C797S Mutation Research Articles

Abstract 1967: IN-119873, the next-generation allosteric EGFR TKI, a potent and highly selective EGFR L858R for the treatment of osimertinib-resistant NSCLC

Abstract EGFR mutations occur in approximately 50% of Asian patients and 20% of Caucasian patients with non-small cell lung cancer (NSCLC). The EGFR L858R mutation constitutes approximately half of the EGFR-mutated patients, and despite receiving first-line or second-line treatment with the 3rd generation EGFR TKI osimertinib, they continue to exhibit a poor prognosis. According to real-world analysis, the leading resistance mechanism to osimertinib is identified as EGFR C797S mutation. Furthermore, osimertinib is still associated with gastrointestinal and skin toxicity, despite its improved selectivity for EGFR WT compared to 1st-generation EGFR TKIs. Here, we show that IN-119873 can overcome C797S-mediated acquired resistance based on the results obtained by various in vitro and in vivo NSCLC models. IN-119873 binds to an EGFR allosteric site separate from the ATP-binding site with a remarkable preference for highly mutant-selective inhibition. It effectively targets EGFR L858R activating mutations while sparing the inhibition of EGFR WT, which sets it apart from ATP competitive inhibitors. Moreover, oral administration of IN-119873 showed excellent anti-tumor efficacy in a dose-dependent manner in a diverse of osimertinib-resistant CDX and PDX models. IN-119873 also demonstrated favorable blood-brain-barrier penetration with promising intracranial efficacy in the H1975-luc brain metastasis model both mono or in combination with osimertinib. As a selective, orally available, and CNS-penetrable next-generation allosteric EGFR-TKI, IN-119873 is currently undergoing IND-enabling studies and has the potential to demonstrate activity in both first-line and resistance settings, either as a monotherapy or in combination with 3rd generation EGFR TKI. Citation Format: Jong Ryoul Choi, Daseul Yoon, Seo Hyun Kim, Do Gyeong Kim, Seock Yong Kang, Seong-Il Choi, Somyi Park, Hyun Kyung Lee, Donghyun Kim, Ju Hyun Lee, Hye-Jung Kim, Bong Tae Kim. IN-119873, the next-generation allosteric EGFR TKI, a potent and highly selective EGFR L858R for the treatment of osimertinib-resistant NSCLC [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 1967.

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 1962: PH009-1, a highly potent, selective and brain-penetrable fourth-generation EGFR-TKI that overcome classic and resistant EGFR mutations in NSCLC

Abstract Background: Epidermal growth factor receptor (EGFR) inhibitors, especially 3rd Gen inhibitor such as osimertinib (Osi), have become the first line therapy for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, treatments with 3rd Gen EGFR inhibitors lead to occurrence of acquired C797S mutation in 10-24% of patients. Though studies showed that double mutations 19Del/C797S (DC) and L858R/C797S (LC) could be inhibited by 1st Gen EGFR TKIs, appearance of triple mutations 19Del/T790M/C797S (DTC) and L858R/T790M/C797S (LTC) always leads to treatment failure. In recent years, 4th Gen EGFR inhibitors have been developed such as BLU-945 and PH009-1. PH009-1 had been reported on AACR annual meeting 2023. Methods: Cell activity and selectivity were determined in engineered Ba/F3 cell lines carrying EGFR wild type (WT, stimulated with EGF), 19Del (D), L858R (L), 19Del/T790M (DT), L858R/T790M (LT), DC, LC, DTC and LTC. The in vivo anti-tumor efficacy and PK-PD studies were performed in cell or patient derived xenograft (CDX or PDX) cancer models (HCC827, D; Ba/F3, LC; PDX, DTC). Brain penetration capability was tested in mice orally treated with PH009-1 for 7 days. Systemic safety of PH009-1 was evaluated in 28-day repeated dosing toxicity studies in rats and dogs following GLP guidelines. Results: PH009-1 displayed potent anti-proliferation activity against Ba/F3 cells harboring EGFR mutations (L, D, LC, DC, LT, DT, LTC, DTC) with IC50 of 2.4, 1.3, 5.2, 1.2, 1.1, 1.5, 2.8, 1.9 nM, respectively, but had very weak effect on EGFR WT Ba/F3 cells (IC50, 1135 nM), suggesting excellent potency and selectivity. In contrast, though BLU-945 potently inhibited DT, LT, DTC, LTC with IC50 less than 5 nM, but its activities on L, D, LC, DC were not strong with IC50 of 31.6, 81.2, 46.2, and 52.0 nM. In in vivo efficacy studies, PH009-1 at a dose of 20 mg/kg BID led to potent inhibition on tumor growth and EGFR phosphorylation, and tumor regression was observed at higher doses in all tested models. In HCC827 model with 19Del (first line therapy setting), mice were treated for more than 200 days, and one tumor relapsed in BLU-945 group but not in PH009-1 or Osi group. Also, deaths were observed in Osi group, but not with PH009-1. After treated mice with PH009-1 at efficacious dose for 7 days, concentrations in cerebrospinal fluids (CSF) at 0-8 h post final dose were more than enough (≥1.5 fold) to cover IC80 on all tested mutations. In GLP toxicity studies, no unexpected toxicity was observed. In dogs. the highest tested dose was determined as highest non-severely toxic dose (HNSTD) and based on high plasma exposure, wide safety margin was confirmed with PH009-1. Conclusion: Our data suggested that PH009-1 overcome T790M/C797S resistant mutations, and also have potential to become a potent and safe approach for first line therapy of NSCLC with EGFR mutations. Citation Format: Feng Gao, Jing Wang, Bin Liu, Yongyong Wu, Liandong Jing, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH009-1, a highly potent, selective and brain-penetrable fourth-generation EGFR-TKI that overcome classic and resistant EGFR mutations in NSCLC [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 1962.

