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It has been confirmed that bee venom (BV) can inhibit tumor metastasis of lung cancer cells induced by epidermal growth factor, suggesting the inhibitory role of BV on the regulation of epidermal growth factor receptor (EGFR), and may synergistically promote the anti-lung cancer effect of EGFR tyrosine kinase inhibitor gefitinib. This paper aims to ascertain the therapeutic potentials of BV combined with gefitinib against non-small cell lung cancer (NSCLC) in vitro. As results, the content of the main component melittin in air-dried BV was determined by HPLC. Subsequently, it was found that BV significantly inhibited the proliferation of NSCLC PC-9 and NCI-H1299 cells, but not generated apparent toxicity to human normal lung epithelial BEAS-2B cells. Meanwhile, the combination of BV and gefitinib also significantly inhibited the proliferation of these two cells, and suppressed the migration and invasion of PC-9 cells. By bioinformatics analysis and molecular docking, it was predicted that the main component melittin in BV could act on the cell membrane and transmembrane protein EGFR. Ultimately, Western blot assays showed BV alone or combined with gefitinib significantly decreased the protein expression of phosphorylated EGFR (p-EGFR) and the protein expression ratio of p-EGFR to EGFR, and increased the protein expression ratio of LC3-II to LC3-I in PC-9 cells or epidermal growth factor-activated PC-9 cells. The results demonstrated that BV could prompt the inhibition of gefitinib on proliferation, migration, and invasion of NSCLC cells via EGFR-mediated autophagy, showing the synergistic anti-NSCLC potential when combined with gefitinib., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Overcoming resistance to Osimertinib in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) is clinically challenging because the underlying mechanisms are not fully understood. The murine double minute 2 (MDM2) has been extensively described as a tumor promotor in various malignancies, mainly through a negative regulatory machinery on the p53 tumor suppressor. However, the significance of MDM2 on the sensitivity to Osimertinib has not been described., Methods: Osimertinib resistant cells were generated by standard dose escalation strategy and individual resistant clones were isolated for MDM2 testing. The MDM2 and its mutant constructs (ΔPBD, ΔRING, C464A) were introduced into PC-9, HCC827 and H1975 cells and evaluated for the sensitivity to Osimertinib by MTT assay, colony formation, EdU assay and TUNEL assay. MDM2 expression in resistant cells was manipulated by pharmacological and molecular approaches, respectively. Proteins that were implicated in PI3K/Akt, MAPK/Erk and apoptosis signaling were measured by Western blot analysis. Candidate proteins that interacted with MDM2 were captured by immunoprecipitation and probed with indicated antibodies., Results: In comparison with parental PC-9 cells, the PC-9 OR resistant cells expressed high level of MDM2. Ectopic expression of MDM2 in PC-9, HCC827 and H1975 sensitive cells generated an Osimertinib resistant phenotype, regardless of p53 status. MDM2 promoted resistance to Osimertinib through a PI3K/Akt and MAPK/Erk-independent machinery, in contrast, MDM2 selectively stabilized MCL-1 protein to arrest Osimertinib-induced cancer cell apoptosis. Mechanistically, MDM2 acted as a E3 ligase to ubiquitinate FBW7, a well-established E3 ligase for MCL-1, at Lys412 residue, which resulted in FBW7 destruction and MCL-1 stabilization. Targeting MDM2 to augment MCL-1 protein breakdown overcame resistance to Osimertinib in vitro and in vivo. Finally, the clinical relevance of MDM2-FBW7-MCL-1 regulatory axis was validated in mouse xenograft tumor model and in NSCLC specimen., Conclusion: Overexpression of MDM2 is a novel resistant mechanism to Osimertinib in EGFR mutant NSCLC. MDM2 utilizes its E3 ligase activity to provoke FBW7 destruction and sequentially leads to MCL-1 stabilization. Cancer cells with aberrant MDM2 state are refractory to apoptosis induction and elicit a resistant phenotype to Osimertinib. Therefore, targeting MDM2 would be a feasible approach to overcome resistance to Osimertinib in EGFR mutant NSCLC., Competing Interests: Declarations Consent for publication Written consent was obtained from each participate. Ethical approval and consent to participate Animal care and experiments were reviewed and approved by the Institutional Animal Care and Use Committee (#2023JLHGZRDWLS-00032). The handling and processing of patient tumor samples were done in accordance with Institutional Ethics Committee Review Board Approved protocols (#2023DZGZR-030). Competing interest Wei Liu is an employee of Liaoning Kanghui Biotechnology. Liu analyzed the NGS data and did not have access to interpret the bioinformatic and experimental results. The remaining authors did not have any financial association with Liaoning Kanghui Biotechnology or other biomedical companies., (© 2024. The Author(s).)

