
,
Yang
N
,
Ou
Q
,
Xiang
Y
,
Jiang
T
,
Wu
X
, et al
Investigating novel resistance mechanisms to third-generation EGFR tyrosine kinase inhibitor osimertinib in non-small cell lung cancer patients
.
Clin Cancer Res
2018
;
24
:
3097
107
.
11.
Oxnard
GR
,
Hu
Y
,
Mileham
KF
,
Husain
H
,
Costa
DB
,
Tracy
P
, et al
Assessment of resistance mechanisms and clinical implications in patients with EGFR T790M-positive lung cancer and acquired resistance to osimertinib
.
JAMA Oncol
2018
;
4
:
1527
34
.
12.
Le
X
,
Puri
S
,
Negrao
MV
,
Nilsson
MB
,
Robichaux
J
,
Boyle
T
, et al
Landscape of EGFR-dependent and -independent resistance mechanisms to osimertinib and continuation therapy beyond progression in EGFR-mutant NSCLC
.
Clin Cancer Res
2018
;
24
:
6195
203
.
13.
Piotrowska
Z
,
Isozaki
H
,
Lennerz
JK
,
Gainor
JF
,
Lennes
IT
,
Zhu
VW
, et al
Landscape of acquired resistance to osimertinib in EGFR-mutant NSCLC and clinical validation of combined EGFR and RET inhibition with osimertinib and BLU-667 for acquired RET fusion
.
Cancer Discov
2018
;
8
:
1529
39
.
14.
Brown
BP
,
Zhang
YK
,
Westover
D
,
Yan
Y
,
Qiao
H
,
Huang
V
, et al
On-target resistance to the mutant-selective EGFR inhibitor osimertinib can develop in an allele specific manner dependent on the original EGFR activating mutation
.
Clin Cancer Res
2019
Feb 22
[Epub ahead of print]
.
15.
Liu
J
,
Jin
B
,
Su
H
,
Qu
X
,
Liu
Y
. 
Afatinib helped overcome subsequent resistance to osimertinib in a patient with NSCLC having leptomeningeal metastasis baring acquired EGFR L718Q mutation: a case report
.
BMC Cancer
2019
;
19
:
702
.
16.
Yang
Z
,
Yang
J
,
Chen
Y
,
Shao
YW
,
Wang
X
. 
Acquired EGFR L718V mutation as the mechanism for osimertinib resistance in a T790M-negative non-small-cell lung cancer patient
.
Target Oncol
. 
2019
;
14
:
369
74
.
17.
Callegari
D
,
Ranaghan
KE
,
Woods
CJ
,
Minari
R
,
Tiseo
M
,
Mor
M
, et al
L718Q mutant EGFR escapes covalent inhibition by stabilizing a non-reactive conformation of the lung cancer drug osimertinib
.
Chem Sci
2018
;
9
:
2740
9
.
18.
Soria
JC
,
Ohe
Y
,
Vansteenkiste
J
,
Reungwetwattana
T
,
Chewaskulyong
B
,
Lee
KH
, et al
Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer
.
N Engl J Med
2018
;
378
:
113
25
.
19.
Ramalingam
SS
,
Vansteenkiste
J
,
Planchard
D
,
Cho
BC
,
Gray
JE
,
Ohe
Y
, et al
Overall survival with osimertinib in untreated, EGFR-mutated advanced NSCLC
.
N Engl J Med
2019
;
382
:
41
50
.
20.
Ramalingam
SS
,
Yang
JC
,
Lee
CK
,
Kurata
T
,
Kim
DW
,
John
T
, et al
Osimertinib as first-line treatment of EGFR mutation-positive advanced non-small-cell lung cancer
.
J Clin Oncol
2018
;
36
:
841
9
.
21.
Ramalingam
SS
,
Rukazenkov
Y
,
Todd
A
,
Markovets
A
,
Chmielecki
J
,
Barrett
JC
, et al
LBA50Mechanisms of acquired resistance to first-line osimertinib: Preliminary data from the phase III FLAURA study
.
Ann Oncol
2018
;
29
:
viii740
.
22.