Abstract 1726: Targeted epigenomic control of MYC as a strategy to treat EGFR inhibitor-resistant NSCLC

Abstract EGFR tyrosine kinase inhibitors (EGFRi) have improved outcomes for non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. However, patients receiving EGFRi therapy frequently relapse with an EGFR T790M mutation. While osimertinib, a third generation EGFRi, blocks EGFR T790M activity, resistance eventually develops through various EGFR-dependent and EGFR-independent mechanisms, such as acquiring additional mutations in EGFR (e.g. C797S), activating bypass signaling pathways, or undergoing an epithelial to mesenchymal transition (EMT). MYC is a master transcription factor critical for mediating oncogenic signal transduction and has been implicated in EGFRi resistance, suggesting that MYC targeting may overcome multiple EGFRi resistance mechanisms. To evaluate this, we have developed a NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC epigenomic controller (NSCLC MYC-EC), designed to selectively target regulatory elements in MYC’s insulated genomic domain and downregulate MYC expression. We have previously shown that NSCLC MYC-EC effectively decreases MYC expression pre-transcriptionally and have demonstrated that MYC-EC combination with osimertinib synergistically reduces viability of EGFR mutant NSCLC cells. Here, we demonstrate NSCLC MYC-EC activity in models that have developed EGFRi resistance through EGFR-dependent and -independent mechanisms. To test the effect of NSCLC MYC-EC in NSCLC cells with T790M mutant EGFR, we treated H1975 (L858R, T790M) and PC9-T790M cells (del19, engineered T790M) with MYC-EC alone or in combination with osimertinib. MYC-EC combination with osimertinib enhanced MYC protein downregulation and synergistically reduced cell viability in both models. To evaluate the effect of MYC-EC in osimertinib-resistant cells, we engineered H1975 and PC9-T790M cells with an EGFR C797S mutation. While osimertinib treatment did not impact cell viability in these models, MYC-EC maintained activity, downregulating MYC levels and reducing cell viability. To investigate MYC-EC activity in cells with alternative resistance mechanisms, we generated osimertinib-resistant H1975 cells by stepwise dose escalation. Protein analysis showed highly reduced phospho-EGFR levels, suggesting an EGFR-independent mechanism of survival. RNA-sequencing analysis revealed that EMT pathway was highly activated in these resistant cells. Importantly, this model retained sensitivity to NSCLC MYC-EC treatment, demonstrating that selectively targeting MYC can treat osimertinib-resistant NSCLC cells with a mesenchymal phenotype. Together, these data demonstrate that pre-transcriptional downregulation of MYC through NSCLC MYC-EC treatment effectively inhibits viability of EGFRi-resistant NSCLC through both EGFR-dependent and -independent mechanisms and supports the development of NSCLC MYC-EC in EGFRi-resistant NSCLC. Citation Format: Eugine Lee, Padraich Flahardy, Cameron Vergato, Stephen Siecinski, Justin Chen, Charles O’Donnell, Graeme Hodgson, Defne Yarar, Thomas McCauley. Targeted epigenomic control of MYC as a strategy to treat EGFR inhibitor-resistant NSCLC [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 1726.

