Mutant Epidermal Growth Factor Receptor Research Articles

Discovery of novel EGFR-PROTACs capable of degradation of multiple EGFR-mutated proteins

Although three generations of Epidermal growth factor receptor (EGFR) - TK inhibitors have been approved for the treatment of Non-small-cell lung cancers (NSCLC), their clinical application is still largely hindered by acquired drug resistance mediated new EGFR mutations and side effects. The Proteolysis targeting chimera (PROTAC) technology has the potential to overcome acquired resistance from mutant EGFR through a novel mechanism of action. In this study, we developed the candidate degrader IV-3 by structural modifications of the lead compound 13, which exhibited limited antiproliferative activity against HCC-827 cells. Compared to compound 13, IV-3 exhibited remarkable anti-proliferative activity against HCC-827 cells, NCI–H1975 cells, and NCI–H1975-TM cells (IC50 = 0.009 μM, 0.49 μM and 3.24 μM, respectively), as well as significantly inducing degradation of EGFR protein in these cell lines (DC50 = 17.93 nM, 0.25 μM and 0.63 μM, respectively). Further investigations confirmed that IV-3 exhibited superior anti-tumor activity in all xenograft tumor models through the degradation of mutant EGFR protein. Moreover, IV-3 showed no inhibitory activity against A431 and A549 cells expressing wild-type EGFR, thereby eliminating potential toxic side effects emerging from wild-type EGFR inhibition. Overall, our study provides promising insights into EGFR-PROTACs as a potential therapeutic strategy against EGFR-acquired mutation.

GraphEGFR: Multi-task and transfer learning based on molecular graph attention mechanism and fingerprints improving inhibitor bioactivity prediction for EGFR family proteins on data scarcity.

The proteins within the human epidermal growth factor receptor (EGFR) family, members of the tyrosine kinase receptor family, play a pivotal role in the molecular mechanisms driving the development of various tumors. Tyrosine kinase inhibitors, key compounds in targeted therapy, encounter challenges in cancer treatment due to emerging drug resistance mutations. Consequently, machine learning has undergone significant evolution to address the challenges of cancer drug discovery related to EGFR family proteins. However, the application of deep learning in this area is hindered by inherent difficulties associated with small-scale data, particularly the risk of overfitting. Moreover, the design of a model architecture that facilitates learning through multi-task and transfer learning, coupled with appropriate molecular representation, poses substantial challenges. In this study, we introduce GraphEGFR, a deep learning regression model designed to enhance molecular representation and model architecture for predicting the bioactivity of inhibitors against both wild-type and mutant EGFR family proteins. GraphEGFR integrates a graph attention mechanism for molecular graphs with deep and convolutional neural networks for molecular fingerprints. We observed that GraphEGFR models employing multi-task and transfer learning strategies generally achieve predictive performance comparable to existing competitive methods. The integration of molecular graphs and fingerprints adeptly captures relationships between atoms and enables both global and local pattern recognition. We further validated potential multi-targeted inhibitors for wild-type and mutant HER1 kinases, exploring key amino acid residues through molecular dynamics simulations to understand molecular interactions. This predictive model offers a robust strategy that could significantly contribute to overcoming the challenges of developing deep learning models for drug discovery with limited data and exploring new frontiers in multi-targeted kinase drug discovery for EGFR family proteins.

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.

Targeting EGFR degradation by autophagosome degraders

Several generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been developed for the treatment of non-small cell lung cancer (NSCLC) in clinic. However, emerging drug resistance mediated by new EGFR mutations or activations by pass, leads to malignant progression of NSCLC. Proteolysis targeting chimeras (PROTACs) have been utilized to overcome the drug resistance acquired by mutant EGFR, newly potent and selective degraders are still need to be developed for clinical applications. Herein, we developed autophagosome-tethering compounds (ATTECs) in which EGFR can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of the LC3 ligand GW5074. A series of EGFR-ATTECs have been designed and synthesized. Biological evaluations showed that these compounds could degrade EGFR and exhibited moderate inhibitory effects on certain NSCLC cell lines. The ATTEC 12c potently induced the degradation of EGFR with a DC50 value of 0.98 μM and a Dmax value of 81% in HCC827 cells. Mechanistic exploration revealed that the lysosomal pathway was mainly involved in this degradation. Compound 12c also exhibited promising inhibitory activity, as well as degradation efficiency in vivo. Our study highlights that EGFR-ATTECs could be developed as a new expandable EGFR degradation tool and also reveals a novel potential therapeutic strategy to prevent drug resistance acquired EGFR mutations.

