EGF Receptor Research Articles

Multimodal scanning of genetic variants with base and prime editing

AbstractMutational scanning connects genetic variants to phenotype, enabling the interrogation of protein functions, interactions and variant pathogenicity. However, current methodologies cannot efficiently engineer customizable sets of diverse genetic variants in endogenous loci across cellular contexts in high throughput. Here, we combine cytosine and adenine base editors and a prime editor to assess the pathogenicity of a broad spectrum of variants in the epithelial growth factor receptor gene (EGFR). Using pooled base editing and prime editing guide RNA libraries, we install tens of thousands of variants spanning the full coding sequence of EGFR in multiple cell lines and assess the role of these variants in tumorigenesis and resistance to tyrosine kinase inhibitors. Our EGFR variant scan identifies important hits, supporting the robustness of the approach and revealing underappreciated routes to EGFR activation and drug response. We anticipate that multimodal precision mutational scanning can be applied broadly to characterize genetic variation in any genetic element of interest at high and single-nucleotide resolution.

Effect of biphasic in vitro maturation (CAPA-IVM) on EGF receptor and embryo development of prepubertal goat oocytes according to follicle size

Oocytes spontaneously resume meiosis following their liberation from follicles, preventing full competence acquisition. Biphasic IVM (CAPA-IVM) maintains oocytes in meiotic arrest to improve developmental competence, and it specially affects poorly developed oocytes. We assessed the effect of CAPA-IVM on oocytes from small (<3mm) and large (>3mm) follicles of prepubertal goats. Oocytes were cultured for 6h in pre-IVM with C-type natriuretic peptide (CNP) and estradiol as meiotic inhibitors, and germinal vesicle (GV) rate and chromatin configuration were assessed. Then, oocytes were cultured in conventional IVM (c-IVM) or CAPA-IVM (pre-IVM + c-IVM) and EGF receptor (EGFR) protein expression, intra-oocyte ROS and blastocyst development were assessed. GV rate was higher in CNP groups than control (69% vs 28%, and 67% vs 31%, small and large follicles, respectively), but GV chromatin configuration was similar. In large follicles, EGFR expression was higher in oocytes and cumulus cells after CAPA-IVM, and ROS levels were lower. In small follicles these differences were not observed. c-IVM and CAPA-IVM produced similar blastocyst rates in small (3.7% vs 2.6%, respectively) and large follicles (8.3% vs 2.5%). Overall, CAPA-IVM enhanced EGFR expression for EGF peptide signalling and antioxidant capacity in oocytes from large follicles but oocytes from small follicles were too immature to benefit from it.

Knockdown of Inhibin Beta A Reversed the Epithelial Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance and Enhanced the Therapeutic Effect of Radiotherapy in Non-Small Cell Lung Cancer.

Lung cancer is a malignant tumor with the highest mortality rate worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately half of all lung cancer cases. Inhibin beta A (INHBA) is a ligand of the transforming growth factor-beta superfamily. This study aimed to analyze the function of INHBA in NSCLC resistance cells. Gene Expression Omnibus and The Cancer Genome Atlas databases were used to identify differentially expressed genes (DEGs) in NSCLC resistance cells and patients. DEGs were further analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. A colony formation assay was performed to determine cell growth. Cell migration and invasion were tested by Transwell assay. Epithelial-mesenchymal transition (EMT)-related genes were analyzed using qRT-PCR and Western blot analysis. Using bioinformatics tools, INHBA was demonstrated to be overexpressed in NSCLC resistant cells and patients. Gefitinib treatment affected NSCLC resistant cells. Additionally, INHBA silencing or X-ray treatment suppressed the growth and metastasis of NSCLC resistant cells. Moreover, the combined application of INHBA silencing and X-ray treatment enhances the therapeutic effects. Moreover, EMT has been confirmed to occur in NSCLC resistant cell lines. Both INHBA silencing and X-ray treatment inhibited EMT development. This study demonstrated that INHBA silencing ameliorates EGFR-TKI resistance and enhances the therapeutic effect of radiotherapy in NSCLC.

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.

