Epidermal Growth Factor Receptor C797S Mutation Research Articles

Targeting pyruvate dehydrogenase kinase 1 overcomes EGFR C797S mutation-driven osimertinib resistance in non-small cell lung cancer

Osimertinib, a selective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), effectively targets the EGFR T790M mutant in non-small cell lung cancer (NSCLC). However, the newly identified EGFR C797S mutation confers resistance to osimertinib. In this study, we explored the role of pyruvate dehydrogenase kinase 1 (PDK1) in osimertinib resistance. Patients exhibiting osimertinib resistance initially displayed elevated PDK1 expression. Osimertinib-resistant cell lines with the EGFR C797S mutation were established using A549, NCI-H292, PC-9, and NCI-H1975 NSCLC cells for both in vitro and in vivo investigations. These EGFR C797S mutant cells exhibited heightened phosphorylation of EGFR, leading to the activation of downstream oncogenic pathways. The EGFR C797S mutation appeared to increase PDK1-driven glycolysis through the EGFR/AKT/HIF-1α axis. Combining osimertinib with the PDK1 inhibitor leelamine helped successfully overcome osimertinib resistance in allograft models. CRISPR-mediated PDK1 knockout effectively inhibited tumor formation in xenograft models. Our study established a clear link between the EGFR C797S mutation and elevated PDK1 expression, opening new avenues for the discovery of targeted therapies and improving our understanding of the roles of EGFR mutations in cancer progression.

Abstract LB056: IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors

Abstract Many solid tumors, such as colorectal cancer (CRC) and lung cancer, are dependent on epidermal growth factor receptor (EGFR) pathway for tumor growth. Antibodies that inhibit EGFR signaling, such as Cetuximab, show clinical efficacy but are associated with on-target toxicities that potentially limit the drug efficacy. To enhance the EGFR signaling inhibition and reduce on-target toxicities, we performed bioinformatics analysis for EGFR/TAA co-expression. We found that B7-H3, a member of the B7 family of immune checkpoint proteins, is broadly expressed in many solid tumors and their expression correlates with EGFR expression in multiple cancers, including lung cancer, head and neck cancer, pancreatic cancer and esophageal cancer. Using Innovent’s proprietary Innobody platform, we developed a bispecific antibody, IBI334, against B7-H3 and EGFR. IBI334 has an EGFR arm for signal blockage and is coupled with a fine-tuned B7-H3 arm with optimal affinity and binding domain. With the aid of B7-H3, IBI334 showed enhanced EGFR signal inhibition than Zalutumumab (EGFR mAb) and Amivantamab (c-met/EGFR bsAb). In addition, IBI334 is afucosylated to enhance its antibody-mediated cell cytotoxicity (ADCC) effects as compared to wild type antibody. IBI334 showed significantly better in vivo efficacy than Amivantamab in NCI-H358 (bronchioalveolar carcinoma with KRAS-G12C mutation), NCI-H292 (lymph node metastasis of a pulmonary mucoepidermoid carcinoma), SK-MES1 (lung cancer) and SK-MES1-LTC (lung cancer with EGFR L858R, T790M and C797S mutations) xenograft models and comparable efficacy in NCI-H322-EGFRexon20ins (lung cancer) model. Cetuximab, an EGFR monoclonal antibody, had high on-target toxicity with 50% early mortalities in cynomolgus monkeys at 120/75 mg/kg/week. With the careful design of B7-H3/EGFR bispecific antibody, IBI334 ameliorated the on-target toxicities and showed only slight hardening of the skin at the injection site at 120 mg/kg/week. As a result, IBI334 has a large therapeutic window of >200 folds. In conclusion, IBI334 is a potent B7-H3/EGFR bispecific antibody with broad application in many EGFR-driven solid tumors. The large therapeutic window enables safe and effective cancer treatment at high exposure levels. Citation Format: Jian Guan, Shuaixiang Zhou, Weiwei Wu, Tianyu Zhu, Lei Cao, Ming Wu, Ninghuan Li, Fenggen Fu, Zhengguan Liao, Shuming Lin, Zeyu Liu, Mengjia Zhu, Nana Luo, Ying Zhang, Tiong Sun Chia, Bingliang Chen, Kaijie He. IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB056.

