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Department of Thoracic Oncology, National Cancer Center Hospital East, Kashiwa, JapanDivision of Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, JapanDepartment of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Division of Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, JapanDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
Department of Thoracic Oncology, National Cancer Center Hospital East, Kashiwa, JapanDivision of Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Japan
Corresponding author. Address for correspondence: Susumu S. Kobayashi, MD, PhD, Department of Medicine, Beth Israel Deconess Medical Center, Boston, MA 02215.
Division of Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, JapanDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, JapanDepartment of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
EGFR exon 20 insertion mutations account for 5% to 10% of EGFR-mutated NSCLC. CLN-081 (formerly known as TAS6417), a novel covalent EGFR tyrosine kinase inhibitor, exhibits pan-mutation selective efficacy, including exon 20 insertions, in the clinical setting. Nevertheless, some patients may not respond to CLN-081 and resistance to CLN-081 may emerge over time in others.
Methods
We exposed Ba/F3 cells transduced with EGFR exon 20 insertions (Y764_V765 insHH or A767_S768insSVD) to increasing concentrations of CLN-081 to generate resistant cells and then subjected their complementary DNA to sequencing to identify acquired mutations. We then evaluated effects of small molecules on engineered Ba/F3 cells on the basis of proliferation assays, Western blotting, and xenograft models.
Results
All CLN-081 resistant clones harbored the EGFR C797S mutation. Ba/F3 cells with C797S (Ba/F3-C797S) were resistant to EGFR tyrosine kinase inhibitors targeting EGFR exon 20 insertion mutations, including CLN-081. Pimitespib, a selective heat shock protein 90 inhibitor, induced apoptosis in Ba/F3-C797S cells in vitro and inhibited growth of Ba/F3-C797S tumors in vivo. Ba/F3 cells with A763_Y764insFQEA-C797S remained sensitive to erlotinib.
Conclusions
We conclude that the EGFR C797S mutation confers resistance to CLN-081. Our preclinical data suggest a potential small molecule to overcome CLN-081 resistance, which may benefit patients with lung cancer with EGFR exon 20 insertions.
NSCLC is an aggressive malignancy. The development of small molecules targeting oncogenic driver alterations has largely changed strategies used to treat advanced NSCLC. Approximately 20% of white people and 60% of Asians with lung adenocarcinomas exhibit oncogenic mutations in EGFR.
Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.
Most EGFR-mutant NSCLCs harbor classical sensitizing mutations, such as exon 19 deletion or L858R point mutation in exon 21. First-generation (gefitinib,
Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.
Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.
Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.
) EGFR tyrosine kinase inhibitors (TKIs) were found to have clinical efficacy such as high overall response rates (ORRs) in the case of sensitizing EGFR mutations. The third most common subtype is insertion mutations in exon 20, which accounts for 5% to 10% of EGFR-mutated NSCLC.
Although some heterogeneity exist on the basis of structural modification of exon 20 insertions, in general, cells harboring exon 20 insertions are insensitive to conventional EGFR TKIs, except for A763_Y764insFQEA.
Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer (Science Translational Medicine (2014) 6, (225er1)).
Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials.
Recently, several novel agents have been developed for tumors with EGFR exon 20 insertion mutations. Mobocertinib and amivantamab, a small molecule and a bispecific EGFR and MET antibody, respectively, were approved by the Food and Drug Administration in 2021. CLN-081 (formerly known as TAS6417) is a novel covalent EGFR TKI exhibiting pan-mutation selective efficacy, including exon 20 insertions, and a wide therapeutic window preclinically.
TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically relevant EGFR mutations.
CLN-081 received Breakthrough Therapy Designation from the Food and Drug Administration in 2022, on the basis of robust and durable response observed in a phase 1 and 2a trial enrolling patients with EGFR exon 20-mutant NSCLC. In that trial, CLN-081 had a 41% ORR, 97% disease control rate, and 12 months of median progression-free survival with manageable toxicity.
