Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Resistance Research Articles

Tamarix aphylla derived metabolites ameliorate indomethacin-induced gastric ulcers in rats by modulating the MAPK signaling pathway, alleviating oxidative stress and inflammation: In vivo study supported by pharmacological network analysis.

Nature has proven to be a treasure resource of bioactive metabolites. In this regard, Tamarix aphylla (F. Tamaricaceae) leaves crude extract was investigated for its gastroprotective effect against indomethacin-induced damage to the gastric mucosa. Additionally, phytochemical investigation of the methanolic extract afforded eight flavonoids' derivatives (1-8). On pharmacology networking study, the isolated compounds identified 123 unique targets where only 45 targets were related to peptic ulcer conditions, these 45 targets include 11 targets specifically correlate to gastric ulcer. The protein-protein interaction defined the PTGS2 gene as one of the highly interacted genes and the complete pharmacology network defined the PTGS2 gene as the most represented gene. The top KEGG signaling pathways according to fold enrichment analysis was the EGFR tyrosine kinase inhibitor resistance pathway. As a result, these findings highlighted the significance of using T. aphylla leaves crude extract as an anti-gastric ulcer candidate, which provides a safer option to chemical antisecretory medicines, which are infamous for their negative side effects. Our findings have illuminated the potent anti-inflammatory and antioxidant effects of T. aphylla, which are likely mediated by suppressing IL-1β, IL-6, TNF-α, and MAPK signaling pathways, without compromising gastric acidity.

Evaluation of drug resistance for EGFR-TKIs in Lung Cancer via Multicellular lung-on-a-chip

Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research.

Polyphyllin I ameliorates gefitinib resistance and inhibits the VEGF/VEGFR2/p38 pathway by targeting HIF-1a in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been administered as the first-line therapy for patients with EGFR mutations in LUAD, but it is almost inevitable that resistance to EGFR-TKIs therapy eventually arises. Polyphyllin I (PPI), derived from Paris polyphylla rhizomes, has been shown to have potent anti-cancer properties in a range of human cancer types including LUAD. However, the role of PPI in gefitinib resistance and the underlying mechanism remain elusive. PurposeTo evaluate the antitumor impacts of PPI on gefitinib resistance cells and investigate its molecular mechanism. MethodsCCK-8, wound healing, transwell assay, and xenograft model were performed to determine the anti-cancer effects of PPI as well as its ability to overcome gefitinib resistance. Immunoblotting, co-immunoprecipitation, phospho-RTK antibody array, qRT-PCR, and immunofluorescence were utilized to explore the mechanism by which PPI overrides gefitinib resistance. ResultsPPI inhibited cell survival, growth, and migration/invasion in both gefitinib-sensitive (PC9) and -resistant (PC9/GR) LUAD cells (IC50 at 2.0 μM). Significantly, treatment with PPI at 1.0 μM resensitized the resistant cells to gefitinib. Moreover, cell-derived xenograft experiments revealed that the combination of PPI and gefitinib overcame gefitinib resistance. The phospho-RTK array and immunoblotting analyses showed PPI significant inhibition of the VEGFR/p38 pathway. In addition, molecular docking suggested the interaction between PPI and HIF-1α. Mechanistically, PPI reduced the protein expression of HIF-1α in both normoxia and hypoxia conditions by triggering HIF-1α degradation. Moreover, HIF-1α protein but not mRNA level was elevated in gefitinib-resistant LUAD. We further demonstrated that PPI considerably facilitated the binding of HIF-1α to VHL. ConclusionsWe present a novel discovery demonstrating that PPI effectively counteracts gefitinib resistance in LUAD by modulating the VEGF/VEGFR/p38 pathway. Mechanistic investigations unveil that PPI facilitates the formation of the HIF-1α /VHL complex, leading to the degradation of HIF-1α and subsequent inhibition of angiogenesis. These findings uncover a previously unidentified mechanism governing HIF-1α expression in reaction to PPI, providing a promising method for therapeutic interventions targeting EGFR-TKI resistance in LUAD.

MiRSNP rs188493331: A key player in genetic control of microRNA-induced pathway activation in hypertrophic scars and keloids.

Our study aims to delineate the miRSNP-microRNA-gene-pathway interactions in the context of hypertrophic scars (HS) and keloids. We performed a computational biology study involving differential expression analysis to identify genes and their mRNAs in HS and keloid tissues compared to normal skin, identifying key hub genes and enriching their functional roles, comprehensively analyzing microRNA-target genes and related signaling pathways through bioinformatics, identifying MiRSNPs, and constructing a pathway-based network to illustrate miRSNP-miRNA-gene-signaling pathway interactions. Our results revealed a total of 429 hub genes, with a strong enrichment in signaling pathways related to proteoglycans in cancer, focal adhesion, TGF-β, PI3K/Akt, and EGFR tyrosine kinase inhibitor resistance. Particularly noteworthy was the substantial crosstalk between the focal adhesion and PI3K/Akt signaling pathways, making them more susceptible to regulation by microRNAs. We also identified specific miRNAs, including miRNA-1279, miRNA-429, and miRNA-302e, which harbored multiple SNP loci, with miRSNPs rs188493331 and rs78979933 exerting control over a significant number of miRNA target genes. Furthermore, we observed that miRSNP rs188493331 shared a location with microRNA302e, microRNA202a-3p, and microRNA20b-5p, and these three microRNAs collectively targeted the gene LAMA3, which is integral to the focal adhesion signaling pathway. The study successfully unveils the complex interactions between miRSNPs, miRNAs, genes, and signaling pathways, shedding light on the genetic factors contributing to HS and keloid formation.

