Epidermal Growth Factor Receptor Inhibitors Research Articles

Special entities of the head and neck region: cancers of the nasopharynx, (para)nasal cavities, salivary glands, and the thyroid gland : Post ASCO 2024

Malignancies of the nasopharynx (NPC), the (para)nasal cavities, the salivary glands, and the thyroid gland are distinct to head and neck squamous cell carcinomas (HNSCC) in the oro-/hypopharynx and larynx in terms of etiology, tumor biology, and the therapeutic concept. The contributions to the 2024 American Society of Oncology (ASCO) Annual Meeting provide insight into the latest developments in these "special entities of the head and neck region." Abstracts were examined for their clinical relevance and placed into context with current therapeutic concepts. In the treatment of locally advanced NPC, arandomized phaseIII study showed equivalence of induction (ICT) and adjuvant therapy (AT; NCT03306121). PD-1inhibitors have become established in the palliative therapy of NPC in recent years and could now also play an increasing role in curation: the phaseIII study "Dipper" showed asignificantly better 3‑year event-free survival in patients adjuvantly treated with camrelizumab versus placebo after IT and definitive platinum-containing chemoradiotherapy (dRCT; 89% vs.80%; NCT03427827). The phaseIII study "Beacon" showed complete remission in 30.5% of patients after IT with gemcitabine/cisplatin and the PD‑1 inhibitor tislelizumab (three cycles), arate almost twice as high as with gemcitabine/cisplatin alone (NCT05211232). Intensification of dRCT in NPC using EGFR and VEGF inhibitors appears promising (NCT04447326). Abstracts on salivary gland and nasal and sinus cancers emphasize the importance of targeted therapies. In anaplastic thyroid carcinoma, the combination of aPD‑1 inhibitor and aCTLA4 inhibitor showed a50% response.

Design and Optimization of Quinazoline Derivatives as Potent EGFR Inhibitors for Lung Cancer Treatment: A Comprehensive QSAR, ADMET, and Molecular Modeling Investigation.

The epidermal growth factor receptor (EGFR) is part of a protein family that controls cell growth and development. Due to its importance, EGFR has been identified as a suitable target for creating novel drugs. For this research, we conducted a 2D-QSAR analysis on a set of 31 molecules derived from quinazoline, which exhibited inhibitory activity against human lung cancer. This investigation incorporated principal component analysis (PCA) and multiple linear regression (MLR), leading to the development of QSAR models with strong predictive capabilities (R 2 = 0.745, R 2_adj = 0.723, MSE = 0.061, R 2_test = 0.941, and Q 2_cv = 0.669). The reliability of these models was confirmed through internal, external, Y-randomization, and applicability domain validations. Leveraging the predictions from the QSAR model, we designed 18 new molecules based on the modifications at the N-3 and C-6 positions of the quinazoline ring, with electronegative substituents at these positions fostering optimal polar interactions and hydrophobic contacts within the ATP-binding site of EGFR, significantly enhancing the inhibitory activity against the lung cancer cell line. Subsequently, ADMET predictions were conducted for these 18 compounds, revealing outstanding ADMET profiles. Molecular docking analyses were performed to investigate the interactions between the newly designed molecules-Pred15, Pred17, Pred20, Pred21-and the EGFR protein, indicating high affinity of these proposed compounds to EGFR. Furthermore, molecular dynamics (MD) simulations were utilized to assess the stability and binding modes of compounds Pred17, Pred20, and Pred21, reinforcing their potential as novel inhibitors against human lung cancer. Overall, our findings suggest that these investigated compounds can serve as effective inhibitors, showcasing the utility of our analytical and design approach in the identification of promising therapeutic agents.

Design and Synthesis of Dual Galectin-3 and EGFR Inhibitors Against Liver Fibrosis.