Abstract 544: Reverse engineering strategy for new targets identification

Abstract The issue of drug resistance in cancer is very similar to the field of infectious disease, either existing before treatment (intrinsic) or generated during therapy (acquired), highly affected by cell endogenous factors and/or the surrounding environment. Acquired resistance can be occurred several months after targeted therapy via alternations of drug targets due to the genomic mutations of the targets, epigenetic transcriptional or translational changes, for example the C797S mutation impairs AZ9291 binding to the EGFR kinase domain. To overcome this, novel well-designed chemical compounds targeting new mutations would be urgently needed, as well as the potential new oncogenes/susceptible genes driving the resistance. Considering about the heterogeneity of primary tumor tissues, LIDE has established one functional cost-effective in vitro and in vivo screening method, named reverse engineering strategy for targets identification by using patients-derived conditional reprogramed cancer cell lines (CRs) and IO-FIVE (Immuno-Oncology drugs Five In Vivo Efficacy test). Basically, pro-siRNA library or CRISPR/sgRNA library is carried out in CRs for candidates hunting, alternatively drug-response evaluation among multiple clinical biopsies in mice cancer trials, after filtered with bioinformatic scanning from large groups of patients, and finally it is validated in vivo by CRISPR, shRNA silence or targeted compounds. In addition to these known factors (EGFR C797S, Her2/Met amplification, Ras and MAPK activation), we have discovered that epigenetic factors for DNA modification, protein translation kinases, mitochondrial/peroxisomal transporters and cell membrane lectins may contribute to AZ92921 resistance in vivo. And one previously unknown acyltransferase member has been identified as key driver gene for pan-cancer development by IO-FIVE, and patients with high expression level of this enzyme have had poor survival outcomes, accompany with uncontrolled tumor growth and immunotherapy resistance, which is becoming a promising therapeutic target. Citation Format: Bin Xie, Taimei Zhang, Le Li, Hongkui Chen, Yang Liu, Lintao Bi, Danyi Wen. Reverse engineering strategy for new targets identification [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 544.

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.

Structural Insights into the Binding Mode of a BLU-945 Analog with Wild-Type and Mutant EGFRs

Overcoming the acquired C797S mutation in nonsmall cell lung cancer (NSCLC) represents a great challenge for EGFR tyrosine kinase inhibitors (TKIs). BLU-945 is a promising fourth-generation EGFR TKI undergoing clinical trials, with different binding conformation from other known EGFR TKIs. In this study, we explored the binding mode of a BLU-945 analog (BLU) with wild-type and mutant EGFRs using [Formula: see text]s-scale molecular dynamics. The results show that Met793 at the hinge region is the critical binding site for BLU as observed in other known EGFR inhibitors. The occupancy of hydrogen bonds with Met793 is lower in wild-type EGFRs than in mutant EGFRs. Meanwhile, Thr854 contributes largely to the hydrogen bond formation for triple mutant EGFR compared to wild-type EGFR. Energy decomposition reveals that Leu718 and Leu844 dominate the energy contribution. T790M mutation plays an important role in BLU binding with wild-type and mutant EGFRs. Dynamic network analysis indicates that Arg841 behaves differently in triple mutant EGFR compared to wild-type, single and double mutant EGFRs, confirmed by further analysis of salt-bridge formation. Our study may provide beneficial information for developing fourth-generation EGFR TKIs.

EGFR amplification indicated poor prognosis in EGFR‐mutated lung cancer with leptomeningeal metastases

AbstractBackgroundLeptomeningeal metastasis (LM) is a lethal complication of advanced lung cancer, and due to limited access to the leptomeningeal lesion, we explored the potential role of cerebrospinal fluid (CSF) as a source for liquid biopsy in LM patients of lung cancer with EGFR mutation.Materials and MethodsFrom August 2018 to June 2021, we collected CSF samples of lung cancer patients at First Affiliated Hospital of Zhengzhou University. Next‐generation sequencing was performed to detect the mutations in EGFR genes, and 38 patients detected with EGFR mutations were finally enrolled in further clinical analyses.ResultsTP53 missense mutation (50%) was the most frequently detected concurrent gene in CSF. In those 10 patients whose CSF was obtained upon resistance to first TKI, TP53 missense mutation (40%, n = 4) and EGFR copy number amplification (40%, n = 4) was detected with high frequency; meanwhile, T790M mutation was found only in two patients. Known mechanisms of acquired resistance to third EGFR‐TKIs were found in 31.8% of cases. C797S or C797G mutation was identified in three patients. Possible EGFR‐independent resistant mechanism included MET amplification (4.5%), RET gene fusion (4.5%), PIK3CA missense mutation (4.5%) and CDK4 amplification (4.5%). The median OS was 55 months, the median OSLM was 38 months. EGFR amplification was associated with shortened OS in these EGFR‐mutated lung cancer with leptomeningeal metastases.ConclusionEGFR amplification indicated poor prognosis in EGFR‐mutated lung cancer with leptomeningeal metastases, providing a novel pathogenesis and treatment direction of these patients.

