Epidermal Growth Factor Receptor C797S 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 CT154: Phase 1/2 clinical trial of JIN-A02, a 4th generation EGFR-TKI, in patients with 3rd generation EGFR-TKI resistance in EGFR mutated advanced/metastatic non-small cell lung cancer (NSCLC)

Abstract Background: Epidermal growth factor receptor (EGFR) mutations are the most common driver mutations in NSCLC. EGFR tyrosine kinase inhibitors (TKIs) are the recommended front-line option for EGFR-mutant advanced NSCLC patients, but patients invariably develop acquired resistance and face disease progression. JIN-A02, a 4th generation EGFR-TKI intended for oral administration, selectively and reversibly binds to EGFR mutations, including the C797S and/or T790M mutation that causes resistance to 3rd generation of EGFR-TKIs. Pre-clinical studies with EGFR C797S and/or T790M mutated cell lines showed that JIN-A02 can inhibit cell growth in a dose dependent manner and has a high degree of selectivity over wild-type EGFR. In vivo studies showed that administration of JIN-A02 dose-dependently inhibited tumor growth in the C797S+ xenograft mice model. Moreover, JIN-A02 shown to penetrate blood-brain barrier and exhibit anti-tumor activity in an intracranial tumor model. The strong potency and high selectivity of JIN-A02 for mutant EGFR may offer improved therapeutic window in the clinical setting vs available EGFR-TKIs. Methods: JIN-A02 is being evaluated in this Phase 1/2, multicenter, an open-label trial (NCT05394831) for subjects with advanced NSCLC harboring C797S and/or T790M mutation as a monotherapy. The primary object of this study is to assess safety, tolerability, pharmacokinetics, and anti-tumor effect for determining the recommended phase 2 dose (RP2D) of JIN-A02. The inclusion criteria are that the subject (≥ 18 years) must have an advanced or metastatic NSCLC showed progress disease (PD) after receiving approved standard EGFR-TKI therapies and/or platinum-based anticancer chemotherapy is allowed up to 1 time after using EGFR-TKI treatment with ECOG status 0 or 1. Before enrollment in the study, the EGFR mutation status is determined using either tumor tissue and/or plasma ctDNA. This study comprises three parts, dose escalation (Part A), dose exploration (Part B), and dose expansion (Part C). In Part A, dose escalation, comprised of 5 cohorts ranged from a starting dose of 12.5 mg P.O, QD to a top dose of 150 mg P.O, QD with every three subjects, is carried out where the maximum tolerated dose (MTD) is evaluated in subjects with 28-day cycles. Dose-limiting toxicities (DLT) are evaluated for 21 days. In Part B, dose exploration is carried out to evaluate the safety of JIN-A02 further and to determine the RP2D using 2 preliminary effective dose levels from Part A in subjects. In Part C dose-expansion study, subjects (n=36 for each cohort) are divided into 5 different cohorts based on the EGFR mutation (both or single positive of C797S and T790M), and the anti-tumor activity of JIN-A02 is evaluated according to RECIST v1.1 at RP2D. Citation Format: Sun Min Lim, Byoung Chul Cho, Ji Youn Han, Sang We Kim, Ki Hyeong Lee, Misako Nagasaka, Anna Jo, Ethan Seah, Choonok Kim, Thanyanan Reungwetwattana. Phase 1/2 clinical trial of JIN-A02, a 4th generation EGFR-TKI, in patients with 3rd generation EGFR-TKI resistance in EGFR mutated advanced/metastatic non-small cell lung cancer (NSCLC) [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 CT154.

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.

Abstract 964: Updated results of a prospective observational multicenter study on resistance mechanisms to osimertinib using circulating tumor DNA analyses in EGFR-mutated non-small cell lung cancer patients (ELUCIDATOR study)