Liver fibrosis, mainly arising from chronic viral or metabolic liver diseases, is a significant global health concern. There is currently only one FDA-approved drug (Resmetirom) in the market to combat liver fibrosis. Both galectin-3 and epidermal growth factor receptor (EGFR) play important roles in liver fibrosis, while galectin-3 may interact with EGFR. Galectin-3 inhibitors, typically lactose or galactose derivatives may inhibit liver fibrosis. We hypothesized that targeting both galectin-3 and EGFR may have better effect against liver fibrosis. Here, EGFR inhibitor erlotinib was used in a series of designed galectin-3 inhibitors after hybridization with the pharmacophore structure in reported galectin-3 inhibitors to impede hepatic stellate cells (HSCs) activation by a typical method of click chemistry. Bioactivity test results showed that compound 29 suppressed TGF-β-induced upregulation of fibrotic markers (α-SMA, fibronectin-1, and collagen I). The preferred compound 29 displayed better binding to galectin-3 (KD = 52.29 μM) and EGFR protein (KD = 3.31 μM) by SPR assay. Further docking studies were performed to clarify the possible binding mode of compound 29 with galectin-3 and EGFR. Taken together, these results suggested that compound 29 could be a potential dual galectin-3 and EGFR inhibitor as leading compound for anti-liver fibrosis new drug development.

Development of a FUT8 Inhibitor with Cellular Inhibitory Properties.

Core fucosylation is catalyzed by α-1,6-fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N-glycans. Given the association of FUT8 with various diseases, including cancer, selective FUT8 inhibitors applicable to in vivo or cell-based systems are highly sought-after. Herein, we report the discovery of a compound that selectively inhibits FUT8 in cell-based assays. High-throughput screening revealed a FUT8-inhibiting pharmacophore, and further structural optimization yielded an inhibitor with a KD value of 49 nM. Notably, this binding occurs only in the presence of GDP (a product of the enzymatic reaction catalyzed by FUT8). Mechanistic studies suggested that this inhibitor generates a highly reactive naphthoquinone methide derivative at the binding site in FUT8, which subsequently reacts with FUT8. Furthermore, prodrug derivatization of this inhibitor improved its stability, enabling suppression of core fucose expression and subsequent EGFR and T-cell signaling in cell-based assays, paving the way for the development of drugs targeting core fucosylation.

Targeting the EGFR and Spindle Assembly Checkpoint Pathways in Oral Cancer: A Plausible Alliance to Enhance Cell Death.

Background/Objectives: Head and neck cancer (HNC) is among the most common cancer types globally, with its incidence expected to increase significantly in the coming years. Oral squamous cell carcinoma (OSCC), the predominant subtype, exhibits significant heterogeneity and resistance to treatment. Current therapies, including surgery, radiation, and chemotherapy, often result in poor outcomes for advanced stages. Cetuximab, an EGFR inhibitor, is widely used but faces limitations. This study explores the combined inhibition of EGFR and mitotic proteins to enhance treatment efficacy. Methods: We analyzed the effects of co-treating OSCC cells with small molecules targeting MPS-1 (BAY1217389), Aurora-B (Barasertib), or KSP (Ispinesib), alongside Cetuximab. The rationale is based on targeting EGFR-mediated survival pathways and the mitotic checkpoint, addressing multiple cell cycle phases and reducing resistance. Results: Our findings indicate that inhibiting MPS-1, Aurora-B, or KSP enhances Cetuximab's therapeutic potential, promoting increased cancer cell death. Additionally, we examined EGFR, MPS-1, Aurora-B, and KSP expression in OSCC patient samples, revealing their clinicopathologic significance. Conclusions: This combinatorial approach suggests a promising strategy to improve treatment outcomes in OSCC.