Elucidation of the Molecular Mechanism of Compound Danshen Dripping Pills Against Angina Pectoris based on Network Pharmacology and Molecular Docking.

Compound Danshen dripping pills (CDDP), a traditional Chinese medicine, has had an extensive application in the treatment of angina pectoris (AP) in China. However, research on the bioactive ingredients and underlying mechanisms of CDDP in AP remains unclear. In the present study, we explored the major chemical components and potential molecular mechanisms linked to the anti-angina effects of CDDP through the application of network pharmacology and molecular docking. The potential targets of active ingredients in CDDP were sourced from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and the Swiss Target Prediction Database (STPD). Additionally, targets related to angina pectoris (AP) were retrieved from various databases, including Gene Cards, DisGeNET, Dis Genet, the Drug Bank database (DBD), and the Therapeutic Target Database (TDD). Protein- protein interaction [1] networks were also established, and core targets were identified based on their topological significance. GO enrichment analysis and KEGG pathway analysis were conducted using the R software. Interactions between active ingredients and potential targets selected through the above process were investigated through molecular docking. Seventy-six active ingredients were selected with the following criteria: OB ≥ 30%, DL ≥ 0.18. 383 targets of CDDP and 1488 targets on AP were gathered, respectively. Afterwards, 194 common targets of CDDP and anti-AP targets were defined, of which 12 were core targets. GO enrichment analysis indicated that CDDP acted on AP by response to lipopolysaccharide, regulating the reactive oxygen species and metal ion metabolism, and epithelial cell proliferation. In addition, KEGG enrichment analysis indicated that the signaling pathways were notably enriched in lipid and atherosclerosis, fluid shear stress and atherosclerosis, IL-17 signaling pathway, EGFR tyrosine kinase inhibitor resistance, PI3K-Akt signaling pathway, and TNF signaling pathway. Moreover, the molecular docking manifested excellent binding capacity between the active ingredients and targets on AP. This study comprehensively illustrated the bioactive, potential targets, and molecular mechanisms of CDDP against AP, offering fresh perspectives into the molecular mechanisms of CDDP in preventing and treating AP.

Revealing the active ingredients and mechanism of P. sibiricumm in non-small-cell lung cancer based on UPLC-Q-TOF-MS/MS, network pharmacology, and molecular docking

The alcohol extraction of P. sibiricum has exhibited significant inhibitory effects on the production of free radicals and the proliferation of non-small-cell lung carcinoma (NSCLC) A549 cells. Despite the diverse components found in alcohol extraction of P. sibiricum and its multiple targets, the active components and associated targets remain largely unidentified. Hence, there is a need for additional investigation into the pharmacodynamic elements and mechanisms of action. This study aimed to analyze and identify the components responsible for the anti-tumor activity of alcohol extraction from P. sibiricum using UPLC-Q-TOF-MS/MS for the first time. Subsequently, the targets of the active components were predicted using the SwissTargetPrediction database, whereas the targets for NSCLC were sourced from the Online Mendelian Inheritance in Man database (OMIM) and the GeneCards database. Next, the targets of chemical composition were integrated with disease targets via Venny online. GO and KEGG pathway enrichment analyses were performed utilizing DAVID. Subsequently, a network analysis of “components-targets-pathways” was established using Cytoscape 3.8.2 and assessed with the “network analyzer” plug-in. Molecular docking was conducted utilizing Autodock 1.5.6. The study aimed to examine the anti-proliferative impacts and underlying mechanisms of alcohol extraction from P. sibiricum on NSCLC through in vivo and in vitro investigations utilizing an animal model of transplanted tumor, CCK8 assay, cell scratch test, RT-qPCR, and western blotting. The study unveiled that 17 active components extracted from P. sibiricum alcohol demonstrated anti-non-small cell lung cancer (NSCLC) effects through the modulation of 191 targets and various significant signaling pathways. These pathways include Endocrine resistance, PI3K/AKT, Chemical carcinogenesis-receptor activation, Proteoglycans in cancer, EGFR tyrosine kinase inhibitor resistance, AMPK signaling pathway, and other related signaling pathways. Network analysis and molecular docking results indicated that specific compounds such as (25S)-26-O-(β-d-glucopyranosyl)-furost-5-en3β,22α,26-triol3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside, Timosaponin H1, Deapi-platycodin D3, (3R)-5,7-dihydroxy-6,8-dimethyl-3-(4′-hydroxybenzyl)-chroman-4-one, Disporopsin, Funkioside F, Kingianoside E, Parisyunnanoside H, and Sibiricoside B primarily targeted 17 key proteins (BCL2, EGFR, ESR1, ESR2, GRB2, IGF1R, JUN, MAP2K1, MAPK14, MAPK8, MDM2, MMP9, mTOR, PIK3CA, RAF1, RPS6KB1, and SRC) collectively. In conclusion, the alcohol extraction of P. sibiricum demonstrated inhibitory effects on cell proliferation, induction of apoptosis, and inhibition of metastasis through various pathways.