Exploring the spatial effects influencing the EGFR/ERK pathway dynamics with machine learning surrogate models

The fate of cells is regulated by biochemical reactions taking place inside of them, known as intracellular pathways. Cells display a variety of characteristics related to their shape, structure and contained fluid, which influences the diffusion of proteins and their interactions. To gain insights into the spatial effects shaping intracellular regulation, we apply machine learning (ML) to explore a previously developed spatial model of the epidermal growth factor receptor (EGFR) signaling. The model describes the reactions between molecular species inside of cells following the transient activation of EGF receptors. To train our ML models, we conduct 10,000 numerical simulations in parallel where we calculate the cumulative activation of molecules and transcription factors under various conditions such as different diffusion speeds, inactivation rates, and cell structures. We take advantage of the low computational cost of ML algorithms to investigate the effects of cell and nucleus sizes, the diffusion speed of proteins, and the inactivation rate of the Ras molecules on the activation strength of transcription factors. Our results suggest that the predictions by both neural networks and random forests yielded minimal mean square error (MSEs), while linear generalized models displayed a significant larger MSE. The exploration of the surrogate models has shown that smaller cell and nucleus radii as well, larger diffusion coefficients, and reduced inactivation rates increase the activation of transcription factors. These results are confirmed by numerical simulations. Our ML algorithms can be readily incorporated within multiscale models of tumor growth to embed the spatial effects regulating intracellular pathways, enabling the use of complex cell models within multiscale models while reducing the computational cost.

A multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.

The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor receptor (EGFR), and this was mediated by the upregulation of caveolin 1 (CAV1). This drug resistance feedback loop could be mitigated by the synergistic action of agents targeting PI3K-AKT-mTOR and EGFR for HGSOC with CAV1 and EGFR expression. Using this workflow could enable the personalized testing of patient-derived tumor samples at single-cell resolution.

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.

Heparin-binding EGF-like growth factor: mechanisms of biological activity and potential therapeutic applications

The diphtheria toxin receptor on sensitive mammalian cells is known as the membrane anchored precursor of heparin-binding EGF-like growth factor (HB-EGF). When the precursor is cleaved by metalloproteinases, a soluble form (sHB-EGF) is formed that can bind to the EGF receptors, resulting in activation of signaling pathways that regulate cell proliferation, differentiation, migration, and inhibition of apoptosis. The ability of HB-EGF to cause both positive and negative consequences for organism underscores the complexity of its biological functions and the need for a nuanced understanding of its role in health and disease. In this review the data on the HB-EGF structure, biological activity, involvement in the mechanism of diphtheria toxin action, wound healing, tumor progression as well as the methods of HB-EGF delivery are summarized. Keywords: cell proliferation, diphtheria toxin, EGF receptor, heparin-binding EGF-like growth factor, signal transduction, wound healing

4-Anilinoquinazoline Derivatives as the First Potent NOD1-RIPK2 Signaling Pathway Inhibitors at the Nanomolar Range.

Inflammation is a defense mechanism that restores tissue damage and eliminates pathogens. Among the pattern recognition receptors that recognize danger or pathogenic signals, nucleotide oligomerization domains 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses, and inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 versus NOD2 pathway inhibitors at the nanomolar range based on a 4-anilinoquinazoline scaffold. We demonstrated that NOD1 inhibition occurs through the inhibition of receptor interacting protein kinase 2 (RIPK2), which is involved in its downstream signaling pathways. Compound 37 demonstrates no cytotoxicity, a selectivity for RIPK2 over epithelial and vascular endothelial growth factor receptors (EGFR/VEGFR), and a capacity to reduce pro-inflammatory cytokine IL-8 secretion. The structure of the RIPK2-compound 37 complex was resolved by crystallography. The 4-anilinoquinazoline scaffold offers novel perspectives to design NOD1-RIPK2 signaling inhibitors.

Precise Preparation of Supramolecular Spherical Nucleic Acids for Nucleolin-Targeted Gene Delivery.

Molecular spherical nucleic acids (m-SNAs) are a second generation of spherical nucleic acids (SNAs), which are of significance in potential application of targeted delivery of nucleic acids or gene regulation due to their defined molecular structures. Nevertheless, m-SNAs typically involve a single DNA sequence which greatly limits its functions as either targeting purpose or gene regulation. In response, we proposed here a third generation, supramolecular spherical nucleic acids (Supra-SNAs) with two different sequences to achieve both above-mentioned functions. Specifically, we constructed a series of supramolecular self-assembly structures by coupling a cell membrane receptor (i.e., nucleolin)-recognizing aptamer (AS1411)-modified adamantine as targeting probe and human epithelial growth factor receptor 2 (HER2) antisense-functionalized β-cyclodextrin to specifically inhibit the overexpression of HER2 proteins for gene regulations. In comparison to the m-SNA precursors, such Supra-SNA structures exhibited enhanced levels of resistance to nuclease degradation, cellular uptake, gene regulation capabilities and tumor retention capacity. We demonstrated that Supra-SNAs exhibited optimal cell suppression rates and cell apoptosis via a phosphatidylinositol 3-kinase/protein kinase B signaling pathway. The well-defined molecular structures provide an attractive platform for investigating interrelationship between structure and property at the molecular level.