Bleeding in Lung Cancer Patients Receiving Therapeutic Anticoagulation While Being Treated with VEGF and EGFR Inhibitors

Bleeding in Lung Cancer Patients Receiving Therapeutic Anticoagulation While Being Treated with VEGF and EGFR Inhibitors

Design, in silico studies and biological evaluation of novel chalcones tethered triazolo[3,4-a]isoquinoline as EGFR inhibitors targeting resistance in non-small cell lung cancer

A novel series of six [1,2,4]triazolo[3,4-a]isoquinolin-3-yl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-ones (3a–3f) was designed and synthesized. They were characterized based on spectral and elemental analyses. In silico studies were also committed to provide insights and a better understanding of their structural features. The six compounds were screened for their antiproliferative activity using the MTT assay against five human cancer cell lines, namely, A549, HCT116, PC3, HT29, and MCF-7 in parallel with the non-cancerous human lung cell line WI-38. The results showed that 3e and 3f have potential cytotoxic activities, especially on A549 cells with IC50 = 2.3 µM and 1.15 µM, respectively. Meanwhile, they recorded a minimal cytotoxic effect on WI-38 cells. Concerning the molecular mechanism of action, the present study showed the inhibitory effect of the six compounds against total EGFR. The most potent EGFR inhibitors were 3e and 3f with IC50 = 0.031 µM and 0.023 µM, respectively. The selectivity index of 3f for EGFRT790M was 1.81 times more selective than that of lapatinib. In addition, 3e and 3f initiated cell cycle arrest at the G2/M and pre-G1 phases along with the downregulation of anti-apoptotic protein Bcl2 and the upregulation of pro-apoptotic proteins: p53, Bax, and caspases 3, 8, and 9. Further studies are recommended to evaluate animal models’ promising anticancer activity and molecular mechanism of triazolo[3,4-a]isoquinoline derivatives 3e and 3f.

Synthesis of S-alkylated oxadiazole bearing imidazo[2,1-b]thiazole derivatives targeting breast cancer: In vitro cytotoxic evaluation and in vivo radioactive tracing studies

Breast cancer is the most common invasive cancer diagnosed in women, accounting for most cancer-related fatalities globally. Numerous investigations have revealed that breast cancer is characterized by abnormal expression and maintenance of EGFR levels. In terms of structural study and optimization of several EGFR inhibitors, two series of oxadiazole bearing imidazo[2,1-b]thiazole derivatives were designed and synthesized as potential EGFR inhibitors and assessed for their antitumor activity at NCI-USA. Four derivatives 3b, 3c, 3d and 3e elicited remarkable GI% against MDA-MB-468, T-47D and MCF-7 breast cancer cell lines. Thereafter, MTT assay was performed to reveal that compounds 3b (IC50 = 2.27 µM) and 3d (IC50 = 1.46 µM) showed promising cytotoxic activity against MCF-7 and MDA-MB-468 cell lines, respectively, compared to their reference drugs. Compounds 3b, 3d and 3e revealed good selectivity toward tumor cells with reasonable safety profile when tested against the normal cell line MCF-10a. In vitro EGFR inhibitory assay demonstrated that compounds 3b (IC50 = 0.099 µM) and 3d (IC50 = 0.086 µM) exhibited comparable inhibitory activity to the standard drug erlotinib (IC50 = 0.046 µM). A flow cytometric analysis demonstrated that derivatives 3b and 3d arrested the cell cycle at the S phase in MCF-7 and MDB-MB-468, respectively. Furthermore, the most active derivative 3d was subjected to in vivo radioactive studies. In-vivo biodistribution of 99mTc-3d complex showed a notable elevated accumulation in the targeted sarcoma muscle, indicating the targeting capacity of compound 3d in the tumor of sarcoma mice model. The binding mode of compounds 3b and 3d with EGFR was studied by molecular docking and was further inspected by molecular dynamic simulations. Both compounds were shown to be stable during the course of simulation and demonstrated a plausible interaction pattern with the EGFR binding pocket.

Precision-cut liver slices as an ex vivo model to evaluate antifibrotic therapies for liver fibrosis and cirrhosis.