Abstract 4474: Novel analogs of 4-substituted pyrrolo[2,3-d]pyrimidines as dual-targeted mEGFR/AURKA inhibitors for lung cancer

Abstract Small molecule, mutant epidermal growth factor receptor kinase (mEGFR) inhibitors such as osimertinib and erlotinib constitute the recommended therapy for mEGFR-positive non-small cell lung cancer (NSCLC). However, mEGFR inhibitors are ineffective in NSCLC over time due to mutations within mEGFR and redundant signaling pathways such as mutant KRAS (mKRAS) that bypass mEGFR inhibition. Simultaneous inhibition of multiple kinases has been suggested to provide synergistic effects on tumor growth inhibition and tumor resistance. Studies have shown that resistance to mEGFR inhibitors was overcome when mEGFR inhibitors were combined with aurora kinase (AURK) inhibitors. Additionally, mEGFR inhibitors were found to retain their anticancer effects in mKRAS-positive NSCLC cells if given concurrently with AURK inhibitors. Kurup et al. previously reported a series of 4-substituted pyrrolo[2,3-d]pyrimidines as dual mEGFR/AURKB inhibitors. Novel analogs of 1-5 have been designed to incorporate AURKA inhibition while maintaining mEGFR inhibition. Molecular modeling was utilized to guide selectivity for AURKA over AURKB. This study led to the identification of novel dual-targeted mEGFR/AURKA inhibitors with demonstrated inhibition of mKRAS+ NSCLC and mEGFR+ NSCLC. The design, synthesis, kinase inhibitory activities, and anticancer effects for these novel analogs will be presented. Structure-activity relationships for dual mEGFR/AURKA inhibition will be defined. Citation Format: Sonali Kurup, Gabe Carpenter, Samantha Satawa, Felix Amissah, Erika Lisabeth, Matthew Giletto, Edmund Ellsworth. Novel analogs of 4-substituted pyrrolo[2,3-d]pyrimidines as dual-targeted mEGFR/AURKA inhibitors for lung cancer [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 4474.

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 3290: FTO mediates tumor growth and resistance to erlotinib in NSCLC

Abstract Lung cancer, among the most common cancer types, ranks first in cancer associated deaths. Nearly 85% of the lung cancer cases are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR), an established marker for NSCLC, is frequently overexpressed on the cell surface. Amplifying mutations often render these receptors activated in a ligand independent manner, therefore, therapies such as erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), target these activating mutations and inhibit tumor growth. Nevertheless, patients acquire resistance to EGFR TKI within 9-14 months of treatment. Recent studies show that fat mass and obesity- associated (FTO) gene, initially identified to increase the body mass index, plays a major role in cancer development and acquired therapy resistance in several cancer types. FTO demethylates m6A mRNA and modulates tumorigenesis, and drug resistance. In this study, we explored the functions of FTO in EGFR TKI resistance in NSCLC. To study the expression of FTO in erlotinib resistant cells, we used wildtype-EGFR NSCLC cell lines (H2170, H358) and mutant EGFR NSCLC cell lines (H1975, PC9) that were rendered resistant to erlotinib. Our RT-qPCR results demonstrated that FTO was upregulated by 1.7-3.0 fold in erlotinib resistant H2170, H358, PC9 and H1975 cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting, and by 1.7 to 2.6 fold in the nucleus of erlotinib resistant H1975, H2170 and PC9 cell lines by immunofluorescence. Furthermore, we investigated FTO inhibition with Dac51 (FTOi) on cell viability and cell migration properties. MTT cell viability assay showed a 9-17% decrease in cell viability when treated with a combination of Dac51 and erlotinib compared to Dac51 alone in erlotinib resistant H2170 and PC9 cells, respectively. The combinatory treatment showed an additive effect compared to the Dac51 and erlotinib treatments alone. Wound healing assay revealed an 18% open wound area after 48-hour treatment with both Dac51 and erlotinib compared to 0.1% after treatment with erlotinib alone in erlotinib resistant H2170 cells. To evaluate the effect of siRNA mediated FTO (siFTO) knockdown on cell viability and energy metabolism, MTT and luciferase assays were performed. MTT assay showed a 21-28% decrease in viability in erlotinib resistant H1975, H2170, PC9 cells, demonstrating increase in the erlotinib efficacy, as compared to the mock siRNA treatment. Luciferase assay showed an inverse relationship between the FTO and ATP, where the latter was upregulated by 1.3 to 3.4 folds in erlotinib resistant H1975, H2170 and PC9 cells, when treated with siFTO, suggesting its involvement in the energy requirements of the NSCLC cells. In conclusion, our study suggests FTO was upregulated in erlotinib resistant NSCLC cells on both the gene and protein levels. FTO could mediate resistance to EGFR TKI possibly through cell proliferation and migration and help with prognosis of NSCLC patients. Citation Format: Aayush Rastogi, Rong Qiu, Neelu Puri. FTO mediates tumor growth and resistance to erlotinib 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 3290.