The Activity, Composition and Molecular Mechanism of Bothriospermum zeylanicum (Hornem.) Fisch. Et Mey Alleviation of Nonsmall Cell Lung Cancer

Objective: To study the effect of the ethyl acetate extract of Bothriospermum zeylanicum (Hornem.) Fisch., the active ingredients and mechanism of Et Mey (EAF) in nonsmall cell lung cancer (NSCLC) were explored. Methods: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to measure the effect of EAF on the proliferation of A549, HL-60, SMMC-7721, MDA-MB-231, and SW480 tumor cells. The components were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS), and the blood components were retrieved from the constituents absorbed into the blood and metabolites of traditional Chinese medicine (DCABM-TCM) database. The mechanism was determined by network pharmacology and verified by molecular docking. Results: The IC50 values for the proliferation of tumor cell strains were 41.13, 56.72, 36.69, 48.63, and 44.48 μg/mL, respectively. A concentration of 100 μg/mL had the strongest inhibitory effect on A549 cells. A total of 33 chemical components were identified by LC-MS, 7 of which have been reported to have certain anti-NSCLC effects, and a total of 6 blood components were identified using the DCABM-TCM database. Network pharmacology analysis revealed that anti-NSCLC activity mainly involves EGFR tyrosine kinase inhibitor resistance, the PI3K-Akt signaling pathway, the AGE-RAGE signaling pathway in diabetic complications, the HIF-1 signaling pathway and the VEGF signaling pathway. The molecular docking results verified that hesperetin, luteolin, and pinocembrin bind well to the AKT1, IL6, EGFR, BCL2, and CASP3 target proteins. Conclusion: The ethyl acetate fraction of B. zeylanicum (Hornem.) Fisch. Et Mey had obvious ant-non NSCLC activity, which may be related to resistance to EGFR tyrosine kinase inhibitors and the PI3K-Akt signaling pathway in diabetic complications, and its active ingredients may be hesperetin, luteolin, and pinocembrin.

Research of the dynamic regulatory mechanism of Compound Danshen Dripping Pills on myocardial infarction based on metabolic trajectory analysis

BackgroundMyocardial infarction (MI) is a serious cardiovascular disease, which presents different pathophysiological changes with the prolongation of the disease. Compound danshen dripping pills (CDDP) has obvious advantages in MI treatment and widely used in the clinic. However, the current studies were mostly focused on the endpoint of CDDP intervention, lacking the dynamic attention to the disease process. It is of great value to establish a dynamic research strategy focused on the changes in pharmacodynamic substances for guiding clinical medication more precisely. PurposeIt is aimed to explore the dynamic regulating pattern of CDDP on MI based on metabolic trajectory analysis, and then clarify the variation characteristic biomarkers and pharmacodynamic substances in the intervention process. MethodsThe rat MI model was successfully prepared by coronary artery left anterior descending branch ligation, and then CDDP intervention was given for 28 days. Endogenous metabolites and the components of CDDP in serum were measured by LC/MS technique simultaneously to identify dynamic the metabolic trajectory and screen the characteristic pharmacodynamic substances at different points. Finally, network pharmacology and molecular docking techniques were used to simulate the core pharmacodynamic substances and core target binding, then validated at the genetic and protein level by Q-PCR and western blotting technology. ResultsCDDP performed typical dynamic regulation features on metabolite distribution, biological processes, and pharmacodynamic substances. During 1-7 days, it mainly regulated lipid metabolism and inflammation, the Phosphatidylcholine (PC(18:1(9Z/18:1(9Z)) and Sphingomyelin (SM(d18:1/23:1(9Z)), SM(d18:1/24:1(15Z)), SM(d18:0/16:1(9Z))) were the main characteristic biomarkers. Lipid metabolism was the mainly regulation pathway during 14-21 days, and the characteristic biomarkers were the Lysophosphatidylethanolamine (LysoPE(0:0/20:0), PE-NMe2(22:1(13Z)/15:0)) and Sphingomyelin (SM(d18:1/23:1(9Z))). At 28 days, in addition to inflammatory response and lipid metabolism, fatty acid metabolism also played the most important role. Correspondingly, Lysophosphatidylcholine (LysoPC(20:0/0:0)), Lysophosphatidylserine (LPS(18:0/0:0)) and Fatty acids (Linoelaidic acid) were the characteristic biomarkers. Based on the results of metabolite distribution and biological process, the characteristic pharmacodynamic substances during the intervention were further identified. The results showed that various kinds of Saponins and Tanshinones as the important active ingredients performed a long-range regulating effect on MI. And the other components, such as Tanshinol and Salvianolic acid B affected Phosphatidylcholine and Sphingomyelin through Relaxin Signaling pathway during the early intervention. Protocatechualdehyde and Rosmarinic acid affected Lysophosphatidylethanolamine and Sphingomyelin through EGFR Tyrosine kinase inhibitor resistance during the late intervention. Tanshinone IIB and Isocryptotanshinone via PPAR signaling pathway affected Lysophosphatidylcholine, Lysophosphatidylserine, and Fatty acids. ConclusionThe dynamic regulating pattern was taken as the entry point and constructs the dynamic network based on metabolic trajectory analysis, establishes the dynamic correlation between the drug-derived components and the endogenous metabolites, and elucidates the characteristic biomarkers affecting the changes of the pharmacodynamic indexes, systematically and deeply elucidate the pharmacodynamic substance and mechanism of CDDP on MI. It also enriched the understanding of CDDP and provided a methodological reference for the dynamic analysis of complex systems of TCM.