Considering the lack of successful treatment options and poor prognosis for cirrhosis and cirrhosis-induced HCC, new platforms to investigate antifibrotic therapies are urgently needed. Precision-cut liver slice (PCLS) is a powerful ex vivo culture model that can supplement and potentially replace the traditional models. PCLS were prepared from 4 different murine cirrhotic models (choline-deficient, l-amino acid-defined, high-fat diet, thioacetamide, diethylnitrosamine, and carbon tetrachloride) and compared with in vivo murine experiments, in vitro hepatic stellate cells, and human cirrhotic PCLS. PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGF receptor inhibitor, significantly inhibited profibrogenic gene expressions in PCLS from choline-deficient, l-amino acid-defined, high-fat diet or thioacetamide-induced cirrhotic rats. Erlotinib treatment of PCLS from diethylnitrosamine or carbon tetrachloride-induced cirrhotic rats inhibited the expression of profibrogenic genes, which was consistent with the impact of erlotinib on these genes in in vivo diethylnitrosamine or carbon tetrachloride-induced cirrhosis. In addition, in hepatic stellate cells at PCLS from normal mice, erlotinib treatment inhibited TGF-β1-upregulated expression of Acta2. Similar expression results were observed in in vitro hepatic stellate cells. Expression of key regulators of fibrosis progression and regression were also significantly altered. Changes in profibrogenic gene expression under erlotinib treatment were also corroborated with human cirrhotic PCLS. Responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed in vivo and in vitro. These results were verified in human cirrhotic PCLS. PCLS is an excellent model for assessing antifibrotic therapies that are aligned with the principles of replacement, reduction, and refinement (3Rs), and it will benefit preclinical and clinical research for human fibrosis and cirrhosis.

A Concise Review of Synthetic Strategy, Mechanism of Action, and SAR Studies of Phthalazine Derivatives as Anticancer Agent

Background: Colorectal cancer is the third foremost cause of death in women and men. Globally, about 1.94 million colon cancer cases were diagnosed and around 0.93 million patients died in the previous year. Introduction: Several drugs have been permitted by the Food And Drug Administration (FDA) for the treatment of colorectal cancer. The main difficulties of current drugs are the expansion of resistance issues, target selectivity issues and toxicity issues. The existing therapies, such as surgery and hormonal therapy, are in use but exhibit numerous adverse effects, such as pharmacokinetic issues and pharmacodynamic issues. Hence, hereby is a crucial requirement of novel moieties that are peaceable and efficient in the handling of colorectal cancer. Methods: Phthalazine derivatives have expanded admiration over a few years due to their efficient anticancer significance. These Phthalazine derivatives exhibit anticancer activity by targeting various mechanisms such as apoptosis induction, tubulin polymerization inhibition, EGFR inhibition, and aurora kinase inhibition. Results: In this study, we have focused on the Structural Activity relationship, numerous synthetic strategies and mechanism of action of phthalazine derivatives for potential treatment of cancer. Conclusion: Among some of phthalazine derivative compounds not only induced antiproliferative activity even also improved bioavailability and reduced side effects, like 4-(phthalazine-1-yl) aniline with (IC50 = 0.22 ± 0.11 μM), and 4-phthalazin-1-yl-amino) benzonitrile (IC50 = 1.20 μM), 4-((5- methyl-pyrazole-3-yl) amino)-2-phenylphthalazin-1-one (IC50 = 0.031 μM) and 4-((5-methylpyrazole- 3-yl) amino)-2-(p-tolyl)phthalazin-1-one (IC50 = 0.065 μM). Therefore, this study would be the inspiration for the betterment of human health.

Chemical Composition, In vitro and In silico Evaluation of Essential Oil Extracted from Mentha Piperita L. for Lung Cancer