Abstract 6027: 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts

Abstract Background: About 80% of colorectal cancer (CRC) and 50 % of Triple-Negative breast cancer (TNBC) patients overexpress epidermal growth factor receptor (EGFR). Mutations in the KRAS oncogene (present in 40% of mCRC) lead to constitutive over-activation of EGFR and drive de novo resistance to anti-EGFR drugs. Here, we propose to target KRAS-mutant/BRAF mutant EGFR positive mCRC and TNBC using 225Ac-labeled matuzumab, a humanized anti-EGFR monoclonal antibody. Methods: p- -SCN-macropa was used to conjugate matuzumab for radiolabeling with 225Ac and SCN-deferoxamine was used to conjugate matuzumab for labelling with 89Zr. The radioimmunoconjugates were characterized by flow cytometry, HPLC and iTLC. in vitro cytotoxicity was evaluated in EGFR-positive mCRC, TNBC cell lines and 3D spheroids with different levels of EGFR density. Tumor growth was monitored using digital caliper. Mice were treated with either 10 MBq of 89Zr-matuzumab for imaging or three doses of 13 KBq/dose administered of 225Ac-matuzumab 10 days apart. In vivo study endpoint was tumor volume greater than or equal to 1500 mm3. Results: Flow cytometry showed about 95% binding to the cells in all EGFR-positive colorectal cell lines (DLD-1, SW620, SNU-C2B, HT-29) and breast cancer cell lines (MDA-MB-468, MDA-MB-231). in vitro studies showed enhanced cytotoxicity of 225Ac-matuzumab compared with matuzumab. IC50 in the MDA-MB-468 cell line for 225Ac-matuzumab (1.86 ± nM) was 35 times more effective than matuzumab (65.25± 7 nM). Similar trends were observed in the other KRAS-mutant mCRC cell lines and breast cancer cell lines. In 3D spheroid models, the IC50 of 225Ac-matuzumab (3.0 ± 2 nM) in MDA-MB-468, was 20.79 times more effective than matuzumab (62.3 ± 1.38 nM). Similar trends were observed for all the other spheroid models as well. High uptake of 89Zr-matuzumab was observed in both mCRC and TNBC xenografts. 225Ac-matuzumab slowed tumor growth rate in MDA-MB-468 and MDA-MB-231 tumors models compared with control antibody or non-treated controls for 30 days. This study is still ongoing and will last for 2 more months. Conclusion: 225Ac-matuzumab shows very promising outcomes in KRAS-mutant mCRC models and breast cancer models and warrants further investigation. Citation Format: Florence Anjong Tikum, Humphrey Fonge, Fabrice Njotu, Hanan Babeker, Jessica K. Pougoue, Nikita Henning, Alireza Doroudi. 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts [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 6027.

Abstract 3264: Characterization of EGFR ectodomain mutation in acquired resistance to cetuximab in colorectal cancer