Abstract 4668: Characterization of fourth-generation EGFR inhibitors in binding experiments with C797S mutant EGFR and cell-based assays with osimertinib-resistant non-small cell lung cancer cell lines

Abstract Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that inhibits the EGFR kinase domain by irreversible binding of the cysteine-797 residue (C797). Osimertinib was originally designed to target the T790M mutation, which is clinically the most frequently observed resistance mechanism to treatment with first- or second-generation EGFR inhibitors. Osimertinib has a high selectivity for the classical EGFR driver mutations (exon 19 deletion and L858R) compared to wild-type EGFR. Due to this favorable selectivity profile, osimertinib has become the standard first-line therapy for non-small cell lung cancer (NSCLC). Common resistance mechanisms following osimertinib treatment are MET oncogene amplification, and cysteine-to-serine substitution at position 797 (C797S) of EGFR, which precludes binding of osimertinib. Fourth-generation EGFR inhibitors and combinations with other targeted agents are potential strategies to overcome osimertinib resistance or to increase its efficacy. Three fourth-generation EGFR inhibitors were profiled in biochemical and cell-based assays: BDTX-1535, BLU-945, and JBJ-09-063. The binding kinetics for wild-type and C797S mutant EGFR were determined in surface plasmon resonance binding experiments on a Biacore 1S+ (Cytiva). The effect on osimertinib-resistant cells, generated using two different approaches, was studied in cell viability assays. In the first model, subclones of the NSCLC cell line PC-9, harboring an exon 19 deletion activating EGFR mutation, were selected for osimertinib resistance by in vitro drug selection [1]. In the second model, the EGFR C797S mutation was introduced into the NSCLC cell line NCI-H1975, which expresses both the L858R activating mutation and the T790M resistance mutation, by gene-editing with CRISPR-Cas9. Synergies with fourth-generation EGFR inhibitors were determined by profiling combinations in a 6-by-6 matrix, and quantifying synergistic effects using the Excess over Bliss score. We will present a detailed comparison of the binding kinetics of the three fourth-generation EGFR inhibitors on wild-type and C797S-mutant EGFR, and their ability to compete with osimertinib for binding to the kinase domain of EGFR. The different fourth-generation inhibitors show various selectivities for the various EGFR mutants and differences in their ability to spare wild-type EGFR. Profiling results of the three fourth-generation EGFR inhibitors in the osimertinib-resistant cell lines and on a panel of more than one hundred human cancer cell lines will be presented. Reference: [1] Bertran-Alamillo et al. Nature Communications 10, 1812; 2019 Citation Format: Janneke J. Melis, Kirsten Kevenaar, Jeffrey J. Kooijman, Yvonne Grobben, Jacob Ytsma, Jordi Bertran-Alamillo, Miguel-Angel Molina-Vila, Nicole Willemsen-Seegers, Guido J. Zaman. Characterization of fourth-generation EGFR inhibitors in binding experiments with C797S mutant EGFR and cell-based assays with osimertinib-resistant non-small cell lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4668.

Design and synthesis of 4th generation EGFR inhibitors against human triple (Del19/T790M/C797S) mutation

Epidermal growth factor receptor (EGFR)-targeted therapy is used to treat EGFR mutation-induced non-small cell lung cancer (NSCLC). However, its efficacy does not last beyond a certain period due to the development of primary and secondary resistance. First and second-generation inhibitors (e.g., gefitinib, erlotinib, and afatinib) induce EGFR T790M mutations, while third-generation inhibitors (e.g., osimertinib) induce C797S as a major target resistance mutation. Therefore, the C797S mutation is being actively researched. In this study, we investigated the structure-activity relationship of several synthesized compounds as fourth-generation inhibitors against the C797S mutation. We identified a compound 13k that displayed nanomolar potency and high selectivity. Moreover, we used a triple mutant xenograft mouse model to evaluate the in vivo efficacy of 13k in inhibiting EGFR C797S, which demonstrated exceptional profiles and satisfactory EGFR C797S inhibition efficacy. Based on its excellent in vitro and in vivo profiles, compound 13k can be considered a promising candidate for treating EGFR C797S mutations.

Diarylheptanoid 35d overcomes EGFR TKI resistance by inducing hsp70-mediated lysosomal degradation of EGFR in EGFR-mutant lung adenocarcinoma