Background: Mentha piperita, a naturally occurring herb, is utilized in medicinal formulations. It possesses abundant bioactive elements, including flavonoids and phenolic acid compounds,that exhibit various properties such as antioxidants, anti-inflammatory and anti-cancer. Objective: In the present study, chemical constituents of essential oil extracted from Mentha piperita were analyzed and identified through GC-MS. In vitro antiproliferative activity was performed on A549 lung cancer cell line lines. In silico study was conducted by Schrodinger’s Maestro’s software to identify chemical constituents in the plant as potential EGFR (Epidermal Growth Factor Receptors) inhibitors Methods: Hydro-distilled essential oil was analyzed by GC-MS to identify chemical components based on the retention index and mass fragmentation pattern, which was then tested for its antiproliferative activity by MTT assay against human lung cancer cell lines. All the identified constituents were investigated in silico for their affinity towards EGFR (Epidermal Growth Factor Receptors). Results: A total of thirty constituents were identified where D-carvone (56.69%), L-limonene (12.36%), squalene (3.36%), cis-carveol (2.93%), and α-amorphene (2.36%) were observed as major constituents of the essential oil. The essential mentha oil also exhibited antiproliferative activity against lung cancer cell lines with an IC50 value of 86.05 µg/ml. Furthermore, from the in silico study, five constituents were identified to have a better affinity for EGFR (Epidermal Growth Factor Receptors) than that of the standard drug Osimertinib. Conclusion: In the present study, the aerial part of the plant Mentha piperita was hydrodistilled.Thirty phytoconstituents were identified through GC-MS data. An in-silico study was performed using Schrodinger software, and a further in vitro study was performed in which essential oil showedgood antiproliferative activity against the A549 cancer cell line.

Oral solid dosage form using alternate crystalline neratinib maleate anhydrous form: Pharmaceutical, bioequivalent, and clinical perspectives.

Neratinib (an active pharmaceutical ingredient [API]) is an irreversible pan-human epidermal growth factor receptor (HER) inhibitor used to treat HER2-positive breast cancer. The dosage form (marketed in the United States, China, Europe, and other regions) is a tablet for oral administration, and the brand product (NERLYNX) has patent protection for the crystalline neratinib maleate monohydrate form. This paper describes the product development using stable crystalline neratinib maleate anhydrous form, stability study, bioequivalence (BE) study of the products, and discussion of the adverse effects observed in the clinical study.

Inhibition of EGFR Pathway Suppresses M1 Macrophage Polarization and Osteoclastogenesis, Mitigating Titanium Particle-Induced Bone Resorption.

The polarization of macrophages towards the pro-inflammatory M1 phenotype and osteoclast overactivation play a significant role in the pathogenesis of aseptic loosening of orthopedic implants. This study sought to examine the expression and activation of macrophages and osteoclasts in implant biopsies with respect to epidermal growth factor receptor (EGFR) signaling and to assess the potential of EGFR inhibition in mitigating titanium particle-induced bone resorption in a cranial resorption murine model. Bone marrow-derived macrophages (BMDMs) were stimulated with Tumor Necrosis Factor-alpha (TNF-α) and Interferon-gamma (IFN-γ) initially. Subsequently, Osteoclast differentiation was initiated after Gefitinib was added to the treatment groups. Male C57BL/6 mice were treated with Gefitinib or 0.5% Carboxymethyl Cellulose-Sodium (CMC-Na) by oral gavage daily for two weeks. A sham group received no further intervention, while the other groups had titanium particles implanted. Tissues were collected and analyzed by measurements such as micro-computed tomography (micro-CT) analyses, histology, immunofluorescence stainings, cell viability assays, assays for resorption pit formation, Reverse Transcription-Polymerase Chain Reactions (RT-PCRs), and Western blots were conducted. The study demonstrated a significant upregulation of EGFR in response to titanium particle exposure. Inhibition of EGFR phosphorylation with gefitinib effectively reduced bone degradation at osteolytic sites in a murine model. Gefitinib treatment led to a notable reduction in M1 macrophage polarization, as indicated by immunofluorescence staining and Western blot analysis of macrophage markers. Mechanistically, selective EGFR inhibitors mitigate osteoclastogenesis and osteoclast resorption by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling pathways. Our findings provide compelling evidence of the essential role of EGFR-related pathways in M1 polarization, osteoclast activation, and ensuing periprosthetic osteolysis. Overall, EGFR presents a novel target for addressing bone resorption-associated conditions triggered by particles or modulated by macrophages and osteoclasts.

Machine Learning Models to Predict Bone Metastasis Risk in Patients With Lung Cancer.