Abstract Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States (US). Individuals with surgically unresectable stage four CRC have a five-year overall survival rate of 13%. Targeting of the receptor tyrosine kinase (RTK), EGF receptor (EGFR) with therapeutic monoclonal antibodies (cetuximab, panitumumab) is approved for use in wild-type (WT) KRAS/NRAS/BRAF (RAS/RAF) advanced CRC. However, these antibodies as a single agent have response rates of 12-17% and expected progression-free survivals of only 3-4 months. Accordingly, resistance may be present at the outset (de novo resistance) or develop during treatment (acquired resistance). Here, we report the identification of an EGFR ectodomain missense mutation acquired in vitro in response to treatment with anti-epidermal growth factor receptor (EGFR) antibody cetuximab. Using 2D and 3D drug sensitivity assays, our results demonstrate that this EGFR ectodomain mutation in addition to being resistant to cetuximab and panitumumab, also could not be targeted by the oligoclonal antibody cocktail MM-151. To determine whether this EGFR ectodomain mutation is necessary and sufficient to impart cetuximab resistance, we engineered CRC lines to inducibly overexpress mutant EGFR and showed that this was sufficient to induce cetuximab resistance. Further structural, and functional characterization is underway for this ectodomain mutation that confers resistance to cetuximab and will be shared during the meeting. These findings necessitate the need for additional treatment strategies for a subset of subjects with mCRC who do not respond to treatment with cetuximab/panitumumab and MM-151. Citation Format: Neeraj Joshi, Ramona Graves Deal, Galina Bogatcheva, James N. Higginbotham, Marisol Ramirez, Benjamin P. Brown, Qi Liu, Jens Meiler, Bhuminder Singh. Characterization of EGFR ectodomain mutation in acquired resistance to cetuximab in colorectal cancer [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 3264.

Next-generation sequencing reveals genetic heterogeneity and resistance mechanisms in patients with EGFR-mutated non-small cell lung cancer treated with afatinib.

Afatinib, an irreversible ErbB family inhibitor, is widely used as first-line treatment in advanced lung adenocarcinoma patients harbouring mutant epidermal growth factor receptor (EGFR). With the advancements in next-generation sequencing (NGS), comprehensive research into the clinical impact of co-occurring genetic mutations and the molecular mechanisms of acquired resistance is required for afatinib users. From January 2010 to December 2019, we enrolled patients with advanced lung adenocarcinoma with EGFR mutations using afatinib as first-line treatment, and we retrospectively collected pre- and post-afatinib treatment specimens from these patients for NGS testing. Of the 362 enrolled patients, 73 samples (68.9%) from 56 patients successfully returned complete NGS reports. In pre-afatinib treatment specimens, the most frequent co-occurring alterations were TP53, MUC16, USH2A, SNYE1, RECQL4 and FAT1; however, they were not related to progression-free survival. Small cell lung cancer transformation, EGFR p.T790M, amplification of MET, ERBB2, KRAS, EGFR, cell cycle-regulated genes and MDM2, and PTEN alterations were identified as acquired resistance mechanisms. EGFR p.T790M (p=0.0304) and APC alterations (p=0.0311) in post-afatinib specimens were significantly associated with longer overall survival, while MET amplification was significantly associated with poor overall survival (p=0.0324). The co-occurrence of TP53 alterations was significantly associated with shorter overall survival (p=0.0298). Our results show that the frequent co-occurring alterations in advanced EGFR-mutated lung adenocarcinoma did not influence the effectiveness of afatinib. EGFR p.T790M is not only the major resistance mechanism to afatinib but also related to favourable survival outcomes. MET amplification and TP53 mutations were associated with poorer overall survival.

Investigating the activity of Quinazoline derivatives against T790 mutant EGFR receptors employing computer-aided drug design

The uncontrolled growth of cancer cells is caused by their innate tendency to multiply without limits. In addition to colonizing adjacent tissues, they are capable of forming aggregates in isolated anatomical sites, thereby significantly increasing their invasiveness. Over time, malignant tumors that progressively destroy essential tissues and organs are ultimately lethal. It is well known that growth factor-driven signaling influence malignancy. The transmembrane receptor tyrosine kinases of growth factors influence the survival, differentiation, and proliferation of cells. To expedite cell division, neurotransmitters and proteins resembling epidermal growth factor (EGF) stimulate EGF receptor (EGFR) family members including HER2-4 and EGFR. The development and growth of numerous varieties of human cancer cells, as well as the morphogenesis of numerous animal species (nematodes to humans), are influenced by this signaling module, which is remarkably conserved. The EGFR family, consisting of over thirty ligands and four receptors, forms a complex and advanced human signal transduction network. The T790M mutation in the EGFR kinase enhances treatment resistance by increasing the affinity for ATP. This work used a molecular design tool to evaluate quinazoline derivatives with the highest IC50 values against EGFR and analyze the molecular interactions in the binding site. The goal was to uncover novel compounds with promising potential.

Tissue and Plasma-Based Highly Sensitive Blocker Displacement Amplicon Nanopore Sequencing for EGFR Mutations in Lung Cancer.