Machine Learning-Based Virtual Screening and Identification of the Fourth-Generation EGFR Inhibitors.

Epidermal growth factor receptor (EGFR) plays a pivotal regulatory role in treating patients with advanced nonsmall cell lung cancer (NSCLC). Following the emergence of the EGFR tertiary CIS C797S mutation, all types of inhibitors lose their inhibitory activity, necessitating the urgent development of new inhibitors. Computer systems employ machine learning methods to process substantial volumes of data and construct models that enable more accurate predictions of the outcomes of new inputs. The purpose of this article is to uncover innovative fourth-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) with the aid of machine learning techniques. The paper's data set was high-dimensional and sparse, encompassing both structured and unstructured descriptors. To address this considerable challenge, we introduced a fusion framework to select critical molecule descriptors by integrating the full quadratic effect model and the Lasso model. Based on structural descriptors obtained from the full quadratic effect model, we conceived and synthesized a variety of small-molecule inhibitors. These inhibitors demonstrated potent inhibitory effects on the two mutated kinases L858R/T790M/C797S and Del19/T790M/C797S. Moreover, we applied our model to virtual screening, successfully identifying four hit compounds. We have evaluated these hit ADME characteristics and look forward to conducting activity evaluations on them in the future to discover a new generation of EGFR-TKI.

Next-generation EGFR tyrosine kinase inhibitors to overcome C797S mutation in non-small cell lung cancer (2019-2024).

Lung cancer is a leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for the major portion (80-85%) of all lung cancer cases. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are commonly used as the targeted therapy for EGFR-mutated NSCLC. The FDA has approved first-, second- and third-generation EGFR-TKIs as therapeutics options. Osimertinib, the third-generation irreversible EGFR-TKI, has been approved for the treatment of NSCLC patients with the EGFRT790M mutation. However, due to the EGFRC797S mutation in the kinase domain of EGFR, resistance to osimertinib is observed and that limits the long-term effectiveness of the drug. The C797S mutation is one of the major causes of drug resistance against the third-generation EGFR TKIs. The C797S mutations including EGFR double mutations (19Del/C797S or L858R/C797S) and or EGFR triple mutations (19Del/T790M/C797S or L858R/T790M/C797S) cause major resistance to the third-generation EGFR-TKIs. Therefore, the discovery and development of fourth-generation EGFR-TKIs to target triple mutant EGFR with C797S mutation is a challenging topic in medicinal chemistry research. In this review, we discuss the discovery of novel fourth-generation EGFR TKIs, medicinal chemistry approaches and the strategies to overcome the C797S mutations. In vitro activities of EGFR-TKIs (2019-2024) against mutant EGFR TK, anti-proliferative activities, structural modifications, binding modes of the inhibitors and in vivo efficacies in animal models are discussed here.

BLU-945, a potent and selective next-generation EGFR TKI, has antitumor activity in models of osimertinib-resistant non-small-cell lung cancer.

Despite the availability of several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), most patients with non-small-cell lung cancer (NSCLC) eventually develop resistance to these agents. Notably, EGFR_C797S mutations confer resistance to the third-generation EGFR-TKI osimertinib and no approved post-osimertinib targeted pharmacology options are currently available. BLU-945 is a novel, reversible, and orally available next-generation EGFR-TKI that selectively targets EGFR-activating (EGFRm) and resistance mutations (including EGFR_C797S) with nanomolar potency while sparing wild-type EGFR in vitro. In vitro activity of BLU-945 as a single agent and in combination with osimertinib was tested in engineered EGFR-mutant cell lines as well as patient-derived cells and patient-derived organoids. In vivo activity was evaluated in osimertinib-resistant patient-derived xenograft mouse models. Three patient cases from the global, first-in-human, phase I/II SYMPHONY trial (NCT04862780) demonstrating the clinical efficacy of BLU-945 were reported. In vitro BLU-945 demonstrated inhibited cell viability and growth of EGFR-mutant/osimertinib-resistant cell lines. BLU-945 demonstrated in vivo tumor shrinkage in osimertinib-resistant models of NSCLC (osimertinib second line: EGFR_L858R/C797S and third line: EGFR_ex19del/T790M/C797S and L858R/T790M/C797S) both as monotherapy and in combination with osimertinib. BLU-945 also demonstrated tumor shrinkage in patients from the SYMPHONY trial. Our findings demonstrate the preclinical and early clinical activity of BLU-945 in EGFRm NSCLC progressing on previous EGFR-TKIs.