Targeted therapies using epidermal growth factor receptor (EGFR) inhibitors have markedly improved survival rates and quality of life for patients with EGFR-mutant lung adenocarcinoma (LUAD). Despite these advancements, resistance to EGFR inhibitors remains a significant challenge, limiting the overall effectiveness of the treatment. This study explored the synergistic effects of combining Paeoniae Radix (PR) with first-generation EGFR-tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, to overcome this resistance. Transcriptomic analysis of EGFR-mutant LUAD cell lines revealed that PR treatment could potentially reverse the gene signatures associated with resistance to EGFR-TKIs, primarily through the suppression of the Aurora B pathway. Experimental validation demonstrated that combining PR with erlotinib and gefitinib enhanced drug responsiveness by inhibiting Aurora kinase activity and inducing apoptosis in LUAD cells. Additionally, gene expression changes confirmed these combined effects, with the suppression of the Aurora B pathway and upregulation of the apoptotic pathway, which was accompanied by increased expression of multiple pro-apoptotic genes. Our findings contribute to the development of natural product-based therapeutic strategies to mitigate drug resistance in LUAD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Acquired resistance to osimertinib limits its clinical efficacy in non-small cell lung cancer (NSCLC) with EGFR mutations. The widespread recognition of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (Chinese yew) as a natural anti-cancer medication is well-established. However, the specific contribution of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu in addressing resistance to osimertinib is still uncertain., Aim of the Study: Based on the biological behaviors and lipid metabolism, we investigated whether aqueous extract of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (AETC) could enhance the antitumor effect of osimertinib in NSCLC with an investigation on the precise mechanisms., Materials and Methods: The effect of AETC on enhancing osimertinib sensitivity was assessed via cell viability measurements, levels of reactive oxygen species (ROS), apoptosis, and lipid levels. Western blotting was used to verify the mechanisms of AETC responsible for overcoming the resistance to osimertinib via ERK1/2 overexpression and knockdown models. In vivo validation was conducted using subcutaneous xenografts from osimertinib-resistant cells in nude mice., Results: Osimertinib-resistant cells exhibited altered cholesterol biosynthesis, which was induced by ERK1/2 activation. The combination of AETC and osimertinib can synergistically decrease the levels of ROS in cells, enhance apoptosis, and inhibit the growth of osimertinib-resistant cells. Mechanistic experiments demonstrated that AETC can downregulate the key regulators of cholesterol biosynthesis by regulating ERK1/2, inhibiting the endogenous synthesis rate of cholesterol, and suppressing the level of lipids in osimertinib-resistant cells and xenograft tumors when combined with osimertinib, ultimately reversing resistance to osimertinib., Conclusions: The resistance to osimertinib is significantly influenced by cholesterol biosynthesis, highlighting its pivotal role in this context. AETC can enhance osimertinib sensitivity via ERK/SREBP-2/HMGCR-mediated cholesterol biosynthesis. These results provide a promising therapeutic target and potential treatment option for resistance to osimertinib., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate the clinicopathological features, molecular pathology characteristics, and prognosis of non-small cell lung carcinoma (NSCLC) exhibiting co-expression of p40 and thyroid transcription factor1 (TTF1). Methods: Clinical and pathological data of six NSCLC cases with co-expression of p40 and TTF1 diagnosed at the First People's Hospital of Xiaoshan District, Hangzhou, China from January 2016 to December 2023 were collected. Relevant literature was also reviewed. Results: NSCLC with co-expression of p40 and TTF1 commonly occurred in male smokers and had been in stage Ⅲ-Ⅳ when diagnosis. Microscopic examination revealed that the tumor cells were arranged in solid nests and sheets with marked atypia and visible mitotic figures. There was no prominent evidence of keratinization or glandular formation. The tumor cells diffusely co-expressed p40 and TTF1, exhibiting a dual immunophenotype characteristic of both squamous cell carcinoma and adenocarcinoma. Molecular testing of four NSCLC co-expressing p40 and TTF1 revealed the presence of common EGFR mutations, as well as mutations of NRAS (mutation rate of 2.09%), EML4-ALK (mutation rate of 24.77%), and PIK3CA (exon 10 c.1658 G>C p.S553T, mutation rate of 4.32%). All six tumors were poorly differentiated, highly invasive, and associated with poor prognosis. Four of the six patients experienced widespread metastasis and died within 7 to 30 months after the diagnosis or initial treatment. Conclusions: NSCLC with co-expression of p40 and TTF1 exhibits distinct clinicopathological features, immunophenotypes, molecular alterations, and clinical outcomes, characterized by rapid progression and poor prognosis. Pathologists should be vigilant in recognizing this entity to avoid misdiagnosis and missed diagnosis.

Objective: Recent evidence suggests that elevated levels of PD-L1 expression may be linked to early resistance to TKI and reduced survival in NSCLC with EGFR mutations. This study aimed to characterize the clinical and molecular features of EGFR-mutated lung adenocarcinomas and determine the prognostic significance associated with high PD-L1 expression., Materials and Methods: We conducted a retrospective chart review of 103 consecutive patients with advanced EGFR-mutated NSCLC, who received treatment between 01/01/2016 and 30/12/2020, at our institution., Results: Among the tumors, 17% (n = 18) exhibited high PD-L1 expression (≥50% tumor proportion score), which was associated with a lower prevalence of common EGFR mutations (56% vs. 82%, p = 0.03) and a higher frequency of complex EGFR mutations (28% vs. 7%, p = 0.02). Univariate analysis did not reveal any significant differences in first-line response, progression-free survival, or overall survival between the PD-L1 ≥50% and <50% groups. However, multivariate analysis demonstrated that PD-L1 ≥50% was independently associated with shorter survival (HR = 2.57; 95%CI[1.20-5.55]; p = 0.02), along with male gender (HR = 2.77; 95%CI[1.54-4.19]; p<0.005), presence of liver metastases (HR = 5.80; 95%CI[2.86-11.75]; p<0.005) or brain metastases (HR = 1.99; 95%CI[1.13-3.52]; p = 0.02), and poor general condition at diagnosis (ECOG 3 and 4) (HR = 10.69; 95% CI[4.42-25.85]; p<0.005). Additionally, a trend towards a higher frequency of de novo resistance was observed in the PD-L1 >50% group (7% vs. 17%, p = 0.19)., Conclusion: High PD-L1 expression was more commonly found in lung adenocarcinomas with uncommon and complex EGFR mutations. Furthermore, high PD-L1 expression independently predicted poor survival. These findings warrant validation through prospective studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Slomka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To overcome C797S mutation, the latest and most common resistance mechanism in the clinical treatment of third-generation EGFR inhibitor, a novel series of substituted 6-(2-aminopyrimidine)-indole derivatives were designed and synthesized. Through the structure-activity relationship (SAR) study, compound 11eg was identified as a novel and potent EGFR L858R/T790M/C797S inhibitor (IC 50  = 0.053 μM) but had a weak effect on EGFR WT (IC 50  = 1.05 μM). 11eg significantly inhibited the proliferation of the non-small cell lung cancer (NSCLC) cells harboring EGFR L858R/T790M/C797S with an IC 50 of 0.052 μM. 11eg also showed potent inhibitory activity against other NSCLC cell lines harboring main EGFR mutants. Furthermore, 11eg exhibited much superior activity in arresting cell cycle and inducing apoptosis of NSCLC cells with mutant EGFR C797S . It blocked cellular EGFR signaling. Importantly, 11eg markedly suppressed the tumor growth in in vivo xenograft mouse model with good safety. Additionally, 11eg displayed good microsomal stability. These results demonstrated the potential of 11eg with novel scaffold as a promising lead compound targeting EGFR C797S to guide in-depth structural optimization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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 IC 50  = 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 IC 50  = 0.031 µM and 0.023 µM, respectively. The selectivity index of 3f for EGFR T790M 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., (© 2024. The Author(s).)