Precise Preparation of Supramolecular Spherical Nucleic Acids for Nucleolin‐Targeted Gene Delivery

AbstractMolecular spherical nucleic acids (m‐SNAs) are a second generation of spherical nucleic acids (SNAs), which are of significance in potential application of targeted delivery of nucleic acids or gene regulation due to their defined molecular structures. Nevertheless, m‐SNAs typically involve a single DNA sequence which greatly limits its functions as either targeting purpose or gene regulation. In response, we proposed here a third generation, supramolecular spherical nucleic acids (Supra‐SNAs) with two different sequences to achieve both above‐mentioned functions. Specifically, we constructed a series of supramolecular self‐assembly structures by coupling a cell membrane receptor (i.e., nucleolin)‐recognizing aptamer (AS1411)‐modified adamantine as targeting probe and human epithelial growth factor receptor 2 (HER2) antisense‐functionalized β‐cyclodextrin to specifically inhibit the overexpression of HER2 proteins for gene regulations. In comparison to the m‐SNA precursors, such Supra‐SNA structures exhibited enhanced levels of resistance to nuclease degradation, cellular uptake, gene regulation capabilities and tumor retention capacity. We demonstrated that Supra‐SNAs exhibited optimal cell suppression rates and cell apoptosis via a phosphatidylinositol 3‐kinase/protein kinase B signaling pathway. The well‐defined molecular structures provide an attractive platform for investigating interrelationship between structure and property at the molecular level.

Immuno-PET Imaging of EGFR with 64Cu-NOTA Panitumumab in Subcutaneous and Metastatic Nonsmall Cell Lung Cancer Xenografts.

Objective: About 65-90% of nonsmall cell lung cancer (NSCLC) express the epithelial growth factor receptor (EGFR) as a transmembrane protein that is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Identifying EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including the full-length human IgG2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64Cu-labeled panitumumab with immuno-PET in subcutaneous and metastatic EGFR-positive NSCLC xenografts. Methods: Bifunctional chelating agent 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA-NCS) was attached to panitumumab. The number of chelators per panitumumab was determined using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The incorporation efficiency of 64Cu into NOTA-panitumumab was measured by using radio-TLC. EGFR-expressing epithelial-like H1299-luc+ NSCLC cells were used for in vitro and in vivo experiments. Cell uptake of [64Cu]Cu-NOTA-panitumumab was measured in the presence and absence of panitumumab. Subcutaneous and metastatic H1299-luc tumor models were grown in male NSG mice. The presence of tumors at lung and metastatic sites was analyzed by [18F]FLT PET. Immuno-PET with [64Cu]Cu-NOTA-panitumumab was performed as static PET imaging at 2, 24, and 48 h postinjection in both tumor models. Proof-of-target was confirmed by blocking experiments with panitumumab. Detailed ex vivo biodistribution experiments were performed in both animal tumor models to confirm biodistribution profiles obtained by immuno-PET imaging. Results: MALDI analysis confirmed the attachment of ∼1.5 NOTA per antibody. Radiolabeling efficiency with [64Cu]CuCl2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [64Cu]Cu-NOTA-panitumumab in H1299 cells demonstrated increasing uptake over time, reaching 29.1 ± 2.9% radioactivity(Bq)/mg protein (n = 3) and plateauing at 45 min. Addition of 25 μg of panitumumab reduced radioligand uptake to 1.22 ± 0.06% radioactivity/mg protein (n = 3). PET imaging revealed high uptake of [64Cu]Cu-NOTA-panitumumab in subcutaneous tumors: Standardized uptake values (SUV)mean reached 4.70 ± 0.42 and 5.37 ± 0.40 (n = 5) after 24 and 48 h postinjection, respectively. Administration of 1 mg panitumumab reduced tumor uptake significantly to 1.94 ± 0.22 and 1.66 ± 0.08 (n = 4; p < 0.001). In the metastatic model, the following SUVmean were analyzed from liver and lung lesions: 5.55 ± 0.34 and 6.28 ± 0.46 (both n = 23 lesions from 6 mice) after 24 and 48 h postinjection, which was also significantly reduced to 2.53 ± 0.39 and 2.31 ± 0.15 (both n = 16 lesions from 4 mice; p < 0.001) after injection of 1 mg panitumumab. Detailed ex vivo biodistribution confirmed immuno-PET analysis in both models. Panitumumab reduced radioactivity uptake into subcutaneous tumors from 11.01 ± 0.72 (n = 4) to 3.67 ± 0.33% ID/g (n = 5; p < 0.001), and in metastatic liver lesions from 29.44 ± 8.14 (n = 4) to 8.35 ± 1.30% ID/g (n = 5; p < 0.001), respectively. Conclusions: [64Cu]Cu-NOTA-panitumumab was successfully used for immuno-PET imaging of EGFR-expressing subcutaneous and metastatic NSCLC tumors. This result represents the basis for developing radiotheranostics for targeting EGFR in cancers and for selecting the right patients for the right treatment at the right time.