Niederst
MJ
,
Hu
H
,
Mulvey
HE
,
Lockerman
EL
,
Garcia
AR
,
Piotrowska
Z
, et al
The allelic context of the C797S mutation acquired upon treatment with third-generation EGFR inhibitors impacts sensitivity to subsequent treatment strategies
.
Clin Cancer Res
2015
;
21
:
3924
33
.
23.
Ercan
D
,
Choi
HG
,
Yun
CH
,
Capelletti
M
,
Xie
T
,
Eck
MJ
, et al
EGFR mutations and resistance to irreversible pyrimidine-based EGFR inhibitors
.
Clin Cancer Res
2015
;
21
:
3913
23
.
24.
Chic
N
,
Mayo-de-Las-Casas
C
,
Reguart
N
. 
Successful treatment with gefitinib in advanced non-small cell lung cancer after acquired resistance to osimertinib
.
J Thorac Oncol
2017
;
12
:
e78
e80
.
25.
Rangachari
D
,
To
C
,
Shpilsky
JE
,
VanderLaan
PA
,
Kobayashi
SS
,
Mushajiang
M
, et al
EGFR-mutated lung cancers resistant to osimertinib through EGFR-C797S respond to 1(st) generation reversible EGFR inhibitors but eventually acquire EGFR-T790M/C797S in preclinical models and clinical samples
.
J Thorac Oncol
2019
;
14
:
1995
2002
.
26.
Wang
Z
,
Yang
JJ
,
Huang
J
,
Ye
JY
,
Zhang
XC
,
Tu
HY
, et al
Lung adenocarcinoma harboring EGFR T790M and in trans C797S responds to combination therapy of first- and third-generation EGFR TKIs and shifts allelic configuration at resistance
.
J Thorac Oncol
2017
;
12
:
1723
7
.
27.
Nishino
M
,
Suda
K
,
Kobayashi
Y
,
Ohara
S
,
Fujino
T
,
Koga
T
, et al
Effects of secondary EGFR mutations on resistance against upfront osimertinib in cells with EGFR-activating mutations in vitro
.
Lung Cancer
2018
;
126
:
149
55
.
28.
Politi
K
,
Zakowski
MF
,
Fan
PD
,
Schonfeld
EA
,
Pao
W
,
Varmus
HE
. 
Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors
.
Genes Dev
2006
;
20
:
1496
510
.
29.
Regales
L
,
Balak
MN
,
Gong
Y
,
Politi
K
,
Sawai
A
,
Le
C
, et al
Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors
.
PLoS One
2007
;
2
:
e810
.
30.
Tichelaar
JW
,
Lu
W
,
Whitsett
JA
. 
Conditional expression of fibroblast growth factor-7 in the developing and mature lung
.
J Biol Chem
2000
;
275
:
11858
64
.
31.
Odegaard
JI
,
Vincent
JJ
,
Mortimer
S
,
Vowles
JV
,
Ulrich
BC
,
Banks
KC
, et al
Validation of a plasma-based comprehensive cancer genotyping assay utilizing orthogonal tissue- and plasma-based methodologies
.
Clin Cancer Res
2018
;
24
:
3539
49
.
32.
Ballard
P
,
Yates
JW
,
Yang
Z
,
Kim
DW
,
Yang
JC
,
Cantarini
M
, et al
Preclinical comparison of osimertinib with other EGFR-TKIs in EGFR-mutant NSCLC brain metastases models, and early evidence of clinical brain metastases activity
.
Clin Cancer Res
2016
;
22
:
5130
40
.
33.
Fang
W
,
Gan
J
,
Huang
Y
,
Zhou
H
,
Zhang
L
Acquired EGFR L718V mutation and loss of T790M-mediated resistance to osimertinib in a patient with NSCLC who responded to afatinib
.
J Thorac Oncol
2019
;
14
:
e274
e5
.
34.
Chmielecki
J
,
Foo
J
,
Oxnard
GR
,
Hutchinson
K
,
Ohashi
K
,
Somwar
R
, et al
Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling
.
Sci Transl Med
2011
;
3
:
90ra59
.
35.