Epidermal growth factor receptor (EGFR)-activating mutations are critical factors in the development of EGFR-driven non-small-cell lung cancer (NSCLC), and molecular targeted therapies have focused on inhibiting these mutations. EGFR tyrosine kinase inhibitors (TKIs) have remarkable inhibitory effects on NSCLC with EGFR mutations. However, acquired resistance limits the clinical application of EGFR-TKIs, highlighting the need for discovery of novel therapeutic strategies. 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone is a natural product derived from Garcinia xanthochymus, has shown potential anti-tumor activity, but the underlying mechanism needs further elucidation. In this study, we developed Ba/F3 and NIH/3T3 cells harboring EGFR L858R/T790M/C797S mutation, and then found that 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone exerted inhibitory effects against cells with EGFR L858R/T790M/C797S mutation. Additionally, 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone exhibited potent anti-tumor activity against cells harboring the triple-mutant EGFR by promoting apoptosis and inducing changes in cell cycle distribution. Furthermore, 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone was found to significantly reduce tumor growth via suppressing the phosphorylation of EGFR in tumor tissues. These effects are associated with binding of 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone to EGFR resulting in the suppression of extracellular signal-regulated kinase (Erk) phosphorylation. In conclusion, our results suggest that 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone may be a potential novel candidate for further investigation and treatment of NSCLC with the triple-mutant EGFR., (© 2024. The Author(s).)

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 IgG 2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64 Cu-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 64 Cu 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 [ 64 Cu]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 [ 18 F]FLT PET. Immuno-PET with [ 64 Cu]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 [ 64 Cu]CuCl 2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [ 64 Cu]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 [ 64 Cu]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 SUV mean 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: [ 64 Cu]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.

The inevitable acquired resistance to osimertinib (AZD9291), an FDA-approved third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) for the treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating or T790M resistant mutations, limits its long-term clinical benefit. Telomere maintenance via telomerase reactivation is linked to uncontrolled cell growth and is a cancer hallmark and an attractive cancer therapeutic target. Our effort toward understanding the action mechanisms, including resistance mechanisms, of osimertinib has led to the identification of a novel and critical role in maintaining c-Myc-dependent downregulation of hTERT, a catalytic subunit of telomerase, and subsequent inhibition of telomerase/telomere and induction of telomere dysfunction in mediating therapeutic efficacy of osimertinib. Consequently, osimertinib combined with the telomere inhibitor, 6-Thio-dG, which is currently tested in a phase II trial, effectively inhibited the growth of osimertinib-resistant tumors, regressed EGFRm NSCLC patient-derived xenografts, and delayed the emergence of acquired resistance to osimertinib, warranting clinical validation of this strategy to manage osimertinib acquired resistance., (© 2024 Chen et al.)

Epidermal growth factor receptor (EGFR) kinase has been implicated in the uncontrolled cell growth associated with non-small cell lung cancer (NSCLC). This has prompted the development of 3 generations of EGFR inhibitors over the last 2 decades due to the rapid development of drug resistance issues caused by clinical mutations, including T790M, L858R and the double mutant T790M & L858R. In this work we report the design, preparation and biological assessment of new irreversible 2,4-diaminopyrimidine-based inhibitors of EGFR kinase. Twenty new compounds have been prepared and evaluated which incorporate a range of electrophilic moieties. These include acrylamide, 2-chloroacetamide and (2E)-3-phenylprop-2-enamide, to allow reaction with residue Cys797. In addition, more polar groups have been incorporated to provide a better balance of physical properties than clinical candidate Rociletinib. Inhibitory activities against EGFR wildtype (WT) and EGFR T790M & L858R have been evaluated along with cytotoxicity against EGFR-overexpressing (A549, A431) and normal cell lines (HepG2). Selectivity against JAK3 kinase as well as physicochemical properties determination (logD 7.4 and phosphate buffer solubility) have been used to profile the compounds. We have identified 20, 21 and 23 as potent mutant EGFR inhibitors (≤20 nM), with comparable or better selectivity over WT EGFR, and lower activity at JAK3, than Osimertinib or Rociletinib. Compounds 21 displayed the best combination of EGFR mutant activity, JAK3 selectivity, cellular activity and physicochemical properties. Finally, kinetic studies on 21 were performed, confirming a covalent mechanism of action at EGFR., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Matthew Paul Gleeson reports financial support was provided by King Mongkut’s Institute of Technology Ladkrabang. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