T6496 targeting EGFR mediated by T790M or C797S mutant: machine learning, virtual screening and bioactivity evaluation study

Acquired resistance to EGFR is a major impediment in lung cancer treatment, highlighting the urgent need to discover novel compounds to overcome EGFR drug resistance. In this study, we utilized in silico methods and bioactivity evaluation for drug discovery to identify novel active anticancer agents targeting EGFRT790M/L858R and EGFRT790M/C797S/L858R. Firstly, we employed ROC-guided machine learning to retrieve nearly 7,765 compounds from a collection of three libraries (comprising over 220,000 compounds). Next, virtual screening, cluster analysis, and binding model analysis were employed to identify six potential compounds. Additionally, the kinase assay revealed that these six compounds demonstrated higher sensitivity to EGFR than c-Met. Among these compounds, T6496 inhibited both EGFRT790M/L858R and EGFRT790M/C797S/L858R kinases, with an IC50 of 3.30 and 8.72 μM. Furthermore, we evaluated the antitumor effects of the six selected compounds, and compound T6496 exhibited the strongest anticancer activity against H1975 cell lines, with an IC50 value of 2.7 μM. These results suggest that T6496 may mitigate EGFR resistance caused by T790M or C797S mutations. Moreover, the AO staining assay, JC-1 staining, ROS experiment and hemolytic toxicity evaluation revealed that T6496 could induce apoptosis in H1975 cell lines in a time-dependent and concentration-dependent manner, and is a potential compound for further structural optimization. Communicated by Ramaswamy H. Sarma

Research On Befortinib: Characteristics, Role, And Development of Fourth-Generation EGFR-Tkis

Lung cancer is one of the most common cancers in terms of morbidity and mortality. Among them, non-small cell lung cancer (NSCLC) accounts for 90% of lung cancer patients. Epidermal Growth Factor Receptor (EGFR) mutations are also the most common type of driver gene mutation in advanced NSCLC. In Chinese NSCLC patients, the proportion of EGFR mutations was 28.2%, and this proportion increased to 50.2% in lung adenocarcinoma. All three generations of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR-TKI) are effective therapeutics for lung cancer patients carrying EGFR-sensitive mutations or T790M mutations, each of which has its own advantages and characteristics, as well as its own limitations and adverse reactions. Befortinib, which is new to the market in 2023, has better Median progression-free survival (mPFS). However, three generations of EGFR-TKI cannot completely overcome the resistance mechanism of lung cancer. In addition to the T790M mutation, there are other mutations or mechanisms that cause lung cancer to become resistant to third-generation EGFR-TKI, such as C797S mutation, MET amplification, HER2 amplification, etc. At present, the fourth-generation of EGFR-TKI is still under continuous research and development.

Design, synthesis and antitumor activity of 4-arylamine substituted pyrimidine derivatives as noncovalent EGFR inhibitors overcoming C797S mutation

Clinical researches have shown that epidermal growth factor receptor (EGFR) is a key target for treatment of non-small cell lung cancer (NSCLC). Many EGFR inhibitors were successfully developed as ani-tumor drugs to treat NSCLC patients. Unfortunately, drug resistances were found in clinic. To overcome C797S mutation in EGFR, a novel series of 4-arylamine substituted pyrimidine derivatives were designed and synthesized under the principle of structure-based drug design. Interestingly, compounds 6e and 9i demonstrated the best anti-proliferative activity against A549, NCI–H1975, and HCC827 cells. In particular, the IC50 values against HCC827 cells reached to 24.6 nM and 31.6 nM, which were much lower than human normal cells 2BS and LO2. Furthermore, compounds 6e and 9i showed extraordinary activity against EGFR19del/T790M/C797S (IC50 = 16.06 nM and 37.95 nM) and EGFRL858R/T790M/C797S (IC50 = 11.81 nM and 26.68 nM), which were potent than Osimertinib (IC50 = 52.28 nM and 157.60 nM). Further studies have shown that compounds 6e and 9i could pertain inhibition of HCC827 colony formation, and arrest HCC827 cells at G2/M phase. Moreover, the most promising compound 6e could inhibit the migration of HCC827 cells, induce HCC827 cells apoptosis, and significantly inhibit the phosphorylation of EGFR, AKT and Erk1/2. In vivo xenograft mouse model with HCC827 cells, compound 6e resulted in remarkable tumor regression without obvious toxicity. In addition, molecular docking studies suggested that compound 6e could firmly combine with T790M-mutant, T790 M/C797S-mutant, and L858R/T790 M/C797S-mutant EGFR kinases as ATP-competitive inhibitor.