As one can expect that patients receiving CLN-081 will acquire on- or off-target resistance over time, it is imperative to identify mechanisms underlying that resistance. Nevertheless, clinical mechanisms of resistance to CLN-081 have yet to be identified. Here, we used preclinical cell line models to identify the EGFR C797S mutation as a potential acquired on-target resistant mechanism. We also sought to identify strategies to overcome resistance mechanisms, anticipating the emergence of this resistance in the clinical setting.
Materials and Methods
Cell Lines and Culture
Ba/F3 cells were acquired from the RIKEN BioResource Center. All cell lines were authenticated by short tandem repeat profiling before purchase or use in this study. Cells were tested for Mycoplasma contamination (using a MycoAlert Mycoplasma Detection Kit, Basel, Switzerland) and confirmed negative. All cell lines were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Biowest, Nuaillé, France) and 1% penicillin and streptomycin at 37°C with 5% CO2. WEHI and Bosc23 cells were kindly provided by Dr. Daniel G. Tenen (Harvard Medical School).
Reagents
CLN-081 was provided by Taiho Pharmaceutical Co., Ltd. Mobocertinib and pimitespib were purchased from MedChemExpress. Poziotinib, osimertinib, afatinib, and erlotinib were purchased from LC Laboratories.
Genetically Engineered Cell Lines
Ba/F3 cells harboring A763_Y764insFQEA, Y764_V765 insHH, or A767_S768insSVD were established as we previously reported.
The C797S (c.2390G>C) mutation was introduced in MIGR1 constructs harboring A763_Y764insFQEA, Y764_V765 insHH, or A767_S768insSVD using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). Ba/F3 cells harboring A763_Y764insFQEA+C797S, Y764_V765 insHH+C797S, or A767_S768insSVD+C797S cells were generated by retroviral transduction, as described.
Ba/F3 stably transduced cells were maintained in RPMI supplemented with 10% fetal bovine serum, 5% WEHI conditional medium (as a source of interleukin-3), and 1% penicillin/streptomycin. Oligonucleotides used for mutagenesis and primers used for sequencing in this study are listed in Supplementary Table 1.
Generation of Resistant Clones Through Chronic Exposure to CLN-081
Ba/F3 cells harboring Y764_V765 insHH or A767_S768insSVD were cultured in increasing CLN-081 concentrations, from 10 nM to 2000 nM. Resistant clones were established using limiting dilution.
Cell Viability Assay
Ba/F3 cells (10,000 cells per well) were seeded in 96-well plates, allowed to adhere overnight, treated with inhibitors of interest for 48 hours, and then evaluated for viability using either a CellTiter-Glo2 Cell Viability Assay (Promega, WI) or a Cell Counting Kit-8 (Fujifilm, Japan), according to manufacturers’ instructions. Plates were captured using the Spectra Max Paradigm (Molecular Devices, CA) with SoftMax Pro software (version 7.10). Concentration that inhibits 50% (IC50) values were calculated with a nonlinear regression model (four parameter) using GraphPad Prism software (version 9.0.2).
Apoptosis Assay
Apoptosis was assessed using an annexin V-FITC apoptosis detection kit (Nacalai Tesque, Kyoto, Japan). Briefly, Ba/F3 cells were treated with erlotinib or CLN-081 for 24 hours and stained with annexin V-FITC and propidium iodide (PI) antibodies for 15 minutes at room temperature. Stained cells were analyzed on FACSCanto II (BD Biosciences, Franklin Lakes, NJ). Data were further analyzed using FlowJo software (version 10.7.1). The proportion of cells in early (annexin V+/PI−) or late (annexin V+/PI+) apoptotic phases was analyzed.
Cells were lysed in sodium dodecyl sulfate sample buffer and boiled for 5 minutes. Lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted on polyvinylidene fluoride membranes (Millipore, Burlington, MA). Antibodies and dilutions used in the experiments are listed in Supplementary Table 1. Images were captured using ImageQuant LAS 4000 (GE Healthcare, IL) and assessed using ImageJ software version 1.53 (National Institutes of Health, Bethesda, MD). All images were assembled and figures were generated using Affinity Designer version 1.10.4 (Serif, Nottingham, United Kingdom).