Regales
L
,
Gong
Y
,
Shen
R
,
de Stanchina
E
,
Vivanco
I
,
Goel
A
, et al
Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer
.
J Clin Invest
2009
;
119
:
3000
10
.
36.
Politi
K
,
Fan
PD
,
Shen
R
,
Zakowski
M
,
Varmus
H
. 
Erlotinib resistance in mouse models of epidermal growth factor receptor-induced lung adenocarcinoma
.
Dis Model Mech
2010
;
3
:
111
9
.
37.
Pirazzoli
V
,
Ayeni
D
,
Meador
CB
,
Sanganahalli
BG
,
Hyder
F
,
de Stanchina
E
, et al
Afatinib plus cetuximab delays resistance compared to single-agent erlotinib or afatinib in mouse models of TKI-naive EGFR L858R-induced lung adenocarcinoma
.
Clin Cancer Res
2016
;
22
:
426
35
.
38.
Yosaatmadja
Y
,
Silva
S
,
Dickson
JM
,
Patterson
AV
,
Smaill
JB
,
Flanagan
JU
, et al
Binding mode of the breakthrough inhibitor AZD9291 to epidermal growth factor receptor revealed
.
J Struct Biol
2015
;
192
:
539
44
.
39.
Stamos
J
,
Sliwkowski
MX
,
Eigenbrot
C
. 
Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor
.
J Biol Chem
2002
;
277
:
46265
72
.
40.
Yang
JC
,
Sequist
LV
,
Geater
SL
,
Tsai
CM
,
Mok
TS
,
Schuler
M
, et al
Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6
.
Lancet Oncol
2015
;
16
:
830
8
.
41.
McFadden
DG
,
Politi
K
,
Bhutkar
A
,
Chen
FK
,
Song
X
,
Pirun
M
, et al
Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma
.
Proc Natl Acad Sci U S A
2016
;
113
:
E6409
E17
.
42.
Tricker
EM
,
Xu
C
,
Uddin
S
,
Capelletti
M
,
Ercan
D
,
Ogino
A
, et al
Combined EGFR/MEK inhibition prevents the emergence of resistance in EGFR-mutant lung cancer
.
Cancer Discov
2015
;
5
:
960
71
.
43.
Eberlein
CA
,
Stetson
D
,
Markovets
AA
,
Al-Kadhimi
KJ
,
Lai
Z
,
Fisher
PR
, et al
Acquired resistance to the mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models
.
Cancer Res
2015
;
75
:
2489
500
.
44.
Dragani
TA
,
Manenti
G
,
Pierotti
MA
. 
Genetics of murine lung tumors
.
Adv Cancer Res
1995
;
67
:
83
112
.
45.
Del Re
M
,
Tiseo
M
,
Bordi
P
,
D'Incecco
A
,
Camerini
A
,
Petrini
I
, et al
Contribution of KRAS mutations and c.2369C >T (p.T790M) EGFR to acquired resistance to EGFR-TKIs in EGFR mutant NSCLC: a study on circulating tumor DNA
.
Oncotarget
2017
;
8
:
13611
9
.
46.
Schoenfeld
AJ
,
Chan
JM
,
Kubota
D
,
Sato
H
,
Rizvi
H
,
Daneshbod
Y
, et al
Tumor analyses reveal squamous transformation and off-target alterations as early resistance mechanisms to first-line osimertinib in EGFR-mutant lung cancer
.
Clin Cancer Res
. 
2020
Jan 7
[Epub ahead of print]
.
47.
Ichihara
E
,
Westover
D
,
Meador
CB
,
Yan
Y
,
Bauer
JA
,
Lu
P
, et al
SFK/FAK signaling attenuates osimertinib efficacy in both drug-sensitive and drug-resistant models of EGFR-mutant lung cancer
.
Cancer Res
2017
;
77
:
2990
3000
.
48.
Galvani
E
,
Sun
J
,
Leon
LG
,
Sciarrillo
R
,
Narayan
RS
,
Sjin
RT
, et al
NF-kappaB drives acquired resistance to a novel mutant-selective EGFR inhibitor
.
Oncotarget
2015
;
6
:
42717
32
.