One of the problems resulting from recurrent hyperactivated or mutant epidermal growth factor receptors (EGFR) in non-small cell lung cancer (NSCLC) is therapeutic resistance. Consequently, this leads to increased expression of oncogenic proteins and reduces the efficacy of EGFR tyrosine kinase inhibitors (TKIs). This study assessed antiviral drug efficacy as potential anti-EGFR agents for NSCLC. We used structure-based virtual screening to evaluate 66 antiviral drugs thoroughly. The top 6 antiviral drugs exhibiting impressive binding energies (i. e. surpassing a threshold of -8.5 kcal mol -1 ) were identified. Subsequent bioactivity analysis and ADMET profiling were performed to select the most promising candidates, followed by a molecular dynamic simulation. Among the selected antiviral regimens, dolutegravir demonstrated the highest docking score (-9.8 kcal mol -1 ), followed by rilpivirine and ensitrelvir, surpassing other candidates and our reference EGFR TKI. Further molecular dynamics simulations revealed promising dynamic interactions of dolutegravir, ensitrelvir, and rilpivirine with the EGFR target as compared with afatinib. Our findings highlight the repositioning potential of antiviral drugs for anti-EGFR drug discovery, supported by their robust docking scores, ADMET profiles, dynamic interactions, and binding free energies. The results open up new avenues for advanced NSCLC therapy. Further in vitro investigations are warranted to evaluate their efficacy and safety., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Objective: To explore the specific pharmacological molecular mechanisms of Laoke Formula (LK) on treating advanced non-small cell lung cancer (NSCLC) based on clinical application, network pharmacology and experimental validation., Methods: Kaplan-Meier method and Cox regression analysis were used to evaluate the survival benefit of Chinese medicine (CM) treatment in 296 patients with NSCLC in Tianjin Medical University Cancer Institute and Hospital from January 2011 to December 2015. The compounds of LK were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the corresponding targets were performed from Swiss Target Prediction. NSCLC-related targets were obtained from Therapeutic Target Database and Comparative Toxicogenomics Database. Key compounds and targets were identified from the compound-target-disease network and protein-protein interaction (PPI) network analysis, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were used to predict the potential signaling pathways involved in the treatment of advanced NSCLC with LK. The binding affinities between key ingredients and targets were further verified using molecular docking. Finally, A549 cell proliferation and migration assay were used to evaluate the antitumor activity of LK. Western blot was used to further verify the expression of key target proteins related to the predicted pathways., Results: Kaplan-Meier survival analysis showed that the overall survival of the CM group was longer than that of the non-CM group (36 months vs. 26 months), and COX regression analysis showed that LK treatment was an independent favorable prognostic factor (P=0.027). Next, 97 components and 86 potential targets were included in the network pharmacology, KEGG and GO analyses, and the results indicated that LK was associated with proliferation and apoptosis. Moreover, molecular docking revealed a good binding affinity between the key ingredients and targets. In vitro, A549 cell proliferation and migration assay showed that the biological inhibition effect was more obvious with the increase of LK concentration (P<0.05). And decreased expressions of nuclear factor κB1 (NF-κB1), epidermal growth factor receptor (EGFR) and AKT serine/threonine kinase 1 (AKT1) and increased expression of p53 (P<0.05) indicated the inhibitory effect of LK on NSCLC by Western blot., Conclusion: LK inhibits NSCLC by inhibiting EGFR/phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, NFκB signaling pathway and inducing apoptosis, which provides evidence for the therapeutic mechanism of LK to increase overall survival in NSCLC patients., (© 2024. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Lung cancer is a complex and heterogeneous disease, which has been associated with various molecular alterations, including the overexpression and mutations of the epidermal growth factor receptor (EGFR). In this study, designed a library of 1843 benzimidazole-1,2,3-triazole hybrids and carried out pharmacophore-based screening to identify potential EGFR inhibitors. The 164 compounds were further evaluated using molecular docking and molecular dynamics simulations to understand the binding interactions between the compounds and the receptor. In-si-lico ADME and toxicity studies were also conducted to assess the drug-likeness and safety of the identified compounds. The results of this study indicate that benzimidazole-1,2,3-triazole hybrids BENZI-0660, BENZI-0125, BENZI-0279, BENZI-0415, BENZI-0437, and BENZI-1110 exhibit dock scores of -9.7, -9.6, -9.6, -9.6, -9.6, -9.6 while referencing molecule -7.9 kcal/mol for EGFR (PDB ID: 4HJO), respectively. The molecular docking and molecular dynamics simulations revealed that the identified compounds formed stable interactions with the active site of EGFR, indicating their potential as inhibitors. The in-silico ADME and toxicity studies showed that the compounds had favorable drug-likeness properties and low toxicity, further supporting their potential as therapeutic agents. Finally, performed DFT studies on the best-selected ligands to gain further insights into their electronic properties. The findings of this study provide important insights into the potential of benzimidazole-1,2,3-triazole hybrids as promising EGFR inhibitors for the treatment of lung cancer. This research opens up a new avenue for the discovery and development of potent and selective EGFR inhibitors for the treatment of lung cancer.Communicated by Ramaswamy H. Sarma.

Resistance to osimertinib represents a significant challenge for the successful treatment of non-small cell lung cancer (NSCLC) harboring activating mutations in EGFR. N6-methyladenosine (m6A) on mRNAs is critical for various biological processes, yet whether m6A regulates osimertinib resistance of NSCLC remains unknown. In this study, we demonstrated that developing osimertinib-resistant phenotypes depends on m6A reduction resulting from downexpression of m6A methyltransferase METTL14 in EGFR-mutant NSCLCs. Both in vitro and in vivo assays showed that specific knockdown of METTL14 was sufficient to confer osimertinib resistance and that elevated expression of METTL14 rescued the efficacy of osimertinib in the resistant NSCLC cells. Mechanistically, METTL14 promoted m6A methylation of pro-apoptotic Bim mRNA and increased Bim mRNA stability and expression, resulting in activating the Bim-dependent pro-apoptotic signaling and thereby promoting osimertinib-induced cell apoptosis. Analysis of clinical samples revealed that decreased expression of METTL14 was observed in osimertinib-resistant NSCLC tissues and significantly associated with a poor prognosis. In conclusion, our study reveals a novel regulatory mechanism by which METTL14-mediated m6A methylation of Bim mRNA inhibited osimertinib resistance of NSCLC cells. It offers more evidences for the involvement of m6A modification in regulation of osimertinib resistance and provides potential therapeutic targets for novel approaches to overcome the tolerance of osimertinib and other EGFR tyrosine kinase inhibitors. Implications: This study offers more evidences for the involvement of METTL14-mediated N6-methyladenosine modification in regulation of osimertinib resistance and provides potential therapeutic targets for novel approaches to overcome the tolerance of osimertinib and other EGFR tyrosine kinase inhibitors., (©2024 American Association for Cancer Research.)

Almost 80% of lung cancer diagnoses each year correspond to non-small cell lung cancer (NSCLC). The percentage of NSCLC with EGFR overexpression ranges from 40% to 89%, with squamous tumors showing the greatest rates (89%) and adenocarcinomas showing the lowest rates (41%). Therefore, in NSCLC therapy, blocking the EGFR-driven pathway by inhibiting the intracellular tyrosine kinase domain of EGFR has exhibited significant improvement. In this view, several small molecules particularly pyrimidine/fused pyrimidine scaffolds were intended for molecular hybridization to develop EGFR-TK inhibitors. However, the associated limitation such as resistance and genetic mutation along with adverse effects, constrained the long-term treatment and effectiveness of such medication. Therefore, in recent years, pyrimidine derivatives were uncovered as potential EGFR TKIs. The present review summarised the research progress of EGFR TKIs to dazed structure-activity relationship, biological evaluation, and comparative docking studies of pyrimidine compounds. We have added the comparative docking analysis followed by the molecular simulation study against the four different PDBs of EGFR to strengthen the already existing research. Docking analysis unfolded that compound 14 resulted as noticeable with all different PDB and managed to interact with some of the crucial amino acid residues. From a future perspective, researchers must develop a more selective inhibitor, that can selectively target the mutation. Our review will support medicinal chemists in the direction of the development of novel pyrimidine-based EGFR TKIs.Communicated by Ramaswamy H. Sarma.