Xenograft Experiments
Athymic nude mice (BALB/cAJcl-Foxn1nu, CLEA, Tokyo, Japan) were housed on a 12:12 light/dark cycle, and temperature was kept at 23°C to 25°C and humidity at 40% to 60%. Ba/F3 cells transduced with A763_Y764insFQEA-C797S (3 × 106 cells) were transplanted into the flanks of the nude mice to establish tumor xenografts. To evaluate pimitespib efficacy, we used the nude mice ranging from 4 to 5 weeks of age and bearing A767_S768insSVD-C797S or A763_Y764insFQEA-C797S tumors. Pimitespib was formulated in 0.5% hydroxypropyl methylcellulose in H2O, CLN-081 was formulated in 0.1N hydrochloric acid solution, and erlotinib was formulated in 5% DMSO plus 45% polyethylene glycol 300 in H2O. When the mean tumor volume reached 100 to 200 mm3, mice were orally treated with pimitespib (14 mg/kg once daily), CLN-081 (100 mg/kg once daily), erlotinib (50 mg/kg once daily), or vehicle control once daily five times a week. Tumor size and body weight were measured as indicated. Tumor volume was calculated using the following formula: 1/2 × (length × width2).
All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Cancer Center Japan (K20-009).
Statistics and Reproducibility
Each experiment was repeated three or more times with similar results, unless otherwise noted. Statistical analysis was conducted on data from three or more biologically independent experimental replicates. Student’s t test (two-tailed) was used to determine statistical significance between the two groups. The threshold for significance was p less than 0.05. All statistical analyses were conducted using GraphPad Prism software (version 9.0.2).
Results
Establishment of CLN-081–Resistant Ba/F3 Clones
To identify mechanisms conferring CLN-081 resistance, we used Ba/F3 cells harboring Y764_V765 insHH (Ba/F3-insHH) or A767_S768insSVD (Ba/F3-insSVD) and established CLN-081–resistant clones (Ba/F3-insHH-CR or Ba/F3-insSVD-CR, respectively) by treating cells with gradually increasing CLN-081 concentrations. After two months of drug escalation, cells grew in media containing 2000 nM CLN-081. The cells were then subjected to limiting dilution, and six clones were established for each insertion. Relative to the parental lines, the resistant clones exhibited 4-fold to 10-fold higher IC50 than that of the parent cells in Ba/F3-insHH and exhibited 500- to 13,000-fold higher IC50 in Ba/F3-insSVD (Fig. 1A). Sanger sequencing revealed that all Ba/F3-insHH-CR or Ba/F3-insSVD-CR clones acquired the C797S (c.2390G>C) point mutation (Fig. 1B).
Figure 1Chronic exposure to CLN-081 induces the C797S mutation in Ba/F3 cells harboring EGFR exon 20 insertions. (A) Parental and CLN-081–resistant Ba/F3 clones (Ba/F3-CR) expressing Y764_V765insHH or A767_S768insSVD were seeded on 96-well plates and treated with various concentrations of CLN-081 for 48 hours. Cell viability was measured using the Cell Counting Kit-8. Results are illustrated as means ± SD from three independent experiments. CLN-081 sensitivity of Ba/F3-insHH-CR or Ba/F3-insSVD-CR cells was higher than that of the parental cells. (B) Sequencing chromatogram of RT-PCR products from parental and CLN-081–resistant Ba/F3 clones expressing insHH or insSVD. Note that all resistant clones exhibit the C797S (c.2390G>C) point mutation in both insertions. #, number; RT-PCR, reverse-transcriptase polymerase chain reaction.