The epidermal growth factor receptor (EGFR) mutation is a widely prevalent oncogene driver in non-small cell lung cancer (NSCLC) in East Asia. The detection of EGFR mutations is a standard biomarker test performed routinely in patients with NSCLC for the selection of targeted therapy. Here, our objective was to develop a portable new technique for detecting EGFR (19Del, T790M, and L858R) mutations based on Nanopore sequencing. The assay employed a blocker displacement amplification (BDA)-based polymerase chain reaction (PCR) technique combined with Nanopore sequencing to detect EGFR mutations. Mutant and wild-type EGFR clones were generated from DNA from H1650 (19Del heterozygous) and H1975 (T790M and L858R heterozygous) lung cancer cell lines. Then, they were mixed to assess the performance of this technique for detecting low variant allele frequencies (VAFs). Subsequently, formalin-fixed, paraffin-embedded (FFPE) tissue and cell-free DNA (cfDNA) from patients with NSCLC were used for clinical validation. The assay can detect low VAF at 0.5% mutant mixed in wild-type EGFR. Using FFPE DNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and Cobas real-time PCR were 98.46%, 100%, and 100%, respectively. For cfDNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and droplet digital PCR were 94.74%, 100%, and 100%, respectively. The BDA amplicon Nanopore sequencing is a highly accurate and sensitive method for the detection of EGFR mutations in clinical specimens.

High-fat diet induces C-reactive protein secretion, promoting lung adenocarcinoma via immune microenvironment modulation

ABSTRACT To understand the effects of a high-fat diet (HFD) on lung cancer progression and biomarkers, we here used an inducible mutant epidermal growth factor receptor (EGFR)-driven lung cancer transgenic mouse model fed a regular diet (RD) or HFD. The HFD lung cancer (LC-HFD) group exhibited significant tumor formation and deterioration, such as higher EGFR activity and proliferation marker expression, compared with the RD lung cancer (LC-RD) group. Transcriptomic analysis of the lung tissues revealed that the significantly changed genes in the LC-HFD group were highly enriched in immune-related signaling pathways, suggesting that an HFD alters the immune microenvironment to promote tumor growth. Cytokine and adipokine arrays combined with a comprehensive analysis using meta-database software indicated upregulation of C-reactive protein (CRP) in the LC-HFD group, which presented with increased lung cancer proliferation and metastasis; this was confirmed experimentally. Our results imply that an HFD can turn the tumor growth environment into an immune-related pro-tumorigenic microenvironment and demonstrate that CRP has a role in promoting lung cancer development in this microenvironment.

Targeting lung cancer with clinically relevant EGFR mutations using anti-EGFR RNA aptamer

A significant fraction of non-small cell lung cancer (NSCLC) cases are due to oncogenic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Anti-EGFR antibodies have shown limited clinical benefit for NSCLC, whereas tyrosine kinase inhibitors (TKIs) are effective, but resistance ultimately occurs. The current landscape suggests that alternative ligands that target wild-type and mutant EGFRs are desirable for targeted therapy or drug delivery development. Here we evaluate NSCLC targeting using an anti-EGFR aptamer (MinE07). We demonstrate that interaction sites of MinE07 overlap with clinically relevant antibodies targeting extracellular domain III and that MinE07 retains binding to EGFR harboring the most common oncogenic and resistance mutations. When MinE07 was linked to an anti-c-Met aptamer, the EGFR/c-Met bispecific aptamer (bsApt) showed superior labeling of NSCLC cells invitro relative to monospecific aptamers. However, dual targeting invivo did not improve the recognition of NSCLC xenografts compared to MinE07. Interestingly, biodistribution of Cy7-labeled bsApt differed significantly from Alexa Fluor 750-labeled bsApt. Overall, our findings demonstrate that aptamer formulations containing MinE07 can target ectopic lung cancer without additional stabilization or PEGylation and highlights the potential of MinE07 as a targeting reagent for the recognition of NSCLC harboring clinically relevant EGFRs.