Identification of risk factors of EGFR-TKIs primary resistance in lung adenocarcinoma patients and construction of a risk predictive model: a case-control study.

Lung cancer is one of the malignancies with the highest incidence and mortality rates. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are recommended as the first-line treatment for patients with EGFR-mutated lung adenocarcinoma (LUAD). However, some patients with EGFR-sensitive mutations develop primary resistance to EGFR-TKIs. This study aims to analyze the clinical characteristics of LUAD patients with primary resistance to EGFR-TKIs, identify independent risk factors for primary resistance, and establish a risk predictive model to provide reference for clinical decision-making. We collected data from LUAD patients with EGFR-sensitive mutations (19del/21L858R) who were hospitalized in our institution between 2020 and 2022 and received first-generation EGFR-TKIs with follow-up exceeding 6 months. These patients were categorized into primary resistance and sensitive groups based on treatment outcomes. We compared general clinical data, laboratory tests, and tumor-related characteristics between the two groups, analyzed risk factors for primary resistance to EGFR-TKIs, and constructed a risk predictive model. The model's predictive value was comprehensively assessed using receiver operating characteristic (ROC) curves, calibration curves, and decision curves. Serum neuron-specific enolase (NSE) concentration (P=0.03), serum pro-gastrin-releasing peptide (ProGRP) concentration (P=0.01), and Ki67 expression (P<0.001) were identified as independent risk factors for primary resistance to EGFR-TKIs in LUAD. The combined presence of these three risk factors had the highest predictive value [area under the curve (AUC) =0.975, P<0.001]. We constructed a predictive model for the risk of primary resistance to EGFR-TKIs in LUAD patients, incorporating these three parameters, and represented it through a visually interpretable nomogram. The calibration curve of the nomogram demonstrated its strong predictive ability. Further decision curve analysis indicated the model's clinical utility. Based on a single-center retrospective case-control study, we identified serum NSE concentration, ProGRP concentration, and Ki67 expression as independent risk factors for primary resistance to EGFR-TKIs in LUAD patients. We constructed and validated a risk predictive model based on these findings. This predictive model holds promise for clinical application, aiding in the development of personalized treatment strategies and providing a scientific basis for early identification of primary resistance patients.

비소세포폐암에서 Gefitinib 내성 극복을 위한 효과적인 화합물 합성 및 항암 활성 평가

Lung cancer remains a leading cause of global cancer-related mortality, emphasizing the need for innovative strategies to combat resistance to the first-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), Gefitinib. This study aimed to discover and evaluate small molecule compounds selectively inhibiting Gefitinib resistance. A compound library was screened against adenocarcinoma (A549), EGFR-mutant NSCLC (H1975 and PC9), and Gefitinib-resistant NSCLC (PC9GR) cell lines. The study identified W1 as a promising lead compound, and its derivative, compound W3, exhibited excellent efficacy in PC9 and PC9GR cells. These findings underscore the potential of W3, an 4-anilinopyrimidine derivative, as an inhibitor of Gefitinib resistance in NSCLC. This research contributes valuable insights to the ongoing efforts in developing effective and sustainable treatment options for lung cancer patients facing EGFR-TKI resistance.