To verify whether C797S confers CLN-081 resistance, we generated Ba/F3-insHH-C797S and Ba/F3-insSVD-C797S cells. Cell viability assays revealed that respective CLN-081 IC50 values for Ba/F3-insHH-C797S and Ba/F3-insSVD-C797S were 1455-fold (80 nM versus 158 μM) and 883-fold (29 nM versus 16.9 μM) higher than values found in the parental cells (Fig. 2A). We also observed that EGFR phosphorylation levels were unchanged in Ba/F3-insHH-C797S or Ba/F3-insSVD-C797S by CLN-081 treatment, whereas Ba/F3 parental cells treated with 0.1 μM CLN-081 had decreased levels of phosphorylated EGFR relative to untreated controls (Fig. 2B). Next, we asked whether other EGFR TKIs could overcome drug resistance caused by the C797S mutation. Cell viability assays revealed that mobocertinib or poziotinib, which are EGFR TKIs targeting EGFR exon 20 insertions, did not inhibit growth of Ba/F3-insHH-C797S cells and Ba/F3-insSVD-C797S cells (Fig. 2C). We also tested first- (erlotinib), second- (afatinib), and third-generation (osimertinib) EGFR TKIs, and none overcame drug resistance in Ba/F3-insHH-C797S and Ba/F3-insSVD-C797S cells (Fig. 2C).
Figure 2The C797S mutation confers resistance to EGFR TKIs targeting EGFR exon 20 insertions. (A) Cell viability assay of Ba/F3-insHH cells, with or without C797S (left), or Ba/F3-insSVD cells, with or without C797S (right), treated with or without varying CLN-081 concentrations for 48 hours. (B) Western blotting revealing phospho- and total EGFR levels after CLN-081 treatment of Ba/F3-insHH, Ba/F3-insHH-C797S, Ba/F3-insSVD, or Ba/F3-insSVD-C797S cells. All cells were incubated for 3 hours with CLN-081 at indicated concentrations. (C) Cell viability assay using Ba/F3-insHH, Ba/F3-insHH-C797S, Ba/F3-insSVD, or Ba/F3-insSVD-C797S cells treated with various concentrations of EGFR TKIs for 48 hours. pEGFR, phosphorylated EGFR; TKI, tyrosine kinase inhibitor.
we hypothesized that HSP90 inhibition could overcome resistance caused by the C797S mutation in tumors with EGFR exon 20 mutations. Western blotting analysis revealed that treatment with the selective HSP90 inhibitor pimitespib at 1 μM decreased levels of total and phosphorylated EGFR in Ba/F3-insSVD cells, in the presence or absence of C797S (Fig. 3A). After 24 hours of pimitespib treatment, Western blotting revealed decreased levels of EGFR and AKT, which are client proteins HSP90, and induction of cleavage of full-length PARP and induction of a nonphosphorylated, stabilized form of Bim in Ba/F3-insSVD-C797S at 1 μM (Fig. 3B). Cell viability assays revealed that pimitespib IC50 values were less than 0.6 μM in Ba/F3-insHH, insHH-C797S, insSVD, and insSVD-C797S cells (Fig. 3C). Pimitespib IC50 values in C797S mutant Ba/F3 cells were consistent with those found in gastrointestinal stromal tumor cells, in which pimitespib-treated patients had prolonged survival relative to placebo controls.
Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial.
To further evaluate pimitespib activity against Ba/F3-insSVD-C797S tumors in vivo, we subcutaneously injected Ba/F3-insSVD-C797S cells into the flanks of nude mice and treated animals with pimitespib, CLN-081, or vehicle for 2 weeks once tumor volumes reached 100 to 200 mm3 (n = 10 for pimitespib and CLN-081; n = 4 for vehicle). Pimitespib treatment inhibited tumor growth (Fig. 3D) and prolonged mouse survival relative to either vehicle or CLN-081 treatment (Fig. 3E).