Epidermal growth factor receptor wild type lung adenocarcinoma (EGFR WT LUAD) still has limited treatment options and unsatisfactory clinical outcomes. Ferroptosis, as a form of cell death, has been reported to play a dual role in regulating tumor cell survival. In this study, we constructed a 3-ferroptosis-gene signature, FeSig, and verified its accuracy and efficacy in predicting EGFR WT LUAD prognosis at both the RNA and protein levels. Patients with higher FeSig scores were found to have worse clinical outcomes. Additionally, we explored the relationship between FeSig and tumor microenvironment, revealing that enhanced interactions between fibroblasts and tumor cells in FeSig high patients causing tumor resistance to ferroptosis. To address this challenge, we screened potential drugs from NCI-60 (The US National Cancer Institute 60 human tumour cell line anticancer drug screen) and Connectivity map database, ultimately identifying 6-mercatopurine (6-MP) as a promising candidate. Both in vitro and in vivo experiments demonstrated its efficacy in treating FeSig high EGFR WT LUAD tumor models. In summary, we develop a novel FeSig for predicting prognosis and guiding drug application., (© 2024. The Author(s).)

Treatment of non-small-cell lung cancer (NSCLC) patients possessing EGFR-activating mutations with tyrosine kinase inhibitors (TKIs) can confer an initial promising response. However, TKI resistance inevitably arises. Numerous TKI resistance mechanisms are identified including EGFR secondary mutations, bypass receptor tyrosine kinase (RTK) signaling, and cellular transition e.g. epithelial-mesenchymal transition (EMT). To increase the knowledge of TKI resistance we performed an epigenetic screen to identify small non-coding (nc) genes with DNA methylation alterations in HCC827 NSCLC EGFR-mutated cells with acquired TKI resistance. We analyzed Infinium Methylation EPIC 850K Array data for DNA methylation changes present in both TKI-resistant HCC827 cells with EMT and MET-amplification. Hereby, we identified that the polymorphic maternal imprinted gene nc886 (vtRNA2-1) has a decrease in promoter DNA methylation in TKI-resistant cells. This epigenetic change was associated with an increase in the expression of nc886. The induction of EMT did not affect nc886 expression. CRISPR/Cas9-mediated distortion of the nc886 sequence increased the sensitivity of HCC827 cells towards TKI. Finally, nc886 sequence distortion hindered MET RTK activation and instead was EMT the endpoint TKI resistance mechanism. In conclusion, the expression of nc886 contributes to TKI resistance in the HCC827 NSCLC cell line by supporting cell survival and selection of the endpoint TKI resistance mechanism. We propose DNA methylation and expression changes for nc886 to constitute a novel TKI resistance contributing mechanism in NSCLC., Competing Interests: Declaration of competing interest ‘The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.’, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In our previous study, we have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove plant and found that CyH inhibited the proliferation of non-small cell lung cancer (NSCLC) cells. Recently, epidermal growth factor receptor (EGFR) activation and programmed cell death 1 ligand (PD-L1) expression have been demonstrated to mediate NSCLC resistance to gefitinib, first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI). Here, we further investigated the effect of CyH on EGFR activation, PD-L1 expression, and gefitinib sensitivity in NSCLC cell lines, A549 (wild-type EGFR), HCC827 (EGFR mutation), and NCI-H1975 (dual EGFR mutations and acquired gefitinib resistance) and animal model. Our results showed that CyH significantly inhibited EGFR activation and PD-L1 expression in NSCLC cells. Additionally, CyH dramatically promoted the inhibitory effect of gefitinib on the proliferation of A549 and HCC827 cells, and enhanced the sensitivity to gefitinib in NCI-H1975 cells. Moreover, CyH increased the inhibitory effect of gefitinib on EGFR activation and PD-L1 expression in HCC827 and NCI-H1975 cells. Animal experiments further demonstrated that CyH significantly promoted the inhibitory effect of gefitinib on the growth of NSCLC and the expression of Ki-67, p-EGFR, and PD-L1 in NCI-H1975 NSCLC xenograft tumors of nude mice. Furthermore, CyH inhibited the activation of JAK3/STAT signaling pathway. Taken together, our findings suggest that CyH promotes the sensitivity to gefitinib in NSCLC cells through the inhibition of EGFR activation and PD-L1 expression., (© 2024. The Author(s).)

Lung squamous cell carcinoma (LUSC) is associated with high mortality and has few therapeutic options. Chemotherapy remains the main treatment for LUSC patients, but multi-drug resistance has become the dominant challenge in the failure of chemotherapy in various cancers. Therefore, the effective therapeutic strategy for LUSC patients is an urgent unmet need. Here, we found vasohibin-2 (VASH2) was a prognostic biomarker for LUSC patients, and VASH2 promoted the malignant biological behaviors of LUSC cells and chemoresistance by increasing the detyrosination of α-tubulin. The high level of detyrosinated-tubulin was negatively associated with patient prognosis. Blocking the tubulin carboxypeptidase (TCP) activity of VASH2 inhibited the xenograft tumor growth and improved the treatment efficacy of paclitaxel in vivo. Results revealed that VASH2-induced increase in tubulin detyrosination boosted the binding of kinesin family member 3C (KIF3C) to microtubules and enhanced KIF3C-dependent endosomal recycling of EGFR, leading to the prolonged activation of PI3K/Akt/mTOR signaling. This study demonstrated that VASH2 was not only a prognostic biomarker but also a promising therapeutic target in LUSC, which offers a novel insight that combination of chemotherapy and EpoY, a TCP inhibitor, may be a promising treatment strategy for LUSC patients., (© 2024. The Author(s).)

Purpose: The aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples., Methods: Four NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex ® -T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning ® Elplasia ® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations)., Results: We successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected., Conclusion: The establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type., (© 2024. The Author(s).)