SUMOylation of AnxA6 facilitates EGFR-PKCα complex formation to suppress epithelial cancer growth

BackgroundThe Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells.MethodsCells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC–MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6K299R plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFRT790M/L858R was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model.ResultsAnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6K299R showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6K299R conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6K299R mutation-expressing cells were much greater than that of AnxA6-expressing cells.ConclusionsBesides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect.4V8MbH9D2-V-dxkGbe_YjXVideo

The relation between epidermal growth factor receptor mutations profiles and smoking patterns in patients with lung adenocarcinoma: A cross‐sectional study

AbstractBackgroundNon‐small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, with smoking being a critical risk factor. The identification of NSCLC patients harboring epidermal growth factor receptor (EGFR) mutations, sensitized to tyrosine kinase inhibitors, has revolutionized treatment plans, resulting in improved clinical responses and reduced chemotherapy toxicity. This study aimed to assess the relationship between EGFR mutations and smoking patterns in patients diagnosed with lung adenocarcinoma referred to major pathologic laboratories.MethodsThis cross‐sectional study included 217 NSCLC patients aged above 18 years. Molecular abnormalities of the EGFR gene were analyzed by polymerase chain reaction amplification of exons 18–21 accompanied by Sanger sequencing. Then, the data were analyzed using the SPSS 26 software. Logistic regression analysis, χ2 test, and Mann–Whitney U test were used to evaluate the relation between EGFR mutations and smoking patterns.ResultsEGFR mutations were identified in 25.3% of patients, predominantly involving deletion in exon 19 (61.8%). For most of the mutant EGFR patients, the majority were nonsmokers (81.8%), and 52.7% were female patients. Besides, the median duration of smoking was 26 years and the median frequency of smoking was 23 pack‐years in the mutant EGFR group, both of which were lower compared to the wild mutant group. Moreover, female gender, current, and heavy smoking were significantly correlated with EGFR mutations based on the univariate logistic regression analysis (p: 0.004, 0.005, and 0.001, respectively).ConclusionsFemale gender and nonsmoker status were strongly associated with positive EGFR mutations. While guidelines traditionally recommended EGFR testing primarily for female nonsmokers with advanced NSCLC, our study in line with the recently published evidence has shown a significant prevalence of positive EGFR mutations among male patients and smokers. Therefore, routine mutation testing is suggested for all NSCLC patients. Considering the limited access to EGFR testing laboratories in developing countries, the results of such epidemiological surveys can assist oncologists in choosing the most suitable treatment plan.

Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

Non-small-cell lung cancer (NSCLC) is a typical inflammation-associated cancer, and lung adenocarcinoma (LUAD) is the most common pathological subtype. Epidermal growth factor (EGF) receptor (EGFR) mutations are the most common driver mutations of LUAD, and they have been identified as important therapeutic targets by EGFR-tyrosine kinase inhibitors (TKIs). The proinflammatory cytokine, interleukin (IL)-17A, and IL-17A-producing cells were reported to be elevated in the tumor microenvironment and peripheral blood of NSCLC patients and to be correlated with tumor progression and poor prognoses. However, the pathophysiological role of IL-17A in NSCLC remains unclear, although some studies suggested its involvement in cancer cell invasion and metastasis. Herein, we observed that expressions of IL-17A and its receptor, IL-17 receptor C (IL-17RC), were elevated in LUAD tissues and were correlated with poor survival in different lung cancer cohorts. In LUAD cells with mutant EGFR, the IL-17A/IL-17RC axis was shown to enhance phosphorylation of EGFR and Met, thereby promoting proliferation and resistance to EGFR-TKIs such as afatinib. In LUAD cells with wild-type (WT) EGFR, we found that the IL-17A/IL-17RC axis enhanced EGF-induced EGFR activation and cell proliferation through causing impairment of EGF-induced EGFR lysosomal degradation. Collectively, our results indicated diverse impacts of the IL-17A/IL-17RC axis on EGFR activation in LUAD cells with WT and mutant EGFR and suggested that developing therapeutic strategies against IL-17A/IL-17RC would be valuable for LUAD treatment.

Arsenic Inhibits Proliferation and Induces Autophagy of Tumor Cells in Pleural Effusion of Patients with Non-Small Cell Lung Cancer Expressing EGFR with or without Mutations via PI3K/AKT/mTOR Pathway.