The aim of this study was to find the most appropriate variables to input into machine learning algorithms to identify those patients with primary lung malignancy with high risk for metastasis to the bone. Patients with either histological or radiological diagnoses of lung cancer were included in this study. The patient cohort comprised 1864 patients diagnosed from 2016 to 2021. A total of 25 variables were considered as potential risk factors. These variables have been identified in previous studies as independent risk factors for bone metastasis. Treatment methods for lung cancer were taken into account during model development. The outcome variable was binary, (presence or absence of bone metastasis) with follow-up until death or 12-month survival, whichever is the sooner. Results showed that American Joint Committee on Cancer staging, the use of EGFR inhibitor, age, T-staging, and lymphovascular invasion were the five input features contributing the most to the model algorithm. High AJCC staging (OR 1.98; p < 0.05), the use of EGFR inhibitor (OR 6.14; p < 0.05), high T-staging (OR 1.47; p < 0.05), and the presence of lymphovascular invasion (OR 4.92; p < 0.05) increase predicted risk of bone metastasis. Conversely, older age reduces predicted bone metastasis risk (OR 0.98; p < 0.05). The machine learning model developed in this study can be easily incorporated into the hospital's Clinical Management System so that input variables can be immediately utilized to give an accurate prediction of bone metastatic risk, therefore informing clinicians on the best treatment strategy for that individual patient.

Functional Hexafluoroisopropyl Group Used in the Construction of Biologically Important Pyrimidine Derivatives.

A series of versatile 4-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)pyridine intermediates have been developed to efficiently produce biaryls, amines, ethers, and thioethers. These hydrolysis-stable ether intermediates exhibit reactivity toward electron-donating groups and nucleophiles in cross-coupling and nucleophilic substitution reactions while surpassing the stability of corresponding aryl halides. In comparison to conventional coupling methods, this protocol offers an alternative pathway for accessing natural product and drug-like compounds without the need for metal catalysts. With assistance of this approach, we successfully obtained a potent P-glycoprotein inhibitor 4k (YS-370), a potent epidermal growth factor receptor inhibitor 4l (YS-363), and a promising lysine-specific demethylase 1 inhibitor 5g.

Epidermal growth factor receptor signaling governs the host inflammatory response to invasive aspergillosis.

The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small-airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole-lung cytokine and chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts.IMPORTANCEWhen A. fumigatus infects the lungs, it invades epithelial cells that line the airways. During this process, the fungus interacts with epithelial cell receptors. This interaction stimulates epithelial cells to endocytose the fungus. It also induces these cells to secrete proinflammatory cytokines and chemokines that recruit phagocytes to the site of infection where they can kill the fungus. Here, we show that in small-airway epithelial cells, the EGFR acts as a sensor for A. fumigatus that triggers the production of chemokines in response to fungal infection. In corticosteroid-immunosuppressed mice, blocking EGFR with the kinase inhibitor gefitinib reduces chemokine production in the lungs. This leads to decreased accumulation of neutrophils and dendritic cells in the lungs, reduced A. fumigatus killing, and increased mortality. These results provide a potential explanation as to why some cancer patients who are treated with EGFR inhibitors develop invasive aspergillosis.

Squamous Cell Carcinoma: From Diagnosis to Effective Treatment-A Comprehensive Guide

Squamous cell carcinoma (SCC) is the second most common malignant skin cancer, and its occurrence is on the rise globally. The main risk factors are cumulative sun exposure, immunosuppression, and inflammation. Early diagnosis and treatment are crucial to prevent metastasis and improve patient outcomes. Advanced diagnostic techniques, such as dermoscopy and reflectance confocal microscopy (RCM), have improved the accuracy of SCC diagnosis. Dermoscopy allows for better identification and targeted biopsy of suspicious areas, while RCM provides noninvasive and precise monitoring of treatment progress. The treatment of SCC has also advanced significantly in recent years. For patients with severe actinic damage and multiple in situ/low-risk SCC, cancer treatment has shown promising results. This approach involves the use of topical agents to target the tumor and the surrounding areas where cancer may develop. Additionally, novel therapies, such as epidermal growth factor receptor inhibitors and immune checkpoint inhibitors have been developed for locally advanced and metastatic SCC. In conclusion, the incidence of SCC has been increasing globally, and early diagnosis and treatment are crucial to prevent metastasis and improve patient outcomes. Advanced diagnostic techn