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) patients often respond to EGFR tyrosine kinase inhibitors (TKIs) initially but eventually develop resistance to TKIs. The switch of EGFR downstream signaling from TKI-sensitive to TKI-insensitive is a critical mechanism-driving resistance to TKIs. Identification of potential therapies to target EGFR effectively is a potential strategy to treat TKI-resistant LUADs. In this study, we developed a small molecule diarylheptanoid 35d, a curcumin derivative, that effectively suppressed EGFR protein expression, killed multiple TKI-resistant LUAD cells invitro, and suppressed tumor growth of EGFR-mutant LUAD xenografts with variant TKI-resistant mechanisms including EGFR C797S mutations invivo. Mechanically, 35d triggers heat shock protein 70-mediated lysosomal pathway through transcriptional activation of several components in the pathway, such as HSPA1B, to induce EGFR protein degradation. Interestingly, higher HSPA1B expression in LUAD tumors associated with longer survival of EGFR-mutant, TKI-treated patients, suggesting the role of HSPA1B on retarding TKI resistance and providing a rationale for combining 35d with EGFR TKIs. Our data showed that combination of 35d significantly inhibits tumor reprogression on osimertinib and prolongs mice survival. Overall, our results suggest 35d as a promising lead compound to suppress EGFR expression and provide important insights into the development of combination therapies for TKI-resistant LUADs, which could have translational potential for the treatment of this deadly disease.

Dual kinase inhibitor for EGFR mutants and ErbB2 limit breast cancer

Mutations in the epidermal growth factor receptor (EGFR) have been found in more than 10% of non-small cell lung cancer (NSCLC) patients in North America. The vast majority of these differences are L858R point mutations in Exon 21. Currently, monoclonal antibodies directed against the extracellular domain of EGFR or small molecule/tyrosine kinase inhibitors (TKI) are the stalwarts of NSCLC therapy. Resistance, however, gradually develops because of the T790 mutation towards first and second generation TKIs. The third generation TKI AZD9291 (Osimertinib) has a high affinity for both activating and the acquired resistant mutation (T790 M) in EGFR, with a low affinity towards wild-type EGFR. Recent research, however, suggests that the EGFR (C797S) mutation in the tyrosine kinase domain is a likely cause of resistance to AZD9291. Another significant transformation mechanism associated with this resistance is erbB2 amplification. Our laboratory has developed a small kinase inhibitor, ER121 (MW: ∼500), that inhibits the erbB2/HER2 tyrosine kinases in addition to the EGFR C797S mutations. We have identified a TKI, ER121 targeting the mutant EGFR(T790 M). Using in vitro and in vivo models, examined the efficacy of ER121 on mutant EGFR cell lines. This has enabled us to establish that ER121 is well tolerated when administered orally and produces significant inhibitory activity against human cancers generated by mutant EGFR and amplified ErbB2.

Histomolecular Resistance Mechanisms to First-Line Osimertinib in EGFR-Mutated Advanced Non-Small Cell Lung Cancer: A Multicentric Retrospective French Study.

Osimertinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) used in first line for the treatment of advanced EGFR-mutated non-small cell lung cancer (NSCLC). The identification of related histomolecular resistance mechanisms to first-line osimertinib is a critical step to define the optimal treatment strategy beyond progression. All consecutive patients treated in the first line with osimertinib for advanced EGFR-mutated NSCLC at 10 hospitals in the Greater Paris area between April 2015 and January 2021 were included. Histomolecular data from plasma and tissue samples taken at progression under osimertinib were collected, and all samples were analyzed using DNA next-generation sequencing. Data on objective response rate (ORR), overall survival (OS), progression-free survival (PFS), and time to treatment discontinuation (TTD) were also collected. Overall, 104 patients were included. Most patients had adenocarcinoma (n=102, 98%) with an exon 19 EGFR deletion (n=54, 52%). Forty-two patients (50%) had central nervous system (CNS) metastasis at the time of osimertinib initiation. ORR was 76%, median PFS and OS were 12.6 months and 52 months, respectively, and TTD was 33 months. At the time of analysis, 44 patients (42%) had tumor progression, and among these patients, 27 (61%) contributive samples were available. The most frequent molecular alterations at progression were mesenchymal epithelial transition factor (MET) amplification (15%; n=4) and EGFR C797S mutation (11%; n=3). Histological transformation was found in one patient (4%). RNA next-generation sequencing was performed in eight patients and showed a CCDC6-RET fusion in one patient (12%). We confirmed the efficacy of osimertinib in patients with advanced EGFR mutation-positive NSCLC. At progression, the most frequent histomolecular alterations were MET amplification and EGFR C797S mutation.

MA07.08 JIN-A02, a Highly Effective 4th Generation EGFR-TKI, Targeting EGFR C797S Triple Mutation in NSCLC

Among non-small cell lung cancer (NSCLC) gene mutations, epidermal growth factor receptor (EGFR) mutations are the most common and occur in approximately 50% of Asian NSCLC.1) Although EGFR-targeted therapeutics have been widely used for the treatment of EGFR-mutant lung cancer, acquired resistance invariably develops after 12-18 months in patients.2) EGFR C797S mutation, located within the tyrosine kinase domain, was recently reported to be a potential mechanism of resistance to irreversible EGFR inhibitors.