Figure 3HSP90 inhibition overcomes CLN-081 resistance conferred by C797S mutation. (A) Western blotting revealing phosphor- and total EGFR levels after pimitespib treatment of Ba/F3-insSVD or Ba/F3-insSVD-C797S cells. All cells were incubated 3 hours with pimitespib at indicated concentrations. (B) Western blotting of factors downstream of EGFR after pimitespib treatment of Ba/F3-insSVD-C797S cells. All cells were incubated 24 hours with pimitespib at indicated concentrations. (C) Cell viability assay of Ba/F3-insHH, Ba/F3-insHH-C797S, Ba/F3-insSVD, or Ba/F3-insSVD-C797S cells treated 48 hours with pimitespib. (D) Effects of pimitespib, CLN-081, or vehicle in mouse xenograft tumor models harboring Ba/F3-insSVD-C797S cells. Nude mice bearing Ba/F3-insSVD-C797S cells were treated with pimitespib (n = 10: 14 mg/kg orally once daily), CLN-081 (n = 10: 100 mg/kg orally once daily), or vehicle (n = 4) for 14 days and monitored for changes in tumor volume. Data are presented as the mean % change in tumor volume ± SD. Asterisks indicate p value: ∗ < 0.05. NS, not significant. (E) Survival of mice (%) described in (D). pAKT, phosphorylated AKT; pEGFR, phosphorylated EGFR; pERK, phosphorylated ERK.
Expectedly, Ba/F3-insFQEA-C797S cells had resistance to CLN-081, mobocertinib, poziotinib, and other EGFR TKIs (Fig. 4A). Nevertheless, Ba/F3-insFQEA-C797S cells remained sensitive to erlotinib (Fig. 4A). The erlotinib IC50 ratio for Ba/F3-insFQEA relative to Ba/F3-insFQEA-C797S cells was 1.57, whereas comparable IC50 ratios of other EGFR TKIs were more than 1000 (Fig. 4B), suggesting erlotinib has inhibitory activity against these mutant cells. Although poziotinib and afatinib had lower IC50 values than erlotinib against Ba/F3-insFQEA-C797S, they are too toxic considering the previously reported IC50 values to Ba/F3-EGFR wild type.
TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically relevant EGFR mutations.
Next, we asked whether erlotinib inhibition of EGFR kinase activity would alter signaling of effectors regulating cell proliferation or apoptosis. To do so, we treated Ba/F3-insFQEA-C797S cells with various concentrations of erlotinib for 16 hours and performed Western blotting with antibodies found in Fig. 4C. That analysis indicated decreased phosphorylation of EGFR, AKT, and ERK relative to untreated controls and induction of cleavage of full-length PARP and induction of a nonphosphorylated, stabilized form of Bim at erlotinib concentrations as low as 0.1 to 1 μM, consistent with the cell viability assay. Accordingly, an apoptosis assay using annexin V staining indicated that erlotinib induced Ba/F3-insFQEA-C797S cell apoptosis (Fig. 4D), suggesting erlotinib may shrink tumors in vivo by apoptosis in this mutant model. To further evaluate erlotinib activity against Ba/F3-insFQEA-C797S cell tumors in vivo, we subcutaneously injected Ba/F3-insFQEA-C797S cells into the flanks of nude mice and treated them with erlotinib or vehicle for 2 weeks once tumor volumes reached 100 to 200 mm3 (n = 6 for each group). Relative to vehicle-treated controls, erlotinib treatment decreased tumor volume (Fig. 4E) and weight (Fig. 4F), consistent with the cell viability assay.
Figure 4Cells harboring EGFR A763_Y764insFQEA remain erlotinib sensitive. (A) Cell viability assay of Ba/F3-insFQEA or Ba/F3-insFQEA-C797S cells treated with various concentrations of EGFR TKIs for 48 hours. (B) IC50 ratios (Ba/F3-insFQEA-C797S relative to Ba/F3-insFQEA) of indicated drugs based on cell viability assays described in (A). (C) Western blotting revealing EGFR and downstream signaling factors after erlotinib treatment of Ba/F3-insFQEA-C797S cells. All cells were incubated 24 hours with pimitespib at indicated concentrations. (D) Apoptosis assay using annexin V staining of Ba/F3-insFQEA-C797S cells treated with CLN-081, erlotinib, or vehicle control for 24 hours. Data are illustrated as means ± SD. Asterisks indicate the p value: ∗∗∗ < 0.005. (E) Effects of erlotinib or vehicle in mouse xenograft tumor models harboring Ba/F3-insFQEA-C797S cells. Nude mice bearing Ba/F3-insFQEA-C797S cells were treated with erlotinib (n = 6: 50 mg/kg orally once daily) or vehicle (n = 6) for 12 days and monitored for changes in tumor volume. Tumor volume is illustrated as the mean ± SD. (F) Weight of tumors in mice analyzed in (E). Values are presented as means ± SD. IC50, Concentration that inhibits 50%; pAKT, phosphorylated AKT; pEGFR, phosphorylated EGFR; pERK, phosphorylated ERK; TKI, tyrosine kinase inhibitor.