Abstract Background: Although osimertinib (Osi) is the key standard therapy for patients (pts) with advanced non-small cell lung cancer (NSCLC) harboring sensitizing epidermal growth factor receptor (EGFR) mutations, most of cases experience the progressive disease (PD) after initial response to the treatment. Therefore, understanding the Osi resistance mechanisms and identifying the poor responders are essential to develop better treatment strategies. Thus, we conducted a prospective observational multicenter study and analyzed serially collected circulating tumor DNA (ctDNA). Here we report the results from updated analyses focusing on the association between genetic information from ctDNA and clinical outcome. Methods: Patient eligibility criteria were as follows: a definitive diagnosis of non-squamous NSCLC confirmed through biopsy or cytology, EGFR exon 19 deletion (19del) or L858R mutation, Osi planned as first-line treatment, and pts who could provide blood specimens. Paired plasma DNA samples were collected before starting Osi as baseline (BL) and at PD and analyzed to investigate Osi resistance by next-generation sequencing (NGS). Correlations between genetic information obtained from ctDNA and clinical outcome such as progression free survival (PFS) and overall survival (OS) were evaluated. The median follow-up time was 33.1 months. Results: One hundred eighty-eight pts were enrolled between May 2019 and January 2021, and 125 (66%) were female, 96 (51%) were EGFR 19del mutation, and 109 (58%) were never smoker. Among those, 178 pts were included in the analyses, and 111 experienced PD. In consistent with previous reports, median PFS (mPFS) and median OS (mOS) were 19.1 months and 36.0 months, respectively. The efficacy of Osi differed between 19del and L858R in both PFS and OS, with 19del being longer outcome [mPFS, 23.3 months vs. 17.2 months, HR: 1.56 (95%CI: 1.08-2.26), mOS, 40.3 months vs. 30.4 months, HR: 1.54 (95%CI: 0.98-2.45)]. Univariate analysis revealed that the presence of TP53 mutations and MET amplification (amp) at BL was a predictive factor of shorter PFS and OS. Multivariate analysis also revealed that the TP53 mutations at BL was a predictive factor of shorter PFS and OS. Plasma samples from 85 pts were available for ctDNA analysis. New adaptive mutations or amp at PD were detected in genes such as PIK3CA in 3 cases, MET amp in 4, TP53 in 4, BRIMP3 in 2, APC in 1, and BRAF in 2. Secondary EGFR C797S resistance mutation was detected in 1 case. Conclusions: TP53 mutation at BL could be predictive of poor response to Osi. MET amp and PIK3CA mutations were detected as the most common resistance mechanisms, and secondary EGFR resistance mutations were observed less frequently than previous reports. Citation Format: Mitsuo Osuga, Akihiro Tamiya, Daijiro Harada, Yasuyuki Mizumori, Shun-ichi Isa, Yoshihiko Taniguchi, Keiichi Nakamura, Tsutomu Shinohara, Hidetoshi Yanai, Katsumi Nakatomi, Masahide Oki, Masahide Mori, Tomohito Kuwako, Koji Yamazaki, Atsuhisa Tamura, Masahiko Ando, Yasuhiro Koh. Updated results of a prospective observational multicenter study on resistance mechanisms to osimertinib using circulating tumor DNA analyses in EGFR-mutated non-small cell lung cancer patients (ELUCIDATOR study) [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 964.

Abstract 4502: Design, synthesis, and evaluation of next-generation EGFR degraders to overcome osimertinib-resistance

Abstract The occurrence of C797S mutation in epidermal growth factor receptor (EGFR) is a leading mechanism of clinically acquired resistance to third-generation EGFR inhibitors, including Osimertinib. L858R/T790M/C797S and del19/T790M/C797S are commonly observed tertiary EGFR mutants identified in Osimertinib-resistant tumors. As of now, no clinically approved treatment exists that specifically targets these mutants. Here, we report the design and synthesis of a series of highly effective next-generation EGFR degraders effectively degrading EGFR C797S-containing triple mutants. Most compounds demonstrated antiproliferation activity in the subnanomolar range when tested on Ba/F3L858R/T790M/C797S and del19/T790M/C797S cells. Not only C797S but our designed degraders also degraded a wide range of EGFR mutants, including Exon19Del and L858R/T790M (DC50 <100nM). One representative Compound, HDBNJ2812, strongly degrades L858R/T790M/C797S and del19/T790M/C797S with DC50 of 34 nM (Dmax 88.5%) and 14 nM (Dmax 99.7%), respectively. This compound potently inhibits the proliferation of Ba/F3L858R/T790M/C797S (GI50 64 nM) and del19/T790M/C797S (GI50 40 nM). HDBNJ2812 demonstrated high inhibitory potential on HCC827 (del19) and H1975 (L858R/T790M) cell lines (GI50 18.9 and 85 nM, respectively). Furthermore, this degrader demonstrates weak cytotoxicity on non-mutant EGFR-expressing cells, such as A431, WI-26 (human lung fibroblast cells), and CHO-K1 (Chinese hamster ovary cells). Additionally, the in vivo PK/PD findings complement this compound's potential to be considered further. HDBNJ2812 may serve as a lead compound to render the greater therapeutic window for treating resistant non-small cell lung cancer patients with EGFR C797S mutants. Citation Format: Mohammad Hassan Baig, Juhan Bok, Dongmin Kim, Sagar Dattatraya Nale, Yun Sung Jo, Changjoong Kim, Taehhwan Park, Jaejune Dong, Byoung Gon Moon. Design, synthesis, and evaluation of next-generation EGFR degraders to overcome osimertinib-resistance [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 4502.