999P JIN-A02, a fourth-generation, highly effective tyrosine kinase inhibitor with intracranial activity, targeting EGFR C797S mutations in NSCLC

Even though epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have improved treatment outcomes for EGFR-driven NSCLC, resistance inevitably emerges and disease often progresses due to brain metastasis. The C797S is the most significant resistance mutation to occur after 3rd generation TKIs treatment. JIN-A02 is an orally available EGFR-TKI targeting C797S mutation with high brain penetration. Cellular activities of JIN-A02 were evaluated on phosphorylation-EGFR expression with AlphaLISA assay and on EGFR mutants with patients-derived cell (PDC) and patients-derived organoid (PDO). Different type of allelic relationship was also assessed in all models by whole exome sequencing. Patients-derived xenografts (PDX) was used to assess antitumor activities of JIN-A02. For in vivo intracranial activity, NCI-H1975 cell line derived tumor xenograft was used. In AlphaLISA assay, JIN-A02 showed high potency in EGFRex19del/T790M/C797S (IC50=4.7 nM, Ba/F3), EGFRL858R/T790M/C797S (IC50=12.8 nM, NCI-H1975 LTC). JIN-A02 strongly inhibited cellular activity of in trans model (IC50=89.7 nM, PDO) and in cis model (IC50=92.1 nM, PDC) of EGFRex19del/T790M/C797S. It appears to be superior to osimertinib (IC50 >4,000 nM for both cells). In addition, JIN-A02 showed comparable potency to osimertinib in EGFRex19del/T790M (IC50=84.4 nM for PC-9GR), and EGFRL858R/T790M (IC50=73.3 nM for NCI-H1975). In the PDX mouse model (YHIM-1094, EGFRex19del/T790M/C797S), JIN-A02 delayed tumor growth at a dose of 30 mg/kg. In the brain metastases model, antitumor activity of JIN-A02 was observed with intracranial implanted NCI-H1975 tumors. JIN-A02 is highly potent EGFR-TKI for various EGFR targeted mutations including in cis and trans EGFRex19del/T790M/C797S and shows intracranial activity. Based on these robust activities for EGFR mutants, JIN-A02 is expected to provide a potential therapeutic opportunity for NSCLC.

Abstract 5457: QLH11811, a selective 4th-generation EGFR inhibitor for osimertinib-resistant EGFR-mutant NSCLC

Abstract Background: The 3rd-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), like osimertinib, provide marked clinical benefit for EGFR-mutant non-small cell lung cancer (NSCLC) patients with extended overall survival vs former EGFR TKIs (e.g., gefitinib). Approximate 10%-24% of NSCLC patients acquired C797S mutation when treated with osimertinib. Unfortunately, limited treatments are available for patients after osimertinib resistance. QLH11811 is a new generation EGFR TKI designed to target the EGFR with ex19del/L858R/T790M/C797S mutations. Here, we disclosed its preclinical data to support its clinical development in EGFR-mutant NSCLC. Methods: The inhibitory activity of QLH11811 on mutated and wild-type EGFR was tested in engineered cell lines and patient-derived organoid (PDO). The in vivo antitumor activity of QLH11811 was evaluated in the patient-derived xenograft (PDX) model with cis EGFR ex19del/T790M/C797S triple mutations, and the H1975 (cis EGFR L858R/T790M/C797S and cis EGFR ex19del/T790M/C797S), PC-9 (EGFR ex19del), and Ba/F3 (EGFR ex19del/C797S) cell line-derived xenograft (CDX) models. The pharmacokinetic (PK) profile was investigated in animals, and the human PK profile was projected using allometric scaling method. Results: QLH11811 displayed potent anti-proliferation activity against Ba/F3 (EGFR ex19del/T790M/C797S, L858R/T790M/C797S, ex19del/C797S, or L858R/C797S), PC-9 (EGFR ex19del/T790M/C797S), H1975 (EGFR L858R/T790M/C797S, L858R/T790M), H3255 (L858R) and HCC827 (ex19del) with IC50 of 2.6, 3.1, 2.4, 4.1, 51, 50, 27, 21, and 11 nM, respectively. QLH11811 also showed excellent selectivity when compared the above values with its IC50 against Ba/F3 (EGFR wild-type, 61 nM) and A431 (EGFR wild-type, 440nM). QLH11811 demonstrated excellent inhibitory activities against seven osimertinib-resistant PDO models. Daily oral QLH11811 significantly inhibited tumor growth at all doses tested (P <0.001) in the PDX model, the H1975, PC-9, and Ba/F3 CDX models. QLH11811 had good PK profile in mice, rats, dogs, and monkeys, with the absolute bioavailability at 71%, 29%, 80% and 42%. The human PK parameters were obtained by allometric scaling method, and the efficacious dose in human was projected to 103 mg, daily. Conclusion: The in vitro and in vivo preclinical data demonstrated QLH11811 is a highly potent and selective 4th-generation EGFR TKI with activity against the osimertinib-resistant NSCLC with EGFR C797S mutation. The preclinical PK data supported the efficacious dose of QLH11811 in human would be 103 mg. Citation Format: Shansong Zheng, Wei Deng, Qingmei Zheng, Yingying Yang, Na Li, Tan Pang, Xueying Feng, Simon Taylor, Lina Ma, Yaqiong Wu, Ziwei Zhao. QLH11811, a selective 4th-generation EGFR inhibitor for osimertinib-resistant EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5457.