Synthesis, activity, and their relationships of 2,4-diaminonicotinamide derivatives as EGFR inhibitors targeting C797S mutation

The C797S mutation is one of the major factors behind resistance to the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. Herein, we describe the discovery of the 2,4-diaminonicotinamide derivative 5j, which shows potent inhibitory activity against EGFR del19/T790M/C797S and L858R/T790M/C797S. We also report the structure–activity relationship of the 2,4-diaminonicotinamide derivatives and the co-crystal structure of 5j and EGFR del19/T790M/C797S.

Abstract A037: Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer

Abstract Molecular therapies targeting EGFR-mutant non-small cell lung cancer (NSCLC) have dramatically improved prognosis for patients with the classical activating mutations, L858R and exon 19 deletion. However, tumors harboring EGFR exon 20 insertions have been historically more difficult to target. Agents such as amivantamab and mobocertinib have recently been granted accelerated approval for treatment of lung cancer patients with exon 20 insertions, but agents with an improved selectivity profile versus wild-type EGFR are still needed. BAY 2927088, currently in phase I clinical trials, is a novel reversible inhibitor that targets EGFR exon 20 insertion mutations with decreased activity towards wild-type EGFR. Although most patients with advanced EGFR-mutant NSCLC eventually progress due to acquired resistance to EGFR inhibitors, the mechanisms of resistance to BAY 2927088 are currently unknown. Using two different methods of in vitro resistance generation by inhibitor treatment of sensitive cell lines, we identified mechanisms of intrinsic and acquired resistance to BAY 2927088 and other EGFR inhibitors. Incubation with a high concentration of inhibitor selected for drug-tolerant persister cells that underwent a transcriptional state transition resembling EMT (epithelial-mesenchymal transition). Conversely, gradual escalation of inhibitor concentrations resulted in acquisition of oncogenic NRAS mutations. In a parallel approach, we performed a deep mutational scanning assay of the EGFR D770_N771insSVD kinase domain, replacing each amino acid with every other possible amino acid, identifying both known and novel mutations that cause resistance to BAY 2927088 and other EGFR inhibitors. As expected, the C797S mutation was enriched as a mechanism of acquired resistance only to irreversible EGFR exon 20 insertion inhibitors. Surprisingly, the T790M gatekeeper mutation was found to cause resistance to all EGFR exon 20 insertion inhibitors tested, including irreversible inhibitors. Several novel resistance mutations clustering predominantly in exons 20 and 21 were also identified. Citation Format: Gizem Karsli Uzunbas, Quinn McVeigh, Akansha Gupta, Stephanie Hoyt, Kristen Doucette, Hasmik Keshishian, Steven Carr, Xiaoping Yang, David Root, Andrew D Cherniack, Franziska Siegel, Stephan Siegel, Matthew Meyerson, Heidi Greulich. Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A037.

Abstract B089: MUC1-C is a common driver of acquired osimertinb resistance in NSCLC

Abstract Background: Osimertinib is an irreversible EGFR tyrosine kinase inhibitor approved for the first-line treatment of patients with metastatic NSCLC harboring EGFR exon 19 deletions or L858R mutations. Patients treated with osimertinib invariably develop acquired resistance by mechanisms involving additional EGFR mutations, MET amplification and other pathways. There is no known involvement of the oncogenic MUC1-C protein in acquired osimertinib resistance. Methods: H1975/EGFR(L858R/T790M) and patient-derived NSCLC cells with acquired osimertinib resistance (EMT, MET amplified, C797S mutation, or PCBP2-BRAF fusion) were investigated for MUC1-C dependence in studies of EGFR pathway activation, clonogenicity and self-renewal capacity. Results: We demonstrate that MUC1-C is upregulated in H1975 osimertinib drug tolerant persister (DTP) cells and is necessary for activation of the EGFR pathway. H1975 cells selected for stable osimertinib resistance (H1975-OR) and MGH700-2D cells isolated from a patient with acquired osimertinib resistance are shown to be dependent on MUC1-C for induction of (i) p-EGFR, p-ERK and p-AKT, (ii) EMT, and (iii) the resistant phenotype. We report that MUC1-C is also required for p-EGFR, p-ERK and p-AKT activation and self-renewal capacity in acquired osimertinib-resistant (i) MET amplified MGH170-1D #2 cells, (ii) MGH121 Res#2/EGFR(T790M/C797S) cells, and (iii) MGH845-1R5/EGFR(19del)/PCBP2-BRAF fusion cells. Importantly, targeting MUC1-C in these diverse models reverses osimertinib resistance. To extend these results, we established MET amplified MGH170-1D #2 tumors in mice and found that treatment with GO-203 inhibitor, which is a cell-penetrating peptide that blocks MUC1-C homodimerization, nuclear localization, and function, results in more pronounced inhibition and that the GO-203+osimertinib combination is more effective than either agent alone. In support of these results, high MUC1 expression is associated with poor prognosis for patients with EGFR mutant NSCLCs and those treated with osimertinib. Conclusions: Our findings demonstrate that MUC1-C is a common effector of osimertinib resistance and is a potential target for the treatment of osimertinib resistant NSCLCs. Citation Format: Naoki Haratake, Hiroki Ozawa, Yoshihiro Morimoto, Nami Yamashita, Tatsuaki Daimon, Atrayee Bhattacharya, Keyi Wang, Ayako Nakashoji, Hideko Isozaki, Aaron Hata, Donald Kufe. MUC1-C is a common driver of acquired osimertinb resistance in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B089.