Abstract 5831: The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma

Abstract In human lung adenocarcinoma that carry Epidermal Growth Factor Receptor (EGFR) sensitizing mutations, targeting the EGFR pathway with the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) has emerged as a promising therapeutic strategy providing initial clinical benefit to many patients. Unfortunately, resistance to EGFR-TKIs eventually develops leading to disease progression. In addition to genetic mutations, transcriptional and epigenetic mediated changes in gene expression remain an important mechanism contributing to EGFR-TKI resistance. Here, we report a novel function of the transcriptional corepressor Transducin-Like Enhancer of Split-1 (TLE1) in mediating EGFR-TKI resistance in EGFR mutant LUAD cells through its survival promoting gene transcriptional program. In EGFR mutant, EGFR TKI sensitive LUAD cells, sole activation of the TLE1 nuclear function attenuates EGFR TKI sensitivity with concomittant reduction of EGFR-TKI-mediated apoptosis. Conversely, downregulation of endogenous TLE1 expression via siRNAs/shRNAs and deletion of TLE1 gene via CRISPR-Cas9 in EGFR-mutant, EGFR-TKI resistant cells partially restore EGFR-TKI sensitivity via induction of EGFR-TKI apoptosis. Mechanistically, the anti-apoptosis promoting gene transcriptional program of TLE1 is dependent on its functional interaction with the transcriptional repressor HES-1, a downstream Notch target which has been associated with relapse in LUAD patients following EGFR-TKI treatment. Collectively, these studies indicate the TLE1 corepressor as a determinant of resistance to EGFR targeted therapy and raise the possibility that inhibiting TLE1 nuclear function may serve as an adjuct therapy to circumvent EGFR-TKI resistance. Citation Format: Hector Ramos Biliran, Xin Yao, Nasir Roberts, Prince Iheukwumere, Renwei Chen, Ma. Carmela Dela Cruz. The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma [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 5831.

Abstract 5658: Genome-scale CRISPR screen in 3D tumor models identifies EGFR-TKI resistance mechanisms in human NSCLC

Abstract INTRODUCTION: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the primary treatment for EGFR-mutated non-small-cell lung cancer (NSCLC). Despite Osimertinib, an FDA-approved EGFR-TKI, showing high selectivity for EGFR-activating mutations, resistance inevitably arises, leading to disease progression. The influence of genes within cancer on Osimertinib response remains unclear. To tackle this challenge, CRISPR screens have been extensively used. However, current CRISPR screens investigating EGFR TKI resistance heavily rely on 2D monolayer cell models, lacking the ability to replicate in vivo tumors accurately. In response, we conducted a genome-scale loss-of-function CRISPR screening employing 3D tumor models embedded in an extracellular matrix across various NSCLC cell lines. Utilizing a comprehensive panel of CRISPR screens in both 3D tumor models and 2D monolayer models, we systematically assessed how genetic alterations impact cell resistance and transformation against treatment with Osimertinib, in diverse cellular contexts. METHOD: Genome-scale CRISPR-Cas9 screens in 10 NSCLC cell lines used 3D tumor models and 2D cells to pinpoint genes pivotal for EGFR TKI resistance. Cas9-expressing cell lines were transduced with a lentivirus carrying 60,000 sgRNAs targeting ~20,000 human genes. Cells were cultured in both models and treated with optimized Osimertinib or DMSO, inducing 20-30% cell growth inhibition for 3 days. Post-treatment, cells were recovered for 4 days. This treatment cycle was repeated 3-4 times in 3D models and 2-3 times in 2D cells. The sgRNA composition in each pool was analyzed using Next Generation Sequencing after treatment cycles in both models. RESULTS: Upon analyzing CRISPR screening data across 10 distinct cell lines to assess drug phenotypes, we confirmed the enrichment of established resistance pathways like PI3K/AKT/mTOR, Hippo, and MAPK signaling pathways in both 2D monolayer cells and 3D tumor models. However, notable discrepancies in EGFR TKI resistance mechanisms were evident between these two models. Specifically, CRISPR screens conducted with 3D tumor models unveiled unique resistance mechanisms, notably the LKB1/AMPK/mTOR pathway (STK11, GSK3B), Ubiquitination (RNF7, ARIH2), and stabilization of E-cadherin adherens junction (NCKAP1, EXOC4) differing from those identified in conventional 2D monolayer model. Furthermore, the 3D tumor models exhibited significantly heightened enrichment of the MAPK and Hippo signaling pathways compared to their drug phenotype score in 2D monolayer cells. CONCLUSION: This study underscores the significance of employing the 3D tumor model in drug phenotype CRISPR screen. This approach recapitulates unique gene dependency patterns and interactions linked to EGFR-TKI resistance in a 3D tumor context, revealing potential targetable vulnerabilities context-dependent. Citation Format: Pranay Agarwal, Soon Youn Choi, Xin Liu, Jina Song, Maya Tureez, Aashka Mehta, Un Jae Baek, Kyuho Han, Hong-Pyo Lee. Genome-scale CRISPR screen in 3D tumor models identifies EGFR-TKI resistance mechanisms in human 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 5658.

Abstract 6110: Computational exploration of genomic differences in Black patients treated with chemoradiation for head and neck squamous cell carcinoma (HNSCC)