Biochemical analysis of EGFR exon20 insertion variants insASV and insSVD and their inhibitor sensitivity

Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer. Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs). Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations. Here, we conducted biochemical analysis of the two most frequent exon 20 insertion variants, V769_D770insASV (insASV) and D770_N771insSVD (insSVD) to better understand their drug sensitivity and resistance. From kinetic studies, we found that EGFR insASV and insSVD are similarly active, but have lower Km, ATP values compared to the L858R variant, which contributes to their lack of sensitivity to 1st-3rd generation EGFR TKIs. Biochemical, structural, and cellular studies of a diverse panel of EGFR inhibitors revealed that the more recently developed compounds BAY-568, TAS6417, and TAK-788 inhibit EGFR insASV and insSVD in a mutant-selective manner, with BAY-568 being the most potent and selective versus wild-type (WT) EGFR. Cocrystal structures with WT EGFR reveal the binding modes of each of these inhibitors and of poziotinib, a potent but not mutantselective inhibitor, and together they define interactions shared by the mutant-selective agents. Collectively, our results show that these exon20 insertion variants are not inherently inhibitor resistant, rather they differ in their drug sensitivity from WT EGFR. However, they are similar to each other, indicating that a single inhibitor should be effective for several of the diverse exon 20 insertion variants.

Alamar Blue assay optimization to minimize drug interference and inter assay viability

Alamar Blue (AB) has become an increasingly popular reagent of choice for cell viability assays. We chose AB over other reagents such as MTT and Cell-Titer Glo due to its cost effectiveness and its ability to be a nondestructive assay. While analyzing the effect of osimertinib, an EGFR inhibitor on the non-small cell lung cancer cell line, PC-9, we noticed unexpected right-shifts of dose response curves as compared to the curves obtained by Cell Titer Glo assay. Here, we describe our modified AB assay method to avoid that right shift in dose response curves.•Removal of the drug containing medium prior to AB addition eliminated the falsely increased readings, giving comparable dose response curves as determined by Cell Titer Glo assay.•Plate-to-plate variability can be minimized by adding an appropriate concentration of a common fluorescence calibration standard to the assay plates to calibrate fluorimeter sensitivity.•Alamar Blue can be used as a continuous longitudinal assay to monitor cell growth or recovery from drug toxicity over time.

Transactivation of the EGF receptor as a novel desensitization mechanism for G protein-coupled receptors, illustrated by dopamine D2-like and β2 adrenergic receptors

Transactivation of epidermal growth factor receptors (EGFR) provides intricate control over multiple regulatory cellular processes that merge the diversity of G protein-coupled receptors (GPCRs) with the robust signaling capacities of receptor tyrosine kinases. Contrary to the typical assertions, our findings demonstrate that EGFR transactivation contributes to the desensitization of GPCRs. Repeated agonist stimulation of certain GPCRs enhanced EGFR transactivation, triggering a series of cellular events associated with GPCR desensitization. This effect was observed in receptors undergoing desensitization (D3R, K149C-D2R, β2AR) but not in those resistant to desensitization (D2R, C147K-D3R, D4R, β2AR mutants lacking GRK2 or GRK6 phosphorylation sites). The EGFR inhibitor AG1478 prevented both desensitization and the associated cellular events. Similarly, these cellular events were also observed when cells were treated with EGF, but only in GPCRs that undergo desensitization. These findings suggest that EGFR transactivation diversifies pathways involved in ERK activation through the EGFR signaling system and also mediates GPCR desensitization. Alongside the widely accepted steric hindrance model, these findings offer new insights into understanding the mechanisms of GPCR desensitization, which occurs through complex cellular processes.