To clarify whether arsenic could exert inhibitory effects on tumor cells in pleural effusions of patients with non-small cell lung cancer (NSCLC), 36 NSCLC pleural effusion samples were collected from Changzheng Hospital and Ruijin Hospital, from 2019 to 2022. The genotype of epidermal growth factor receptor (EGFR) was identified. Tumor cells were isolated and treated with arsenic trioxide (ATO) or/and gefitinib. Additionally, six patients were intrapleurally administrated with ATO. Results showed that 25 samples bore EGFR wild type (WT) and 11 harbored EGFR mutations, including 6 with L858R, 3 with ΔE746-A750, and 2 with T790M. ATO diminished the number of tumor cells from patients with WT and mutant EGFR, down-regulated the expression or phosphorylation of EGFR, pmTOR, PI3K, PTEN, and p4E-BP1, and up-regulated the expression of LC3. Immunofluorescent experiments showed that ATO enhanced LC3 and P62. By contrast, gefitinib was only effective in those harboring EGFR sensitizing mutations. Notably, in patients with intrapleural ATO injection, the pleural effusion underwent a bloody to pale yellow color change, the volume of the pleural effusion was reduced, and the number of the tumor cells was significantly reduced. In conclusion, arsenic is effective against NSCLC with various EGFR genotypes in vitro and in vivo, and potentially circumvents gefitinib resistance.

Inhibition of PLK1 Destabilizes EGFR and Sensitizes EGFR-Mutated Lung Cancer Cells to Small Molecule Inhibitor Osimertinib.

Tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) have significantly prolonged survival in EGFR-mutant non-small cell lung cancer patients. However, the development of resistance mechanisms prohibits the curative potential of EGFR TKIs. Combination therapies emerge as a valuable approach to preventing or delaying disease progression. Here, we investigated the combined inhibition of polo-like kinase 1 (PLK1) and EGFR in TKI-sensitive EGFR-mutant NSCLC cells. The pharmacological inhibition of PLK1 destabilized EGFR levels and sensitized NSCLC cells to Osimertinib through induction of apoptosis. In addition, we found that c-Cbl, a ubiquitin ligase of EGFR, is a direct phosphorylation target of PLK1 and PLK1 impacts the stability of c-Cbl in a kinase-dependent manner. In conclusion, we describe a novel interaction between mutant EGFR and PLK1 that may be exploited in the clinic. Co-targeting PLK1 and EGFR may improve and prolong the clinical response to EGFR TKI in patients with an EGFR-mutated NSCLC.

Structural identification of novel pyrimidine derivatives as epidermal growth factor receptor inhibitors using 3D QSAR, molecular docking, and MMGBSA analysis: a rational approach in anticancer drug design

Non-small cell lung cancer (NSCLC) has evolved into the deadliest in the present scenario. The progression of NSCLC is mainly due to the dysregulation of the tyrosine kinase family's epidermal growth factor receptor (EGFR). Thus, EGFR has been widely studied as a major target in the treatment of NSCLC, but the lack of selectivity and drug resistance limit the use of existing therapeutic agents. Considering the urgent necessity for the advanced development of EGFR inhibitors, we have implemented a three-dimensional structure-activity relationship (3D QSAR), molecular docking, and MMGBSA studies on a series of pyrimidine derivatives. In the 3D QSAR, the comparative molecular field analysis model (CoMFA) was obtained with a correlation coefficient (r<sup>2</sup>) = 0.698, cross-validated correlation coefficient (q<sup>2</sup>) = 0.541, and predictive r<sup>2</sup> (r<sup>2</sup><sub>pred</sub>) = 0.509. The comparative molecular similarity indices analysis (CoMSIA) model also displayed similar results with r<sup>2 </sup>= 0.72, q<sup>2 </sup>= 0.586, and r<sup>2</sup><sub>pred</sub>= 0.495. The statistical parameters fulfill the acceptability criteria of the models. Docking studies revealed the binding interactions of the pyrimidine derivatives with double mutant EGFR<sup>L858R/T790M</sup>. Docking scores of the top two selected compounds 29 and 34 were 92.99 and 92.13, respectively. Analyzing 3D QSAR contour plots and docking results reviewed some important structural attributes of EGFR <sup>L858R/T790M</sup> selective inhibitors, which directed the designing of some new molecules. The designed compounds showed good predictive activity and exhibited higher binding interactions with EGFRL858R/T790M than the reference ligand gefitinib. Moreover, to evaluate the binding of selected top hits from docking and designed compounds, MMGBSA (Molecular Mechanics-Generalized Born Surface Area) analysis was performed, which revealed that the designed compound (N7) showed a good binding affinity with EGFR<sup>L858R/T790M</sup> (dG = -68.59 kcal/mol) as compared to other compounds. Further, in silico ADME predictions revealed the drug-likeness of the designed compounds. Thus, this work will guide researchers in future developments of pyrimidine derivatives as EGFR inhibitors.