A Phase II trial of alternating osimertinib and gefitinib therapy in advanced EGFR-T790M positive non-small cell lung cancer: OSCILLATE

In this phase II, single arm trial (ACTRN12617000720314), we investigate if alternating osimertinib and gefitinib would delay the development of resistance to osimertinib in advanced, non-small cell lung cancer (NSCLC) with the epidermal growth factor receptor (EGFR) T790M mutation (n = 47) by modulating selective pressure on resistant clones. The primary endpoint is progression free-survival (PFS) rate at 12 months, and secondary endpoints include: feasibility of alternating therapy, overall response rate (ORR), overall survival (OS), and safety. The 12-month PFS rate is 38% (95% CI 27.5–55), not meeting the pre-specified primary endpoint. Serial circulating tumor DNA (ctDNA) analysis reveals decrease and clearance of the original activating EGFR and EGFR-T790M mutations which are prognostic of clinical outcomes. In 73% of participants, loss of T790M ctDNA is observed at progression and no participants have evidence of the EGFR C797S resistance mutation following the alternating regimen. These findings highlight the challenges of treatment strategies designed to modulate clonal evolution and the clinical importance of resistance mechanisms beyond suppression of selected genetic mutations in driving therapeutic escape to highly potent targeted therapies.

The D ~ Sense ex-vivo viability assay application in a patient with stage IV lung adenocarcinoma: a case report

BackgroundThe treatment resistance is a problem for lung cancer. In this study, we used a vitro tissue culturing system to select a new therapy strategy for a patient with tyrosine kinase inhibitors (TKIs) resistance.Case presentationA 42-year-old male Asian patient was diagnosed with advanced lung adenocarcinoma harboring an exon 19 deletion in the epidermal growth factor receptor (EGFR) gene. The patient was treated with Gefitinib, resulting in an almost complete remission for over a year. The patient relapsed after 13 months treatment, and received four cycles of chemotherapy. At 20 months, the patient had developed multiple lung metastases and a solitary cerebellar metastasis. An EGFR T790M mutation was identified in the peripheral blood sample. Subsequent treatment with Osimertinib resulted in a complete response of the intracranial metastasis. By 33 months, the patient had developed a mediastinal tumor mass that responded well to local radiotherapy. By 39 months, an EGFR C797S cis-mutation had been identified and the patient was treated with Brigatinib and Cetuximab. By 44 months, the tumor cells from the pleural effusion had been tested for sensitivity against 30 targeted and cytostatic drugs using the D ~ Sense ex-vivo viability assay. The assay identified 8 drugs with moderate to high sensitivity. Combination therapy of Gemcitabin and Lobaplatin had resulted in disease stabilization.ConclusionsThe case showed that individualized treatment aided by D ~ Sense ex-vivo viability assay can be a viable option for patients with advanced lung adenocarcinoma with pleural effusions.

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.

Mechanistic Modeling of Lys745 Sulfonylation in EGFR C797S Reveals Chemical Determinants for Inhibitor Activity and Discriminates Reversible from Irreversible Agents.

Targeted covalent inhibitors hold promise for drug discovery, particularly for kinases. Targeting the catalytic lysine of epidermal growth factor receptor (EGFR) has attracted attention as a new strategy to overcome resistance due to the emergence of C797S mutation. Sulfonyl fluoride derivatives able to inhibit EGFRL858R/T790M/C797S by sulfonylation of Lys745 have been reported. However, atomistic details of this process are still poorly understood. Here, we describe the mechanism of inhibition of an innovative class of compounds that covalently engage the catalytic lysine of EGFR, through a sulfur(VI) fluoride exchange (SuFEx) process, with the help of hybrid quantum mechanics/molecular mechanics (QM/MM) and path collective variables (PCVs) approaches. Our simulations identify the chemical determinants accounting for the irreversible activity of agents targeting Lys745 and provide hints for the further optimization of sulfonyl fluoride agents.

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.

LS-106, a novel EGFR inhibitor targeting C797S, exhibits antitumor activities both in vitro and in vivo.