Durable Remission After Targeted Therapy in BRAF V600E–Mutant Metastatic Colorectal Cancer: Case Report

ABSTRACT BRAF mutation leads to constitutive activation of the MAPK pathway and is associated with the immune-activating molecular subtype of colorectal cancer. Targeted therapy for BRAF V600E–mutant metastatic colorectal cancer (CRC) has significantly improved outcomes for these patients when combined with anti–epithelial growth factor receptor (EGFR) therapy. However, most patients ultimately develop disease progression. We report a case of a patient with metastatic-deficient mismatch repair, BRAF V600E–mutated CRC, who achieved a durable complete response to vemurafenib plus cetuximab and chemotherapy despite initial high burden of disease, including peritoneal involvement. Recent clinical trials of combined anti-EGFR/anti–BRAF V600E therapy in BRAF V600E-mutant CRCs have found a few instances of robust response. Further study of combined targeted and chemotherapeutic regimens and the immunogenic properties of BRAF mutation may yield promising results.

NEU4-mediated desialylation enhances the activation of the oncogenic receptors for the dissemination of ovarian carcinoma.

Glycosylation profoundly influences the interactions between cancer cells and microenvironmental stromal cells during the peritoneal disseminated metastasis of ovarian carcinoma (OC), which is the major cause of cancer-related death. Although the characteristic cancer glycoconjugates are widely used as biomarkers for cancer diagnosis, our knowledge about cancer glycome remains quite fragmented due to the technique limitations in analyzing glycan chains with tremendous structural and functional heterogeneity. Given the dysregulated cancer glycome is defined by the altered glycosylation machinery, here we performed a systematic loss-of-function screen on 498 genes involved in glycosylation for key regulators of OC dissemination. We identified neuraminidase 4 (NEU4), an enzyme capable of hydrolyzing terminal sialic acid from glycoconjugates, as a vital peritoneal dissemination-promoting modifier of OC glycome. In human patients with high-grade serous OC (HGSOC), increased NEU4 was detected in the disseminated OC cells when compared with that in the primary tumor cells, which significantly correlated with the worse survival. Among three alternative splice-generated isoforms of human NEU4, we revealed that only the plasma membrane-localized NEU4 isoform 2 (NEU4-iso2) and intracellular isoform 3 promoted the peritoneal dissemination of OC by enhancing the cell motility and epithelial-mesenchymal transition. We also identified NEU4-iso2-regulated cell surface glycoproteome and found that NEU4-iso2 desialylated the epithelial growth factor receptor (EGFR), in particular at N196 residue, for the hyperactivation of EGFR and its downstream tumor-promoting signaling cascades. Our results provide new insights into how the OC glycome is dysregulated during OC progression and reveal a functionally important glycosite on EGFR for its abnormal activation in cancer.

Targeted nanoliposomes of oncogenic protein degraders: Significant inhibition of tumor in lung-cancer bearing mice