Abstract Background: Black patients treated with chemoradiation for head and neck squamous cell carcinoma (HNSCC) experience significantly worse survival outcomes than White patients treated with the same modality. Our group aims to explore potential sociodemographic and biological factors undermining this differing response to therapy. Here, we present our computational findings of genomic differences in Black compared with White HNSCC patients receiving chemoradiation. Methods: We obtained gene expression, phenotype, and clinical datasets for the Cancer Genome Atlas (TCGA) HNSCC cohort from the UCSC Xena web browser. Of the 528 HNSCC patients, 131 (Black=13, White=118) of those receiving chemoradiation had gene expression, phenotype, and clinical data available. Our analysis included anatomic sites involving the oral cavity, hypopharynx, and larynx, and excluded the oropharynx. We performed differential gene expression analysis in Black compared with White (reference population) patients via edgeR in R studio (-log2FC &amp;lt; -1 or log2FC &amp;gt;1; p value &amp;lt; 0.05; FDR &amp;lt; 0.05). The Database for Annotation, Visualization, and Integrated Discovery (DAVID) webtool was used to identify significantly enriched molecular processes and pathways. Results: 462 genes were found to be significantly differentially expressed between Black and White HNSCC patients receiving chemoradiation. Of these genes, 198 were found to be higher in Black patients, and the most significant included JPH3, PCSK1N, DCUN1D5, CRYGS, and CCL7 (p &amp;lt; 0.005, FDR &amp;lt; 0.005). 264 were found to be lower in Black patients, with the most significant being KBTBD12, CMYA5, NEFH, LDB3, PYGM (p &amp;lt; 0.005, FDR &amp;lt; 0.005). Genes found to be higher in Black patients showed enrichment for processes such as EGFR tyrosine kinase inhibitor resistance (p &amp;lt; 0.005), drug metabolism (p &amp;lt; 0.005), and chemical carcinogenesis (p &amp;lt; 0.005). Genes found to be lower in Black patients showed enrichment for muscle-related processes such as myogenesis (p &amp;lt; 0.005), actin-binding (p &amp;lt; 0.005), muscle protein (p &amp;lt; 0.005), and hypertrophic cardiomyopathy (p &amp;lt; 0.005). Conclusion: In conclusion, using TCGA, we identified differentially expressed genes between Black and White HNSCC patients receiving chemoradiation. Moreover, these findings suggest that biological processes may differ in Black compared with White patients receiving chemoradiation, and this may dictate differing response to therapy. Additional studies will involve further computational analysis using other datasets for validation. Citation Format: Chayil C. Lattimore, Caretia J. Washington, Dejana Braithwaite, Shama D. Karanth, Kristianna M. Fredenburg. Computational exploration of genomic differences in Black patients treated with chemoradiation for head and neck squamous cell carcinoma (HNSCC) [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 6110.

Abstract 3263: VEGFA mediates traditional EGFR-TKI resistance in non-small cell lung cancer with EGFR exon 20 insertions

Abstract Background: Epidermal growth factor receptor (EGFR) exon 20 insertions (Ex20ins) accounted for 4%-12% of EGFR mutations and showed primary resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) which is an urgent issue to be solved. In our previous studies, we found that antiangiogenetic therapy had certain advantages for patients with Ex20ins in the real-world practice. Previous research showed the tumor microenvironment has been associated with resistance to targeted therapy in sensitive mutations. However, the mechanism of resistance of EGFR-TKIs and the advantage of antiangiogenetic therapy in patients with EGFR Ex20ins were still unclear. Methods: In this study, patients with EGFR exon 19 deletion (19del), exon 21 p.L858R (21L858R), and Ex20ins were collected to identify the efficacy of EGFR-TKIs. Stable cell lines expressing were established and used to compare signaling pathway activation. Matrigel angiogenesis assay was conducted in different cell lines. Next, we evaluated the efficacy of various treatments combined with antiangiogenetic agents or not in mouse models. Results: We collected 23 patients with EGFR sensitive mutations and Ex20ins, respectively, and found Ex20ins presented primary resistance to TKIs and poor survival outcomes. Based on the RNA sequence of stable cell lines, it was found that Ex20ins had a higher level of VEGFA expression and enriched in MAPK pathway compared to EGFR 19del and 21L858R. Immunohistochemical studies also revealed that a higher level of VEGFA expression was observed in a cohort of NSCLC patients with Ex20ins mutation compared with 19del or L858R. Matrigel angiogenesis assay has shown consistent result that Ex20ins promoted angiogenesis in vivo. Further experiments in mouse models identified that in the group of Osimertinib, mobocertinib alone, chemotherapy alone, mobocertinib or chemotherapy combination with bevacizumab, Osimertinib achieved the worst efficacy and additional utility of antiangiogenetic agents had a more favorable efficacy. Conclusion: Our study highlights the potential of VEGFA as a mechanism of resistance to EGFR-TKIs and induces immunosuppression microenvironment in NSCLC with Ex20ins. Furthermore, the findings suggested that antiangiogenetic therapy could be a promising therapeutic approach. Keywords: VEGFA, targeted therapy, resistance, EGFR exon 20 insertions, non-small cell lung cancer Citation Format: Yaning Yang, Chengming Liu, Guangjian Yang, Haiyan Xu, Siyu Lei, Yan Wang. VEGFA mediates traditional EGFR-TKI resistance in non-small cell lung cancer with EGFR exon 20 insertions [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 3263.

Network pharmacology, molecular docking, and molecular dynamics simulation analysis reveal the molecular mechanism of halociline against gastric cancer.