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin ( r 2 : 0.438, 0.695, and 0.736, respectively, all P ＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P =0.034). Traf4 -/- cells had a weakened proliferative capacity than traf4 +/+ cells and formed tumors with smaller size ( P ＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4 -/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4 +/+ cells [(62.3±10.3)%, P =0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4 -/- cells were lower than those of the traf4 +/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P ＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P ＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Objectives: EGFR tyrosine kinase inhibitor (EGFR-TKI) therapies such as erlotinib and gefitinib are approved for the treatment of non-small cell lung cancer (NSCLC). However, the high incidence of acquired resistance to these EGFR-TKIs may preclude their effectiveness. Piperlongumine (PPL), an extract from the long pepper fruit ( Piper longum ), has been shown to possess anticancer properties. The purpose of the study was to investigate piperlongumine as an anticancer agent and to study a combination treatment approach with EGFR-TKIs against lung cancer cells., Methods: Anticancer efficacy of PPL, erlotinib (ERL), gefitinib (GEF), and cisplatin (CIS) were investigated in H1299 and H1975 cell lines. Cells were treated with PPL, ERL, GEF, and CIS alone, and in combination, cell viability was determined after 72 h. The mechanism of PPL-induced cytotoxicity was investigated via reactive oxygen species (ROS) induction, and apoptosis induction using acridine orange/ethidium bromide staining and flow cytometry. The effect of treatment on EGFR-mediated oncogenic signaling was investigated by immunoblotting for mitogenic and apoptotic markers., Results: PPL exhibited a potent cytotoxic effect in H1299 and H1975 cells compared to ERL, GEF, and CIS. Combination treatments of PPL with GEF and ERL showed significant reductions in cancer cells compared to control in both cell lines, which were associated with apoptotic induction, but without significant ROS induction. Compared to control, PPL with GEF significantly increased apoptotic cell death in H1975as confirmed with flow cytometry. Treatment with PPL alone and in combination induced anti-mitogenic and apoptotic responses at the molecular level., Conclusion: PPL sensitized lung cancer cells to EGFR-TKI and induced potent cytotoxic effects at low concentrations., Competing Interests: The authors declare that they have no conflicts of interest to report regarding the present study. The authors also declare that the article is an original research work that has not been submitted anywhere else., (© 2024 The Authors.)

A novel series of substituted thiazolo[5,4-b]pyridine analogues were rationally designed and synthesized via a multi-step synthetic pathway, including Suzuki cross-coupling reaction. The anticancer activity of all forty-five synthesized derivatives was evaluated against HCC827, H1975, and A549 cancer cell lines utilizing the standard MTT assay. A significant number of the thiazolo[5,4-b]pyridine derivatives exhibited potent anticancer activity. Notably, compounds 10b, 10c, 10h, 10i, and 10k emerged as the most promising anticancer agents. The lead compound, N-(3-(6-(2-aminopyrimidin-5-yl)thiazolo[5,4-b]pyridin-2-yl)-2-methylphenyl)-2,5-difluorobenzenesulfonamide (10k), displayed remarkable potency with IC 50 values of 0.010 μM, 0.08 μM, and 0.82 μM against the HCC827, NCI-H1975 and A-549 cancer cell lines, respectively, which were comparable to the clinically approved drug Osimertinib. Importantly, the potent derivatives 10b, 10c, 10h, 10i, and 10k exhibited selective cytotoxicity towards cancer cells and showing no toxicity against the normal BEAS-2B cell line at concentrations exceeding 35 μM. Mechanistic studies revealed that the active compound 10k acts as an EGFR-TK autophosphorylation inhibitor in HCC827 cells. Furthermore, apoptosis assays demonstrated that compound 10k induced substantial early apoptosis (31.9 %) and late apoptosis (8.8 %) in cancer cells, in contrast to the control condition exhibiting only 2.0 % early and 1.6 % late apoptosis. Molecular docking simulations of the synthesized compounds revealed that they formed essential hinge interactions and established hydrogen bonding with Cys797, indicating potential target engagement. These findings highlight the potential of the synthesized thiazolo [(Woodburn, 1999; Zigrossi et al., 2022) 5,45,4-b]pyridine derivatives as promising anticancer agents, warranting further investigation for the development of novel targeted therapies against non-small cell lung cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

In search of small molecules for targeted therapy of non-small cell lung carcinoma (NSCLC), an efficient four-step synthetic route was followed for the synthesis of new imidazothiazole-hydrazone hybrids, which were assessed for their cytotoxic effects on human lung adenocarcinoma (A549) and human lung fibroblast (CCD-19Lu) cells. Among them, compounds 4, 6, 13, 16, 17 and 21 exhibited selective cytotoxic activity against A549 cell line. In vitro mechanistic studies were performed to assess their effects on apoptosis, caspase-3, cell cycle, EGFR and Akt in A549 cells. Compounds 6, 16, 17 and 21 promoted apoptotic cell death more than erlotinib. According to the in vitro data, it is quite clear that compound 6 promotes apoptosis through caspase-3 activation and arrests the cell cycle at the G0/G1 phase in A549 cells. Compounds 16 and 17 arrested the cell cycle at the S phase, whereas compounds 4, 13 and 21 caused the cell cycle arrest at the G2/M phase. The most effective EGFR inhibitor in this series was found as compound 13, followed by compounds 17 and 16. Furthermore, Akt inhibitory effects of compounds 16 and 17 in A549 cells were close to that of GSK690693. In particular, it can be concluded that the cytotoxic and apoptotic effects of compounds 16 and 17 are associated with their inhibitory effects on both EGFR and Akt. Molecular docking studies suggest that compounds 16 and 17 interact with crucial amino acid residues in the binding sites of human EGFR (PDB ID: 1M17) and Akt2 (PDB ID: 3D0E). Based on the in silico data, both compounds are predicted to possess favorable oral bioavailability and drug-likeness. Further studies are required to benefit from these compounds as anticancer agents for targeted therapy of NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Introduction: Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils that may modulate different steps of the metastatic cascade. The aim of our study was to investigate how NETs can modulate the adhesion properties of cancer cells and whether cell exposure to NETs can activate the epithelial-to-mesenchymal transition (EMT) program thus enhancing the migratory and invasive properties of tumor cells., Materials and Methods: Different cancer cell lines were subjected to a solid-phase adhesion assay using NET-coated plates with or without the addition of antibodies against α5β1 or CCDC25 receptor. After 1-4 h of incubation, adherent cells were expressed as the percentage of total cell number. To test EMT occurrence, cells were treated with NETs for up to 48 h and then the levels of E-cadherin, vimentin, Snail, Slug, Zeb 1 and Twist 1 along with levels of Notch 1 and cleaved Notch 1 were determined by western blotting. Untreated and NET-treated cells were subjected to migration assays using 24-multiwell plates with transwell and FBS as chemoattractant., Results: Cancer cell adhesion to NET-coated plates varied between 30% and 92.7% and was significantly higher than that obtained in uncoated plates. The addition of antibodies against α5β1 or CCDC25 caused a strong reduction of cell adhesion to NETs. The prolonged exposure of EGFR-driven cancer cell lines to NETs caused the activation of the EMT program through the upregulation and cleavage of Notch 1 and was confirmed by the enhanced expression of EMT markers. The consequent loss of the epithelial phenotype induced a strong reduction of the expression of the oncogene driver. Cell migration was significantly enhanced in NET-treated cells as compared to untreated cells., Discussion: Our findings reveal the dynamic role of NETs that may provide a DNA and fibronectin rich environment for binding of many cancer cells at distant sites where the prolonged exposure to NETs triggers the EMT through the activation of Notch 1 signaling pathway with the subsequent enhancement of migratory and invasive properties of cancer cells. Furthermore, our findings provide an example of how an immune/inflammatory microenvironment may directly modulate the sensitivity of cancer cells to oncogene targeted agents., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dimitrov, Maddalena, Terlizzi, Altobelli, Pellegrino, Mehmood, De Rosa, Iommelli and Del Vecchio.)