Abstract C025: Trial in progress: First-in-human study of BBT-207 in advanced non-small cell lung cancer harboring EGFR mutation after treatment with EGFR TKI

Abstract Background: There is a high unmet need for an effective therapy for the treatment of non-small cell lung cancer (NSCLC) following osimertinib or other 3rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) failure, especially when resistance is driven by the occurrence of the C797S mutation. BBT-207 is a fourth-generation EGFR inhibitor. Preclinically BBT-207 has shown activity against treatment-emergent complex EGFR mutations containing T790M and/or C797S as well as drug-naïve mutants providing a possibility to overcome resistance to prior TKIs. Activity to intracranial lesions is also anticipated from animal models. Methods: Study BBT207-ONC-001 is an open-label, phase 1/2 study evaluating safety, tolerability, pharmacokinetic profile, pharmacodynamic characterization, and preliminary antitumor activity of BBT-207 monotherapy in approximately 92 patients with EGFR-mutant NSCLC. It consists of 3 phases: Phase 1a (dose escalation), phase 1b (recommended phase 2 dose selection), and phase 2 (dose expansion). Key eligibility criteria include patients ≥18 years of age with advanced stage and refractory NSCLC with an activating EGFR mutation, documented partial response, complete response, or durable stable disease after treatment of an EGFR TKI, and previous treatment with all standard therapeutic options and at least one third-generation EGFR TKI. There is no limit on the number of previous treatments received. Phase 1a requires confirmation that the tumor harbors a classical EGFR mutation (19Del or L858R), and phase 1b and phase 2 require confirmation that the tumor or the blood harbors complex EGFR mutations containing C797S. Patients with asymptomatic brain metastases who are on stable dose of corticosteroid are eligible. Primary endpoints are to determine recommended dose range (RDR) which is defined between the minimal reproducibly active dose, and the maximum tolerated dose or maximum administered dose and to characterize the overall safety and tolerability of BBT-207 (phase 1a); to determine the recommended phase 2 dose (phase 1b); to evaluate preliminary antitumor activity with objective response rate (phase 2). Phase 1a dose escalation will be guided by Bayesian logistic regression model. The safety monitoring committee will determine RDR and two recommended dose levels (RDs) within the RDR to be explored in phase 1b. In phase 1b, up to 20 patients will be randomized to each of the two RDs. At the end of phase 1b, a futility test will be performed in the dose level to be treated with the RP2D using Bayesian analysis. Phase 2 will be a non-randomized, open-label phase with up to 20 patients. Patients may receive treatment until disease progression or unacceptable toxicity. Treatment continuation beyond progression will be allowed if potential benefits are justified. Investigational new drug application was cleared by the U.S. FDA. Patient enrollment will begin in approximately August 2023 in the U.S. and Republic of Korea. Clinical trial information: NCT05920135. Citation Format: NaEun Jeon, Alexander Spira, Douglas Orr, Lyudmila Bazhenova, Misako Nagasaka, Jin Seok Ahn, Dong-Wan Kim, Yu Jung Kim, Jimin Cho, Agnes Jung, Eunsoo Jung, Chul-Won Kim, Taiguang Jin, Yong-Hee Lee, Sang-Yoon Lee. Trial in progress: First-in-human study of BBT-207 in advanced non-small cell lung cancer harboring EGFR mutation after treatment with EGFR TKI [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C025.