An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

Design, Synthesis, and Biological Evaluation of Novel EGFR PROTACs Targeting Del19/T790M/C797S Mutation.

The tertiary epidermal growth factor receptor (EGFR) C797S mutation predominates in the acquired mutational resistance in cancer patients to third-generation EGFR inhibitors. Small-molecule inhibitors targeting the EGFR C797S mutation have been developed with good efficiency. However, these compounds may still induce new EGFR mutations to evade the inhibition pathway. One EGFR protein degrader based on an allosteric inhibitor has shown some benefits of degrading the EGFR L858R/T790M/C797S triple mutant. However, the degrader of the other important triple EGFR mutation Del19/T790M/C797S has not been reported. Here we present the design and synthesis of a series of EGFR proteolysis-targeting chimeras (PROTACs) that can rapidly and potently induce EGFR degradation in Ba/F3 cells expressing the EGFRDel19/T790M/C797S mutant. One representative compound 6h time- and dose-dependently induced EGFR degradation with a DC50 of 8 nM. It also showed good antiproliferation activity (IC50 = 0.02 μM) against Ba/F3-EGFRDel19/T790M/C797S cells. 6h may serve as a lead compound to develop therapeutic agents for the treatment of resistant non-small cell lung cancer patients with EGFR C797S mutants.

Growth Suppression in Lung Cancer Cells Harboring EGFR-C797S Mutation by Quercetin.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved treatments for non-small-cell lung cancer (NSCLC) patients harboring activating EGFR mutations. The EGFR C797S mutation is one of the known acquired-resistance mutations to the latest third-generation TKIs. At present, there are no clear options for treating patients who acquire resistance to third-generation TKIs. The acquisition of the EGFR C797S mutation was shown to upregulate the expression of AXL, a receptor tyrosine kinase of the TAM (TYRO3-AXL-MER) family, and the suppression of AXL is effective in reducing the growth of NSCLC cells harboring EGFR C797S. As quercetin was recently shown to inhibit AXL, quercetin may be effective in treating NSCLC cells harboring the EGFR C797S mutation. In this work, the cytotoxic effects of quercetin and its ability to inhibit tumor growth were examined in TKI-resistant NSCLC cells harboring the EGFR C797S mutation. We demonstrated that quercetin exhibited potent cytotoxic effects on NSCLC cells harboring the EGFR C797S mutation by inhibiting AXL and inducing apoptosis. Quercetin inhibited the tumor growth of xenografted NSCLC cells harboring the EGFR C797S mutation and appeared to act synergistically with brigatinib to inhibit of tumor growth in vivo. In summary, herein, we revealed that quercetin is an effective inhibitor for the treatment of non-small-cell lung cancer harboring the EGFR C797S mutation.

Integrated Omics Analysis of Non-Small-Cell Lung Cancer Cells Harboring the EGFR C797S Mutation Reveals the Potential of AXL as a Novel Therapeutic Target in TKI-Resistant Lung Cancer.