Radioresistance contributes to metastasis and recurrence in non-small cell lung cancer (NSCLC) patients. However, the underlying mechanism remains unclear. To provide novel clues, a complete multi-omics map of a radioresistant cancer cell line has been profiled. In this article, a lung adenocarcinoma cell line, radioresistant A549 (RA549), was generated by exposure to a series of irradiation. Subsequently, we adopted transcriptome, quantitative proteome and lysine 2-hydroxyisobutyrylome to construct a differential profile on the transcriptional to post-tanslational levels on A549 and RA549 cell lines, respectively. Our analysis revealed 920 significantly differentially expressed genes and 699 proteins. Furthermore, 2-hydroxyisobutyrylome identified 30,089 Khib modified sites on 4635 proteins, indicating that Khib modifications play vital role in regulating NSCLC radioresistance. Multi-omics combined analysis identified 19 significantly differentially expressed genes/proteins in total. Meanwhile, we found that EGFR, a well-known lung cancer-related receptor, was upregulated at both the protein and Khib modification levels in RA549. Further gain/loss of function experiments showed that Khib modified EGFR level positively correlates with NSCLC cell radioresistance. Taken together, our findings report that Khib-modified proteins enhanced resistance to radiation and represent promising therapeutic targets., (© 2024. The Author(s).)

Mutations in the epidermal growth factor receptor (EGFR) gene are common driver oncogenes in non-small cell lung cancer (NSCLC). Studies have shown that afatinib is beneficial for NSCLC patients with rare EGFR mutations. However, the effectiveness of tyrosine kinase inhibitors (TKIs) against the G719X (G719A, G719C and G719S) mutation has not been fully established. Herein, using the CRISPR method, the EGFR G719X mutant cell lines were constructed to assess the sensitivity of the rare mutation G719X in NSCLC. WZ3146, a novel mutation-selective EGFR inhibitor, was conducted transcriptome sequencing and in vitro experiments. The results showed that WZ3146 induced cytotoxic effects, inhibited growth vitality and proliferation via ERK and AKT pathway in the EGFR G719X mutant cells. Our findings suggest that WZ3146 may be a promising treatment option for NSCLC patients with the EGFR exon 18 substitution mutation G719X., (© 2024. The Author(s).)

Small-cell lung cancer (SCLC) transformation accounts for 3-14% of resistance in EGFR-TKI relapsed lung adenocarcinomas (LUADs), with unknown molecular mechanisms and optimal treatment strategies. We performed transcriptomic analyses (including bulk and spatial transcriptomics) and multiplex immunofluorescence on pre-treated samples from LUADs without transformation after EGFR-TKI treatment (LUAD-NT), primary SCLCs (SCLC-P) and LUADs with transformation after EGFR-TKI treatment (before transformation: LUAD-BT; after transformation: SCLC-AT). Our study found that LUAD-BT exhibited potential transcriptomic characteristics for transformation compared with LUAD-NT. We identified several pathways that shifted during transformation, and the transformation might be promoted by epigenetic alterations (such as HDAC10, HDAC1, DNMT3A) within the tumor cells instead of within the tumor microenvironment. For druggable pathways, transformed-SCLC were proved to be less dependent on EGF signaling but more relied on FGF signaling, while VEGF-VEGFR pathway remained active, indicating potential treatments after transformation. We also found transformed-SCLC showed an immuno-exhausted status which was associated with the duration of EGFR-TKI before transformation. Besides, SCLC-AT exhibited distinct molecular subtypes from SCLC-P. Moreover, we constructed an ideal 4-marker model based on transcriptomic and IHC data to predict SCLC transformation, which obtained a sensitivity of 100% and 87.5%, a specificity of 95.7% and 100% in the training and test cohorts, respectively. We provided insights into the molecular mechanisms of SCLC transformation and the differences between SCLC-AT and SCLC-P, which might shed light on prevention strategies and subsequent therapeutic strategies for SCLC transformation in the future., (© 2024. The Author(s).)

We report a case of 59-year-old female with solitary bilateral renal metastases after surgery of stage IA primary lung adenocarcinoma who underwent next-generation sequencing (NGS) of both lesions. The patient received right upper lobectomy and lymph node dissection, which revealed primary invasive lung adenocarcinoma (pT1cN0M0, stage IA3). Two years following this, positron emission tomography-computed tomography (PET/CT) revealed multiple masses in both kidneys without other distant metastases, and ultrasonography-guided puncture biopsy indicated the presence of metastatic lung adenocarcinoma. The NGS of both the primary and metastatic lesions revealed the co-alteration of epidermal growth factor receptor (EGFR), RB transcriptional corepressor 1 (RB1), and mitogen-activated protein kinase kinase 1 (MAP3K1), which is potentially associated with the risk of renal metastasis in early postoperative non-small cell lung cancer., (© 2024 The Author(s). The Clinical Respiratory Journal published by John Wiley & Sons Ltd.)