Abstract LB_C14: EAI-432: A mutant-selective allosteric EGFR inhibitor for L858R-mutant non-small cell lung cancer

Abstract While osimertinib is highly efficacious in lung cancers with sensitizing mutations in EGFR, there is a major unmet need for agents that overcome resistance to it and other covalent third-generation EGFR TKIs due to the C797S mutation. Furthermore, osimertinib extends overall survival in patients with exon19 deletions but offers little improvement in overall survival in patients with the L858R mutation relative to the first-generation inhibitors. We are developing EAI-432 with the goal of improving outcomes for patients with EGFRL858R+ lung cancer. EAI-432 is a mutant-selective allosteric inhibitor that is effective against L858R and its common resistance variants, L858R/T790M, L858R/C797S, and L858R/T790M/C797S while sparing WT EGFR. The compound induces tumor regressions in a L858R/T790M/C797S mouse xenograft model at low doses (5 mg/kg). In the H1975 (L858R/T790M) xenograft model, EAI-432 induced marked tumor regressions in both subcutaneous and intracranial implants. EAI-432 co-binds with osimertinib, allowing double-drugging of the mutant receptor. The distinct binding site and allosteric mechanism of EAI-432 are expected to confer exquisite kinome-wide selectivity and orthogonal resistance mechanisms as compared with osimertinib and other ATP-competitive TKIs, properties that are highly desirable in a combination agent. The compound has efficacy, mutant-selectivity, and pharmacokinetic properties including brain-penetrance that support its further development as a therapeutic agent for EGFRL858R+ lung cancer alone or in combination with osimertinib. Citation Format: Michael J. Eck, David A. Scott, Pasi Janne, Tyler S. Beyett, Jie Jiang, Praful Gokhale, Thomas W. Gero. EAI-432: A mutant-selective allosteric EGFR inhibitor for L858R-mutant non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C14.

Abstract B168: Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations

Abstract Background: EGFR mutations are well validated clinical targets in non-small cell lung cancer (NSCLC). Osimertinib, a highly-selective EGFR mutation-targeting covalent drug, is increasingly used in the first line setting for patients with NSCLC bearing EGFR L858R mutation or exon 19 deletions (ex19del). In a subset of these patients, co-occurring L858R/C797x or ex19del/C797x mutations (“double mutants”) are emerging as an on-target resistance mechanism, necessitating the need for new therapies, particularly in patients with CNS metastases. Materials and methods: STX-241 was tested across a panel of in vitro biochemical, cell signaling, and proliferation assays for potency against EGFR L858R and ex19del single mutants and the corresponding C797S double mutants. Additionally, STX-241 was tested for in vivo activity in mice bearing human NSCLC cell line xenografts NCI-H3255 (L858R) and PC-9 (ex19del), and in a PC-9-derived ex19del/C797S double mutant knock-in xenograft. Free CNS penetration (Kp,uu) was determined in non-tumor bearing mice. Osimertinib and gefitinib (an approved reversible EGFR inhibitor) were used as benchmark molecules across assays. Results: STX-241, an ATP-competitive reversible EGFR inhibitor representing novel chemical matter, demonstrated potent and selective inhibition of recombinant EGFR L858R/C797S mutant protein relative to wild-type, with increased mutant residence time relative to gefitinib. Potent (high picomolar to low nanomolar) inhibition of L858R, ex19del, L858R/C797S, and ex19del/C797S mutants was observed in proliferation assays using engineered Ba/F3 and human NSCLC cell lines. STX-241 demonstrated >150x selectivity for all tested EGFR mutants relative to wild-type EGFR in engineered Ba/F3 cells and human cancer cells. In these assays, STX-241 selectivity exceeded that of the benchmark reversible EGFR inhibitor gefitinib. Strong potency and double mutant selectivity was also observed for STX-241 in pharmacodynamic assays measuring EGFR pathway activation (pEGFR). STX-241 was well tolerated at doses of 15 mg/kg BID or 50 mg/kg QD in mice, where regression of EGFR exon 19 or 21 mutant NSCLC xenografts was observed, concomitant with EGFR pathway suppression. Notably, using an isogenic pair of PC-9 (EGFR ex19del) NSCLC xenografts that differ only by the presence of a C797S mutation, STX-241 demonstrates no drop off in antitumor activity in the presence of C797S. In mouse pharmacokinetic studies measuring free CNS penetration, Kp,uu measurements were comparable to Osimertinib, run in parallel as a benchmark. Conclusions: STX-241 demonstrates strong potency and selectivity against EGFR L858R/C797S and ex19del/C797S double mutants, as well as robust CNS penetrance. These data warrant further exploration of this potential best-in-class inhibitor in the clinic for patients progressing on Osimertinib via C797x mutation. Citation Format: Raymond A Pagliarini, Benjamin C Milgram, Deanna R Borrelli, Erin O'Hearn, Michael R. Huff, Brendon Ladd, Natasja Brooijmans, Weixue Wang, Petr Kuzmic, Angel Guzman-Perez, Darrin D Stuart. Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B168.