With 60 % of non-small cell lung cancer (NSCLC) expressing epidermal growth factor receptor (EGFR), it has been explored as an important therapeutic target for lung tumors. However, even the well-established EGFR inhibitors tend to promptly develop resistance over time. Moreover, strategies that could impede resistance development and be advantageous for both EGFR-Tyrosine kinase inhibitor (TKI)-sensitive and mutant NSCLC patients are constrained. Based on the critical relationship between EGFR, c-MYC, and Kirsten rat sarcoma virus (K-Ras), simultaneous degradation of EGFR and Bromodomain-containing protein 4 (BRD4) using “Proteolysis Targeting Chimeras (PROTACs)” could be a promising approach. PROTACs are emerging class of oncoprotein degraders but very challanging to deliver in vivo. Compared to individual IC50s, strong synergism was observed at 1:1 ratio of BPRO and EPRO in NSCLC cell lines with diverse mutation. Significant inhibition of cell growth with higher cellular apoptosis was observed in 2D and 3D-based cell assays in nanomolar concentrations. EGFR activation assay revealed 47.60 % EGFR non-expressing cells confirming EGFR-degrading potential of EPRO. A lung cancer specific nanoliposomal formulation of EGFR and BRD4-degrading PROTACs (EPRO and BPRO) was prepared and characetrized. Successful encapsulation of the two highly lipophilic molecules was achieved in EGFR-targeting nanoliposomal carriers (T-BEPRO) using a modified hydration technique. T-BEPRO revealed a particle size of 109.22 ± 0.266 nm with enhanced cellular uptake and activity. Remarkably, parenterally delivered T-BEPRO in tumor-bearing mice showed a substantially higher % tumor growth inhibition (TGI) of 77.6 % with long-lasting tumor inhibitory potential as opposed to individual drugs.

Redox Signaling in Endosomes Using the Example of EGF Receptors: A Graphical Review.

Early endosomes represent first-line sorting compartments or even organelles for internalized molecules. They enable the transport of molecules or ligands to other compartments of the cell, such as lysosomes, for degradation or recycle them back to the membrane by various mechanisms. Moreover, early endosomes function as signaling and scaffolding platforms to initiate or prolong distinct signaling pathways. Accordingly, early endosomes have to be recognized as either part of a degradation or recycling pathway. The physical proximity of many ligand-binding receptors with other membrane-bound proteins or complexes such as NADPH oxidases may result in an interaction of second messengers, like reactive oxygen species (ROS) and early endosomes, that promote the correct recognition of individual early endosomes. In fact, redoxosomes comprise an endosomal subsection of signaling endosomes. One example of such potential interaction is epidermal growth factor receptor (EGFR) signaling. Here we summarize recent findings on EGFR signaling as a well-studied example for receptor trafficking and trans-activation and illustrate the interplay between cellular and endosomal ROS.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.

Potential Use of Extracellular Vesicles for the HER2 Status Assessment in Breast Cancer Patients.

Human epithelial growth factor receptor 2 (HER2)-targeted therapies are effective in patients with HER2-positive breast cancer. Recent advances have shown that HER2-targeted therapies can also be of benefit when treating tumors expressing low levels of HER2, highlighting the importance of identifying the HER2-low subgroup. This clinical trend has opened new therapeutic avenues for patients who were previously ineligible for HER2-targeted therapies. Thus, the development of new diagnostic methods for real-time HER2 profiling is crucial for accurately tailoring the treatment for these patients. We hypothesized that tumor-derived extracellular vesicles (TEVs) could reflect the HER2 profiles of primary tumors and potentially serve as diagnostic tools for HER2 status. This approach was validated using six breast cancer cell lines, which confirmed that the TEVs accurately reflected the HER2 profiles of the tumor cells. TEVs were isolated using an immunoaffinity method, and copy number variation (CNV) in the ERBB2/EIF2C ratio was assessed using droplet digital PCR of DNA from these vesicles. Clinical validation using plasma samples from 33 breast cancer patients further reinforced the diagnostic potential of our method. Pearson's correlation coefficient analysis of the flow cytometry results demonstrated that TEVs reflected HER2 expression in primary cells. To distinguish between HER2-negative and HER2-low patients, the area under the curve (AUC) of the ROC curve in our method was 0.796, with a sensitivity of 53.8% and a specificity of 100%. These findings suggest the clinical utility of extracellular vesicles derived from plasma and emphasize the need for further research to distinguish HER2-negative from HER2-low patients.