In this study, we investigated potential mechanisms that could explain acquired resistance to CLN-081, a novel EGFR inhibitor that targets EGFR exon 20 insertion mutations. Using preclinical models, we found that the EGFR C797S mutation confers acquired resistance to CLN-081, which can be overcome by treatment with pimitespib, a selective HSP90 inhibitor, in vitro and in vivo. In contrast, the C797S mutation in the context of the “classical-like” EGFR FQEA insertion (A763_Y764 insFQEA) failed to render cells resistant to erlotinib.
A recent phase 1/2a study of CLN-081 was found to have promising results, with median progression-free survival of 12 months and an ORR of 41% to NSCLC harboring EGFR exon 20 insertion mutations.
Nevertheless, clinical information defining mechanisms of resistance to CLN-081 has yet to be identified. In terms of other inhibitors targeting EGFR exon 20 insertion mutations, investigation of poziotinib resistance in 14 clinical specimens revealed T790M to be a major on-target acquired mutation.
By analyzing CLN-081–resistant Ba/F3 cells harboring exon 20 insertion mutations (Y764_V765insHH or A767_S768insSVD), we detected an acquired C797S mutation in all resistant clones. The observance that CLN-081, osimertinib, mobocertinib, poziotinib, and afatinib covalently bind to C797 at the EGFR ATP-binding site
strongly suggests that emergence of the C797S mutation may promote clinical resistance to these compounds. Indeed, C797S accounts for approximately 7% of patients with NSCLC resistant to first-line osimertinib,
Preclinical characterization of mobocertinib highlights the putative therapeutic window of this novel EGFR inhibitor to EGFR exon 20 insertion mutations.
Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer (Science Translational Medicine (2014) 6, (225er1)).
exhibits a sensitivity profile similar to major mutations such as exon 19 deletion and exon 21 L858R mutation and is thus classified as a “classical-like” type. We found that cells harboring insFQEA-C797S remained sensitive to erlotinib, suggesting that tumors with insFQEA
Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer (Science Translational Medicine (2014) 6, (225er1)).
could be treated by early generation EGFR inhibitors such as erlotinib, either alone or in combination with CLN-081. Further studies are required to determine the optimal treatment for lung cancers with insFQEA.
HSP90 inhibitors were found to have antitumor activity in preclinical models by destabilizing HSP90 client proteins.
The fact that mutated EGFR proteins are more sensitive to HSP90 inhibition than are wild-type EGFR proteins has prompted investigations of whether HSP90 inhibition would exert an antitumor effect after emergence of EGFR TKI resistance.
Indeed, HSP90 inhibition has antitumor activity in vitro in EGFR-mutant cells resistant to EGFR TKIs owing to EGFR amplification, MET amplification, or HGF-MET activation.
Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors.
Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors.
We revealed that Ba/F3-insSVD cells were sensitive to pimitespib, regardless of C797S. Pimitespib has a high selectivity for HSP90 and maintains antitumor activity with minimal ocular toxicity,
TAS-116, a highly selective inhibitor of heat shock protein 90α and β, demonstrates potent antitumor activity and minimal ocular toxicity in preclinical models.
Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial.
Further clinical investigations are warranted to determine whether pimitespib overcomes resistance to CLN-081 in tumors with EGFR exon 20 insertion mutations.