Halociline, a derivative of alkaloids, was isolated from the marine fungus Penicillium griseofulvum by our group. This remarkable compound exhibits promising antineoplastic activity, yet the precise molecular mechanisms underlying its anticancer properties remain enigmatic. To unravel these mechanisms, we employed an integrated approach of network pharmacology analysis, molecular docking simulations, and molecular dynamics simulations to explore halociline therapeutic targets for gastric cancer. The data from network pharmacology indicate that halociline targets MAPK1, MMP-9, and PIK3CA in gastric cancer cells, potentially mediated by diverse pathways including cancer, lipid metabolism, atherosclerosis, and EGFR tyrosine kinase inhibitor resistance. Notably, molecular docking and dynamics simulations revealed a high affinity between halociline and these targets, with free binding energies (ΔEtotal) of - 20.28, - 27.94, and - 25.97kcal/mol for MAPK1, MMP-9, and PIK3CA, respectively. This study offers valuable insights into the potential molecular mechanism of halociline's inhibition of gastric cancer cells and serves as a valuable reference for future basic research efforts.

The effect of dapagliflozin on anemia in elderly patients with heart failure by bioinformatics analysis.

Anemia associated with heart failure is frequent and can exacerbate the symptoms of heart failure. Dapagliflozin is the first SGLT-2 inhibitor with significant cardiovascular protection. However, the effect of dapagliflozin on anemia in elderly patients with heart failure is unknown. We aimed to study the effect of dapagliflozin on anemia in elderly patients with heart failure by bioinformatics analysis. The target genes were determined, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The protein-protein interaction (PPI) network and modules were constructed. The dapagliflozin-targets network in anemia and heart failure was constructed. Molecular docking experiments between dapagliflozin and its key target AKT1 were performed. We found 1 dapagliflozin related target gene and 2 disease related genes. Totally, 134 target genes of dapagliflozin on anemia in elderly patients with heart failure were determined. The pathways may involve lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, hepatitis B, insulin signaling pathway, fluid shear stress and atherosclerosis, neurotrophin signaling pathway, insulin resistance, toxoplasmosis, colorectal cancer, and EGFR tyrosine kinase inhibitor resistance. The hub genes in network were AKT1, TP53, GAPDH, TNF, CASP3, EGFR, and MAPK3. The structure of dapagliflozin and AKT1 molecular docking was exhibited. The hub genes in network were AKT1, TP53, GAPDH, TNF, CASP3, EGFR, and MAPK3. The structure of dapagliflozin and AKT1 molecular docking was exhibited.

MiR-200c-3p as a novel genetic marker and therapeutic tool for alopecia areata.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression in diverse biological processes. They hold promise as therapeutic candidates for targeting human disease pathways, although our understanding of their gene regulatory mechanism remains incomplete. Alopecia areata (AA) is a prevalent inflammatory ailment distinguished by the infiltration of T cells targeting the anagen-stage hair follicles. The scarcity of effective remedies for AA may stem from limited understanding regarding its precise cellular mechanism. To investigate and examine the importance and role of the miR-200c-3p as a genetic indicator for AA, and its possible impact on disease progression. Case-control study included 65 patients with AA and 65 matched healthy controls. A real-time PCR technique was used to measure the expression of miR-200c-3p for both groups. Bioinformatic tools were used for prediction with genes and gene-gene interaction, and protein-protein interaction. The expression levels of miR-200c-3p were significantly higher in AA patients than in healthy controls. We predicted that miR-200c-3p plays a markable role in the development of AA by its effect on the EGFR tyrosine kinase inhibitor resistance pathway. We were able to identify the influence of miR-200c-3p on both PLCG1 and RPS6KP1 genes which in turn regulate the EGFR tyrosine kinases resistance pathway that displayed the most substantial increase in activity. Our outcomes shed light on the era of the potential theranostic role of this innovative miRNA in AA.

Network Pharmacology Approach and Partial Experimental Validation of Aidi Injection Solution for the Treatment of Colorectal Cancer

Purpose The objective of this research was to employ network pharmacology to analyze and identify the key active components and target points of action of Aidi injection in relation to colorectal cancer. Additionally, this study aimed to experimentally validate the mechanism of action of hinokinin in treating colorectal cancer. Methods This study employed a network pharmacology methodology to identify the primary components and action targets of Aidi injection in public databases; a similar approach was used to identify effective targets for colorectal cancer treatment. The protein‒protein interaction network, along with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, facilitated the prediction of central targets and pathways by which Aidi injection combats colorectal cancer. Molecular docking techniques were harnessed to sift through drugs and their targets for high binding affinity. Experimental validation was carried out to corroborate the findings derived from network pharmacology. Results We identified 37 active constituents of Aidi injection (ADI), 701 potential targets, 768 colorectal cancer (CRC) targets, and 111 overlapping targets. The anti-CRC efficacy of ADI seems to be associated with several pathways: cancer pathways, EGFR tyrosine kinase inhibitor resistance, proteoglycans in cancer, endocrine resistance, central carbon metabolism in cancer, chemo-oncogenic receptor initiation, cancer-specific microRNAs, and signaling pathways such as PI3K-Akt, mitogen-activated protein kinase, prolactin, FoxO, and Ras. Predominantly, ADI exhibited activity against key markers such as EGFR, ERBB2, HSP90AA1, mTOR, HIF1A, CCND1, JUN, AKT1, SRC, and STAT3 to mitigate CRC. Furthermore, Hinokinin has been shown to curtail the proliferation of colorectal cancer cells, amplify apoptosis, and modulate the expression of the mTOR protein. Conclusion Through network pharmacology, we identified 13 shared targets associated with colorectal cancer. Subsequent experimental validation revealed that hinokinin can curtail the proliferation of colorectal cancer cells and enhance their apoptosis, primarily by modulating mTOR expression.