Changes in cytokine profile as a criterion for severity prediction of skin toxicity in cancer patients receiving epidermal growth factor receptor inhibitors

BACKGROUND: Globally, colorectal cancer is the third most diagnosed cancer with an increased incidence of 1.93 million cases in 2020 according to World Health Organization. Approximately one quarter of the initial manifestations of colorectal cancer are metastatic, and 40–50% of individuals with early-stage disease eventually develop metastatic colorectal cancer (mCRC). Targeted drugs, which include epidermal growth factor receptor inhibitors (EGFRi), are a frequent choice in the therapy of metastatic colorectal cancer. Clinical observations and in vivo studies have established a significant association between the administration of EGFRi drugs and skin inflammation, particularly acute in the first 3 months. However, the role of proinflammatory cytokines in the formation of severe skin lesions has not been definitively studied. Here we present clinical evidence of the involvement of a few cytokines in the formation of the pathologic cascade of reactions during EGFR blockade on keratinocyte membranes. AIM: Evaluation of changes in cytokine profile parameters based on the MILLIPLEX Analyte MAP panel to identify predictors of the severity of unwanted skin toxicity associated with epidermal growth factor receptor inhibition. MATERIALS AND METHODS: Blood serum samples from 81 patients aged 18–80 years who were taking EGFRi for oncologic disease and had adverse dermatologic reactions of severity 2 or more were analyzed. The data were analyzed using SPSS software and R 4.3.1 programming language and tidyverse, rstatix packages. Detected changes with p 0.05 were analyzed considering subgroup analysis on the basis of selected cutaneous toxicity severity degrees based on the criteria developed by the US National Cancer Institute NCI-CTC v. 4. RESULTS: Blocking of EGF receptor inhibits the expression and release of vascular endothelial growth factor (VEGF), which is the main inducer of vascular proliferation, the consequence of which is inflammation of vascular endothelium of skin capillaries. Significant increase of IFN-γ and decrease of IFN-α2 level was found in patients with manifestations of skin toxicity of 2 and more severity degree. Also, a significant criterion of severity of the skin process was an increase in the level of TNF-α. At such dissociation there is induction of STING protein (interferon gene stimulator) and increase of TNF-β production that, in its turn, leads to increase of concentration of proinflammatory cytokines G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-15, IL-17A, IL-1β, IL-3, IL-6, IL-8, IP-10, TNF-α, TGF-α, IL-9. This contributes not only to the maintenance of tissue inflammation, but also to the formation of a vicious circle leading to an increase in the severity of class-mediated inflammatory response against the background of further EGFRi administration. CONCLUSION: Determination of predictors of severity of adverse dermatologic reactions is extremely important for predicting the development of severity and further personalized tactics for correction of adverse events.

Abstract C012: Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 12%, with limited treatment options. Oncogenic KRAS mutations drive PDAC tumor progression, which occur in the majority of PDAC tumors. KRAS G12D is the most common KRAS mutation, found in 36% of PDAC cases, which is associated with the worst clinical outcomes. Mutant-selective KRAS G12D inhibitors (KRASi G12D), such as MRTX1133, have recently demonstrated initial responses in KRAS G12D-mutant preclinical models. However, mechanisms of adaptive resistance to KRAS G12D inhibition are largely under-investigated and novel therapeutic strategies to overcome drug resistance are urgently needed. Results: Adaptive RAS signaling reactivation was found to dampen KRAS G12D inhibition in a library of KRAS G12D PDAC cell lines, thereby minimizing drug efficacy. Quantitative downstream analyses of drug combination screening using a KRAS G12D cell line library further identified this adaption to KRAS inhibition was cell subtype specific. Fibroblast growth factor receptor (FGFRs) drove adaptive RAS reactivation specifically in mesenchymal PDAC cells, which are aggressive, and intrinsically resistant to KRASi. In contrast, epithelial growth factor receptor (EGFR) reactivated RAS G12D signaling in epithelial cells, which are sensitive to KRASi G12D. Genetic and pharmacological inhibition of FGFR1 or FGFR2 improved drug efficacy by decreasing FGFR-mediated RAS reactivation specifically in mesenchymal PDAC lines. Pharmacological blockade of EGFR enhanced drug efficacy by inhibiting EGFR-mediated RAS reactivation specifically in epithelial PDAC lines. Furthermore, low-concentration of a novel pan- RAS inhibitor, which inhibits both wild-type and mutant RAS, completely abrogated MAPK signaling and significantly improved efficacy, when combined with KRASi G12D, suggesting a pro-survival dependency on wild-type RAS as a compensatory mechanism in response to KRAS G12D inhibition. Conclusion: Upstream RTK-mediated adaptive RAS signaling re-activation in response to KRAS inhibition in PDAC is cell subtype specific. Complete inhibition of RAS signaling by overcoming adaptive RAS rebound improves efficacy of KRASi G12D. Citation Format: Qingxiang (Nick) Lin, Alvin Morales, Haley Barnes, Ryan Bruce Corcoran. Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C012.