The main limitation of this study is lack of clinical information. The frequency of C797S mutation after CLN-081 treatment is to be investigated using clinical samples. Furthermore, other on-target resistance and off-target resistance mechanisms could be detected in clinical specimens resistant to CLN-081. Nevertheless, we believe that the current results provide important insights into treatment strategy to tackle the C797S resistant mutation using pimitespib.
In summary, our preclinical studies identified acquisition of the C797S mutation as a potential mechanism of acquired resistance to CLN-081. Development of novel strategies to overcome CLN-081 resistance will benefit patients with lung cancer with EGFR exon 20 insertion mutations.
Takuma Hayashida, Jie Liu, Shunta Mori, Shogo Kumagai: Investigation.
Hibiki Udagawa: Methodology and Conceptualization.
Noboru Hattori and Koichi Goto: Writing—review and editing.
Susumu S. Kobayashi: Conceptualization, Investigation, Methodology, Formal analysis, Writing—original draft, Visualization, Supervision, Funding acquisition, and Project administration.
Acknowledgments
This work was supported by Taiho Pharmaceutical Co., Ltd. (C2020-097), the Princess Takamatsu Cancer Research Fund 18-250 (Dr. Kobayashi), JSPS KAKENHI Grant Number JP20K17215 (Dr. Izumi), 16K21746 (Dr. Kobayashi), JPJSBP120207408 (Dr. Kobayashi) and 22H03084 (Dr. Kobayashi), and the National Cancer Center Research and Development Fund 31-A-6 (Dr. Kobayashi).
Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.
Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.
Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer (Science Translational Medicine (2014) 6, (225er1)).
Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials.
TAS6417/CLN-081 is a pan-mutation-selective EGFR tyrosine kinase inhibitor with a broad spectrum of preclinical activity against clinically relevant EGFR mutations.
Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial.
Preclinical characterization of mobocertinib highlights the putative therapeutic window of this novel EGFR inhibitor to EGFR exon 20 insertion mutations.
Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors.
TAS-116, a highly selective inhibitor of heat shock protein 90α and β, demonstrates potent antitumor activity and minimal ocular toxicity in preclinical models.
Disclosure: Dr. Izumi reports receiving grants from Amgen, Ono, Takeda, and Eisai and personal fees from Ono, Chugai, AstraZeneca, Merck, and Takeda. Dr. Kumagai reports receiving personal fees from Merck Sharp & Dohme and Chugai. Dr. Udagawa reports receiving grants from Takeda and Boehringer Ingelheim. Dr. Hattori reports receiving personal fees from Boehringer Ingelheim and AstraZeneca. Dr. Goto reports receiving grants from Merck, Takeda Pharmaceutical, Amgen, Astellas, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Janssen, Kyowa Kirin, Lilly, Medical & Biological Laboratories, Merck Sharp & Dohme, Novartis, Ono, Pfizer, Sumitomo Dainippon, Bayer, Haihe Biopharma, Ignyta, Kissei, Life Technologies Japan, Loxo Oncology, Merus, Pfizer, Spectrum Pharmaceuticals, Sysmex Corporation, Turning Point Therapeutics, and Taiho; grants and personal fees from Amgen, Astellas, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Blueprint Medicines, Chugai, Daiichi Sankyo, Eisai, Eli Lilly, Janssen, Merck, MS, Novartis, Ono, Taiho, and Takeda; and personal fees from Amoy Diagnostics, Bayer, Guardant Health, Thermo Fisher Scientific, Medpace, and Otsuka. Dr. Kobayashi reports receiving research support from Boehringer Ingelheim, MiRXES, Johnson & Johnson, and Taiho Therapeutics and personal fees (honoraria) from AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Chugai Pharmaceutical, and Takeda Pharmaceuticals. The remaining authors declare no conflict of interest.
Cite this article as: Kagawa Y, Hayashida T, Liu J, et al. The EGFR C797S mutation confers resistance to a novel EGFR inhibitor CLN-081 to EGFR exon 20 insertion mutations. JTO Clin Res Rep. 2023;3:100462.
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