Efficacy and potential mechanism of atherosclerosis prevention by the active components of leech based on network pharmacology combined with animal experiments

IntroductionLeeches are flesh-eating and bloodsucking parasitic worms. They are being used as a traditional Chinese medicine for centuries in activating blood and dissolving statis, dreging the meridims and tick. Hirudin, an active peptide product present in leech, has blood anticoagulant property and can assist in the treatment of thrombosis and diseases related to blood circulation. The efficacy and potential mechanism of action of leeches in such diseases should be further explored. Materials and methodsFirst, network pharmacology was used to screen the predicted potential targets of the active constituents of leech and AS. The common targets of the active constituents of leech and AS were obtained using Venn diagram. Further, the drug-active-constituent–target network diagram, protein–protein interaction, and GO and KEGG pathway enrichment analyses were used to construct the active-constituent–AS target–pathway network diagram. Subsequently, the protein–drug molecule docking model was drawn. Finally, the results of network pharmacology were validated using a mouse model of AS. ResultsIn total, 34 active constituents of leech and 1172 AS-related gene targets were selected, took the drug action targets and potential disease targets to get the common targets, and took the top 10 of degree value as the main active constituents for the treatment of atherosclerosis. There were 89 common targets and 12 core targets. The main targets included MAPK, EGFR, PIK3CB, etc. Potential regulatory pathways included cancer pathways, EGFR tyrosine kinase inhibitor resistance, Rap1 signaling pathway, PPAR signaling pathway, PI3K-Akt signaling pathway, C-type lectin receptor signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. Animal experiments using mouse model of AS confirmed that AS plaques were smaller after treatment with leeches. SRC level was measured using western blotting. Expression of SRC in myocardial tissue was remarkably lower in the mice treated with leech than in the mice from model group fed on high-fat chow. ConclusionsTo the best of our knowledge, this is the first study to explore the mechanism of action of the active components of leech in AS prevention. The active components of leeches play a coordinated role in preventing AS through multicomponent, multitarget, and multichannel mechanism of action related to inflammatory response, oxidative stress, and lipid metabolism. This study provided a reference for subsequent cellular and animal experiments.

Integrated Analysis of a ceRNA (lncRNA-miRNA-mRNA) Regulatory Network for Prostate Cancer

Objective. This study was to construct a ceRNA (lncRNA-miRNA-mRNA) regulatory network for prostate cancer (PCa) and to explore the prognostic correlation, key biological functions, and pathways of core RNAs. Methods. Three subgene expression matrices (miRNA, lncRNA, and mRNA expression matrix) were taken from the TCGA database and used in this investigation. Differential expression analysis and differential expression miRNAs were carried out. Next, the ceRNA (lncRNA-miRNA-mRNA) regulatory complex was used to visualize our data and show how they interacted. Ultimately, the primary molecular roles and biological pathways of PCa were identified using enrichment analysis by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). An effect of AC016773.1 on prostate cancer cell proliferation was investigated by knocking out AC016773.1 in an animal model. The interrelationship between AC016773.1 and hsa-mir-25 was validated using RNA immunoprecipitation technology. Results. The lncRNA, miRNA, and mRNA expression matrices obtained from the TCGA database contain 16901, 1881, and 19962 transcripts, respectively. Through differential expression analysis, we obtained 2010 de lncRNA comparative information, 75 lncRNA and miRNA interaction pairs, and 31 targeted de mRNAs. Through the differential expression analysis of RNA nodes in the ceRNA regulatory network, we found that compared with the NP group, in the PCa group, there were 14 lncRNAs upregulated and 25 lncRNAs downregulated, 1 miRNA upregulated and 3 miRNA downregulated, and 10 mRNA upregulated and 21 mRNA downregulated. In KEGG enrichment analysis, the pathways identified by targeted DE-mRNAs were mainly related to calcium signaling pathway, EGFR tyrosine kinase inhibitor resistance, melanoma, PI3K−Akt signaling pathway, and proteoglycans in cancer. In animal models, it was found that knocking down AC016773.1 significantly reduced tumor volume and weight, indicating that AC016773.1 may promote the proliferation of PCa cells. The use of RNA immunoprecipitation technology indicates a direct binding between AC016773.1 and hsa-mir-25. Conclusion. We elucidated the network regulatory relationship of lncRNA-miRNA-mRNA in PCa and further explored the key molecular functions, biological pathways, and prognostic value of targeted DE-mRNAs.