Simple SummaryIn this study, we employed CRISPR/Cas9 editing technology to introduce the EGFR C797S mutation into an NSCLC cell line harboring EGFR L858R/T790M to establish a cellular model for the investigation of the resistance mechanism associated with the acquired C797S mutation and to explore strategies to battle this type of TKI resistance. Transcriptome and proteome analyses revealed that the differentially expressed genes/proteins in the cells harboring the EGFR C797S mutation are associated with elevated expression of AXL. Furthermore, we presented evidence that inhibition of AXL is effective in slowing the growth of NSCLC cells harboring EGFR C797S. Our findings suggest that AXL inhibition could be a second-line or a potential adjuvant treatment for NSCLC harboring the EGFR C797S mutation.Oncogenic mutations of epidermal growth factor receptor (EGFR) are responsive to targeted tyrosine kinase inhibitor (TKI) treatment in non-small-cell lung cancer (NSCLC). However, NSCLC patients harboring activating EGFR mutations inevitably develop resistance to TKIs. The acquired EGFR C797S mutation is a known mechanism that confers resistance to third-generation EGFR TKIs such as AZD9291. In this work, we employed CRISPR/Cas9 genome-editing technology to knock-in the EGFR C797S mutation into an NSCLC cell line harboring EGFR L858R/T790M. The established cell model was used to investigate the biology and treatment strategy of acquired EGFR C797S mutations. Transcriptome and proteome analyses revealed that the differentially expressed genes/proteins in the cells harboring the EGFR C797S mutation are associated with a mesenchymal-like cell state with elevated expression of AXL receptor tyrosine kinase. Furthermore, we presented evidence that inhibition of AXL is effective in slowing the growth of NSCLC cells harboring EGFR C797S. Our findings suggest that AXL inhibition could be a second-line or a potential adjuvant treatment for NSCLC harboring the EGFR C797S mutation.

Genetic alterations in epidermal growth factor receptor-tyrosine kinase inhibitor-naïve non-small cell lung carcinoma.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are an approved first-line therapy against unresectable or advanced non-small cell lung cancer (NSCLC) harboring EGFR gene activating mutations. However, the majority of tumors develop acquired resistance against EGFR-TKIs and some tumors exhibit natural resistance. A number of resistance mechanisms against the latest third-generation EGFR-TKIs have been reported, including tertiary EGFR C797S mutation and several gene alterations activating EGFR or other signaling pathways. The current study aimed to identify the frequency of natural EGFR-TKI resistance in pretreatment NSCLC and to predict the therapeutic effect of EGFR-TKIs. A total of 246 EGFR-TKI-naïve NSCLC patients harboring known EGFR gene mutations were identified. The presence of EGFR C797S and T790M mutations were determined using the peptide nucleic acid-locked nucleic acid PCR clamp method. ERBB2, MET, EGFR, ALK, BRAF, FGFR1, MYC, RET, CCND1, CCND2, CDK4, CDK6, MDM2 and MDM4 gene amplification, which can lead to resistance against any generation EGFR-TKIs, was determined using the multiplex ligation-dependent probe amplification assay. No concurrent C797S mutation with known EGFR mutations were identified. T790M mutation was identified in 12 patients (4.9%). ERBB2 or MET gene amplification was found in some patients (0.0-0.4%). MDM2 gene amplification was associated with tumor recurrence and shorter progression-free survival (PFS) for first- or second-generation EGFR-TKIs. De novo EGFR C797S mutation was not identified. Other resistance mechanisms against EGFR-TKIs were indicated in some patients with EGFR-TKI-naïve NSCLC. MDM2 gene amplification, which can lead to altered cell cycle, was associated with tumor recurrence and shorter PFS in EGFR-TKI therapy.

Abstract B004: Revealing the mechanisms of acquired resistance to osimertinib from re-biopsy specimens in advanced non-small cell lung cancer with EGFR T790M mutation (LOGIK1607)