With the wide clinical use of the third‐generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR‐mutated non–small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth‐generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS‐106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell‐free assay, LS‐106 potently inhibited the kinase activities of EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S with IC50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS‐106 exhibited comparable kinase inhibitory effect to osimertinib on EGFRL858R/T790M and wild‐type EGFR. Results from cellular experiments demonstrated that LS‐106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR19del/T790M/C797S or EGFRL858R/T790M/C797S, and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR19del/T790M/C797S (named PC‐9‐OR cells) using the CRISPR/Cas9 system and found that LS‐106 markedly suppressed the activation of EGFR19del/T790M/C797S and the proliferation of PC‐9‐OR cells. Moreover, cells harboring EGFR19del/T790M/C797S underwent remarkable apoptosis upon LS‐106 treatment. In vivo experiments further demonstrated that oral administration of LS‐106 caused significant tumor regression in a PC‐9‐OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS‐106 as a novel fourth‐generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S–triple‐mutant tumor models.

Abstract P230: A phase 1/2 study of BLU-945, a highly potent and selective inhibitor of epidermal growth factor receptor (EGFR) resistance mutations, in patients with EGFR-mutant non-small cell lung cancer (NSCLC)

Abstract BACKGROUND Activating EGFR mutations such as exon 19 deletion (ex19del) and L858R mutations, exon 19 insertions (ex19ins), and point mutations L861Q, G719X, and S768I are oncogenic drivers in lung adenocarcinoma, the most common NSCLC subtype. Although EGFR-targeted therapies, including first-, second-, and third-generation tyrosine kinase inhibitors (TKIs), have improved outcomes in patients (pts) with EGFR-mutant (EGFRm) NSCLC, resistance to these drugs is inevitable. BLU-945 is an investigational fourth-generation EGFR TKI designed to suppress both activating and on-target resistance EGFR mutations, such as C797S and T790M. Preclinical data showed BLU-945 has nanomolar potency against EGFRm/T790M double and EGFRm/T790M/C797S triple mutants with >450-fold selectivity for these mutants over wild-type EGFR in biochemical and cellular assays, and demonstrated robust preclinical antitumor activity, including in intracranial tumors harboring these mutations, providing the therapeutic rationale for BLU-945 clinical development. METHODS BLU-945-1101 (NCT04862780) is an international, open-label, first-in-human, phase 1/2 study designed to evaluate safety, tolerability, and antitumor activity of BLU-945 in pts with EGFRm NSCLC. Key eligibility criteria include adults with pathologically confirmed metastatic NSCLC with an activating EGFR mutation per local assessment (including ex19del or L858R); Eastern Cooperative Oncology Group performance status 0–2; and previous treatment with ≥1 EGFR-targeted TKI. Pts with asymptomatic brain metastases and on stable doses of corticosteroids are eligible. Key exclusion criteria are any additional known primary driver alterations, including but not limited to pathologic aberrations of EGFR exon 20 (insertions), KRAS, BRAF V600E, NTRK1/2/3, HER2, ALK, ROS1, MET, or RET. Phase 1 primary endpoints are maximum tolerated dose, recommended phase 2 dose (RP2D), and safety. The phase 2 primary endpoint is overall response rate (ORR) by RECIST 1.1. Phase 1 secondary endpoints include ORR, duration of response (DOR), pharmacokinetics (PK), and pharmacodynamics (PD); phase 2 secondary endpoints include DOR, disease control rate (DCR), clinical benefit rate (CBR), progression free survival (PFS), overall survival (OS), time to intracranial progression and intracranial response rate, PK, PD, and safety. Phase 1 dose escalation (N≈60) will be conducted using Bayesian optimal interval design. In the phase 2 dose expansion (N≈61), pts will be enrolled into 3 groups based on mutation profile and treated at the RP2D: primary group 1 for pts with EGFR T790M and C797S (n≈37); exploratory group 2 for pts with EGFR T790M but not C797S (n≈12); exploratory group 3 for pts with EGFR C797S but not T790M (n≈12). Group 1 will use Simon’s 2-stage design to test the null hypothesis of ORR ≤20% against a 1-sided alternative of ≥40%. Pts may receive treatment until disease progression or unacceptable toxicity. Recruitment has started and approximately 30 sites will be open for enrollment across North America, Europe, and Asia. Citation Format: David Spigel, Koichi Goto, D. Ross Camidge, Yasir Elamin, Adrianus J. de Langen, Natasha B. Leighl, Anna Minchom, Zofia Piotrowska, David Planchard, Karen Reckamp, Faris Albayya, Jennifer Green, Sean Kim, Melinda Louie-Gao, Renata Sawtell, Alena Zalutskaya, Byoung Chul Co. A phase 1/2 study of BLU-945, a highly potent and selective inhibitor of epidermal growth factor receptor (EGFR) resistance mutations, in patients with EGFR-mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P230.