Abstract Background: Although treatment with osimertinib confers survival benefits in the second-line treatment of epidermal growth factor receptor (EGFR)-mutated lung cancer with the EGFR T790M mutation, the mechanism of acquired resistance to osimertinib has not been well understood. Here, we planned a multicenter, prospective observational study in order to clarify the possible acquired resistance mechanisms to second-line treatment using osimertinib in Japanese non-small cell lung cancer patients. Methods: EGFR T790M positive advanced non-small cell lung cancer patients were included in this study. We excluded EGFR tyrosine kinase inhibitor naïve patients, patients whose EGFR T790M proved only by liquid biopsy, and patients whose tumor specimens were not adequate for targeted resequencing analysis. Re-biopsy was planned after the patient developed acquired resistance to osimertinib under written informed consent. Nucleic acids were extracted from formalin-fixed paraffin embedded tumor sections and targeted resequencing was performed using a panel consisting of 143 genes (Oncomine Comprehensive Assay, OCA, ThermoFisher Scientific, USA) in a CLIA-certified laboratory (SRL Inc., Japan), and then analyzed using IonReporter software (ThermoFisher Scientific, USA). Results: Between November 2016 and February 2019, 86 patients from 25 institutions were screened. Among 71 patients who were eligible, 31 patients were still on osimertinib and 33 patients developed acquired resistance. Among the 33 patients who developed acquired resistance, 16 patients proceeded to re-biopsy (4 males, 8 Del19 and 7 L858R EGFR mutations). 15/16 samples from re-biopsies and 9/16 samples from pre-osimertinib biopsies proceeded to OCA analyses. Others were excluded because of low quality of extracted nucleic acids. The acquired resistance mechanisms were as follows: T790M maintained in 10 patients with EGFR G796S being found in 1 patient. T790M loss observed in 5 patients with 1 patient having copy number gain in growth-arrest specific gene 6 (GAS6). Another patient possibly had copy number alterations in cell cycle genes. All patients had equal or reduced mutation counts during the therapeutic course, suggesting clonal selections by the therapies. Conclusions: EGFR C797S mutation was not observed in this series. Copy number alteration in GAS6-AXL pathway and cell cycle pathway could be targets of therapy. Citation Format: Atsushi Osoegawa, Masafumi Yamaguchi, Tomomi Nakamura, Ryotaro Morinaga, Kentaro Tanaka, Kosuke Kashiwabara, Kenichi Taguchi, Kazuki Nabeshima, Junji Kishimoto, Kenji Sugio. Revealing the mechanisms of acquired resistance to osimertinib from re-biopsy specimens in advanced non-small cell lung cancer with EGFR T790M mutation (LOGIK1607) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B004. doi:10.1158/1535-7163.TARG-19-B004

Abstract 4451: Evaluation of the therapeutic potential of phosphine oxide pyrazole inhibitors in tumors harboring EGFR C797S mutation

Abstract Osimertinib is a next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with activity against both the activating and the ‘gatekeeper’ T790M EGFR mutations. An acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in approximately 15% and 7% of patients in second-line and first-line treatment respectively. This percentage in the first-line setting will likely evolve as the first line data mature. The C797S mutation leads to the loss of covalent binding of osimertinib to mutant EGFR. The high affinity of the EGFR triple mutant for ATP presents a challenge for reversible inhibitor design, particularly as the loss of the cysteine at position 797 precludes the previously exploited covalent approaches. We have explored various approaches to address this challenge, including an effort to maximise reversible affinity to target the C797S mutation without requiring a covalent bond. We describe herein the therapeutic potential of reversible phosphine oxide pyrazole inhibitors in tumors harboring C797S. Using structure-based design, we were able to design a series of phosphine oxide pyrazole inhibitors that displayed exceptionally high biochemical potency against EGFR C797S mutation, which translated into good activity in cell-based assays. Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease-relevant models to express the C797S mutation to evaluate potency in vitro and in vivo. By modulating the physicochemical properties of our in vitro leads, we were able to achieve good oral exposure of cellularly active EGFR C797S inhibitors such as AZ’7608. We showed that AZ’7608 inhibits signalling pathways and cellular growth of C797S EGFR cell lines in vitro and demonstrated an improved WT EGFR margin. This translated into 52% (p<0.05, at day 14) tumor growth inhibition in vivo when compared to the control group. The efficacy of AZ’7608 is enhanced by its combination with anti-EGFR antibody, showing tumor regression (82%, p<0.001). In addition, we performed pharmacodynamic studies to explore the relationship between efficacy and target/pathway modulation. These studies establish a clear relationship between depth and duration of inhibition of the phosphorylation of EGFR and anti-tumor efficacy. The work presented herein shows a proof of concept for reversible phosphine oxide pyrazole inhibitors to target tumors harboring C797S. The emergence of the C797S EGFR mutation remains a key area of unmet need and warrants further efforts in drug discovery. Citation Format: Nicolas Floch, M. Raymond V. Finlay, Ambra Bianco, Sue Bickerton, Nicola Colclough, Darren A. Cross, Emanuela M. Cuomo, Carine M. Guerot, David Hargreaves, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Amar Rahi, Paul D. Smith, Richard A. Ward. Evaluation of the therapeutic potential of phosphine oxide pyrazole inhibitors in tumors harboring EGFR C797S mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4451.

Standard dose osimertinib for erlotinib refractory T790M-negative EGFR-mutant non-small cell lung cancer with leptomeningeal disease.

Leptomeningeal spread in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations who experience disease progression on TKIs portends a poor prognosis. Mutation profiling of tumour DNA in cerebrospinal fluid (CSF) samples can be used to determine the presence of the EGFR T790M resistance mutation, indicating that osimertinib, a CNS-penetrating 3rd generation TKI may be efficacious. Eight patients on EGFR TKIs who progressed with cytology-proven leptomeningeal disease at our institution were studied. EGFR mutations were profiled in CSF using droplet digital PCR (ddPCR) and compared to matched plasma samples. Clinical characteristics and survival outcomes on subsequent therapies tailored to ddPCR analysis were reported. None of the four patients who developed leptomeningeal disease while receiving 1st generation EGFR TKIs developed the EGFR T790M mutation in CSF. One patient who did not have extra-cranial disease and was EGFR T790M-negative in both plasma and CSF was nevertheless treated with standard-dose osimertinib, and achieved a rapid and durable response lasting 9 months to date. Three patients developed leptomeningeal disease on osimertinib, with one patient developing the EGFR C797S mutation in a cis-allelic conformation with the EGFR T790M mutation in plasma. Standard-dose osimertinib resulted in a clinically meaningful response in a patient with EGFR T790M-negative 1st generation EGFR TKI refractory leptomeningeal disease. Next generation sequencing and ddPCR has a role at identifying the C797S mutation and its allelic conformation with the T790M mutation with clinical implications.

PCC0208027, a novel tyrosine kinase inhibitor, inhibits tumor growth of NSCLC by targeting EGFR and HER2 aberrations

PCC-0208027 is a novel tyrosine kinase inhibitor that has a strong inhibitory effect on epidermal growth factor receptor (EGFR)- or HER2-driven cancers. The aim is to assess the anti-tumor activity of PCC0208027 and related mechanisms in non-small cell lung cancer (NSCLC). We examined the activity of PCC0208027 on various mutated EGFRs, HER2, and HER4. MTT assays, flow cytometry, and Western blotting were used to examine the effects of PCC0208027 on NSCLC cells with different genetic characteristics and relevant molecular mechanisms. Nude mouse xenograft models with HCC827, NCI-H1975, and Calu-3 cells were used to evaluate the in vivo anti-tumor activity of PCC0208027. Results showed that PCC0208027 effectively inhibited the enzyme activity of EGFR family members, including drug-sensitive EGFR mutations, acquired drug-resistant EGFR T790M and EGFR C797S mutations, and wild-type (WT) HER2. PCC0208027 blocked EGFR phosphorylation, thereby downregulating downstream PI3K/AKT and MAPK/ERK signaling pathways and inducing G0/G1 arrest in NSCLC cells. PCC0208027 inhibited tumor growth in mouse xenograft models of HCC827, NCI-H1975, and Calu-3 cells. In summary, our findings suggest that PCC0208027 has the potential to become an oral antineoplastic drug for NSCLC treatment and is worthy of further development.

Resistances to EGFR tyrosine kinase inhibitors in lung cancer-how to routinely track them in a molecular pathology laboratory?

Patients with advanced or metastatic forms of lung cancer with an activating mutation in epidermal growth factor receptor (EGFR) are given tyrosine kinase inhibitors (TKIs) targeted therapies that are more efficient than chemotherapy. These patients are excluded from first-line immunotherapy. After a phase of regression these tumors develop systematically resistance requiring a rapid change in therapy. At present two strategies are being discussed. The first strategy, so called "historical' sequential treatment strategy, is based on the administration of first- or second-generation TKIs until the emergence of therapeutic resistance and, in the case of a EGFR T790M mutation, on the administration of third-generation TKIs. The recently proposed second strategy, so called the "next-generation" TKIs strategy, concerns initial treatment with third-generation TKIs. This latter strategy appears to be promising but needs to be confirmed by data comparing survival curves of patients treated in a sequential manner. Several criteria influence the choice of these strategies, in particular the presence of brain metastases, the potential toxicity and the economic model. The selected therapeutic algorithm has certainly an impact on the activity of laboratories. The sequential approach requires investigation into EGFR T790M resistance mutations, using blood and then possibly a tissue biopsy, or into other mechanisms of resistance in the absence of this mutation. In the case of tumor progression under treatment with third-generation TKIs the EGFR C797S mutation in the cis or trans positions is looked for. In the absence of this latter mutation other mechanisms of resistance are then investigated. We describe here the different methodological approaches used to identify resistance mechanisms linked to treatment with TKIs targeting mutations in EGFR.