Despite initial high response rates to first-line EGFR TKI, all non-small-cell lung cancer (NSCLC) with EGFR-activating mutation will ultimately develop resistance to treatment. Identification of resistance mechanisms is critical to adapt treatment and improve patient outcomes. Here, we show that a PPP3CB transcript that encodes full-length catalytic subunit 2B of calcineurin accumulates in EGFR-mutant NSCLC cells with acquired resistance against different EGFR TKIs and in post-progression biopsies of NSCLC patients treated with EGFR TKIs. Neutralization of PPP3CB by siRNA or inactivation of calcineurin by cyclosporin A induces apoptosis in resistant cells treated with EGFR TKIs. Mechanistically, EGFR TKIs increase the cytosolic level of calcium and trigger activation of a calcineurin/MEK/ERK pathway that prevents apoptosis. Combining EGFR, calcineurin, and MEK inhibitors overcomes resistance to EGFR TKI in both in vitro and in vivo models. Our results identify PPP3CB overexpression as a new mechanism of acquired resistance to EGFR TKIs, and provide a promising therapeutic approach for NSCLC patients that progress under TKI treatment., (© 2024 Gazzeri et al.)

Background/aim: Low selectivity and high frequency of side-effects are the major problems of currently used chemotherapeutics. Among natural compounds, the polyprenylated acylphloroglucinol, guttiferone E, isolated from Brazilian red propolis, has attracted attention due to its marked anticancer properties and was evaluated here for its role against osimertinib-resistant H1975 cells (with double mutations of epidermal growth factor receptor: EGFR L858R/T790M)., Materials and Methods: Guttiferone E was obtained from red propolis using established extraction procedures. Guttiferone E was tested using the H1975 cell line in in vitro (2D and 3D) cell cultures and in vivo in BALB/c athymic nude mice. Live/dead assay was also performed to support the results. Tumor tissues obtained from in vivo studies were used for western blotting. Guttiferone E reduced H1975 cell viability in a concentration-dependent manner. The IC 50 values in 2D and 3D cell lines were 2.56±0.12 μM and 11.25±0.34 μM. Furthermore, at 10 mg/kg intraperitoneally, guttiferone E significantly reduced the tumor volume in tumor xenografts when used alone and in combination with carboplatin. Guttiferone E and carboplatin displayed synergistic inhibition of H1975 cells and animal tumors. Co-treatment of guttiferone E with carboplatin induced more prominent apoptosis than treatment with either drug alone. Guttiferone E treatment induced cleavage of poly-ADP ribose polymerase and induced apoptosis by significantly reducing levels of mammalian target of rapamycin, sirtuin 1, sirtuin 7, superoxide dismutase, programmed death-ligand 1, and programmed cell death 1 in tumor tissues., Conclusion: Our results show guttiferone E to be a promising, novel and potent antitumor drug candidate for osimertinib-resistant lung cancer with EGFR L858R/T790M mutations., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aberrant activation or mutation of the EGFR-PI3K-Akt-mTOR signaling pathway has been implicated in a wide range of human cancers, especially non-small-cell lung cancer (NSCLC). Thus, dual inhibition of EGFR and PI3K has been investigated as a promising strategy to address acquired drug resistance resulting from the use of tyrosine kinase inhibitors. A series of dual EGFR/PI3Kα inhibitors was synthesized using pharmacophore hybridization of the third-generation EGFR inhibitor olmutinib and the PI3Kα selective inhibitor TAK-117. The optimal compound 30k showed potent kinase inhibitory activities with IC 50 values of 3.6 and 30.0 nM against EGFR L858R/T790M and PI3Kα, respectively. Compound 30k exhibited a significant antiproliferative effect in NCI-H1975 cells with a higher selectivity profile than olmutinib. The potential antitumor mechanism, molecular binding modes, and in vitro metabolic stability of compound 30k were also clarified., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: We investigated the unknown mechanisms of osimertinib-resistant EGFR-mutant lung cancer., Methods: An osimertinib-resistant cell line (PC-9/OsmR2) was established through continuous exposure to osimertinib using an EGFR exon 19 deletion (19Del) lung adenocarcinoma cell line (PC-9). EGFR 19Del (M1), L858R/T790M/C797S (M6), and L858R/C797S (M8) expression vectors were introduced into Ba/F3 cells. A second osimertinib-resistant line (M1/OsmR) was established through continuous exposure to osimertinib using M1 cells., Results: SLC1A3 had the highest mRNA expression level in PC-9/OsmR2 compared to PC-9 cells by microarray analysis and SLC1A3 was increased by flow cytometry. In PC-9/OsmR2 cells, osimertinib sensitivity was significantly increased in combination with siSLC1A3. Because SLC1A3 functions in glutamic acid transport, osimertinib with a glutaminase inhibitor (CB-839) or an SLC1A3 inhibitor (TFB-TBOA) increased the sensitivity. Also, CB-839 plus TFB-TBOA without osimertinib resulted in greater susceptibility than did CB-839 or TFB-TBOA plus osimertinib. Comprehensive metabolome analysis showed that the M1/OsmR cells had significantly more glutamine and glutamic acid than M1 cells. CB-839 plus osimertinib exerted a synergistic effect on M6 cells and an additive effect on M8 cells., Conclusion: Targeting glutaminase and glutamic acid may overcome the osimertinib-resistant EGFR-mutant lung cancer., Competing Interests: Declaration of competing interest Dr. Takigawa has received grants and personal fees from Eli Lilly Japan, AstraZeneca, Daiichi-Sankyo Pharmaceutical, Chugai Pharmaceutical, Taiho Pharmaceutical, Pfizer Inc. Japan, Boehringer-Ingelheim Japan, and Ono Pharmaceutical; grants from Kyowa Hakko Kirin, Nippon Kayaku Co. Ltd., and Takeda Pharmaceutical Co. Ltd.; and personal fees from MSD and Bristol-Myers Squibb Company Japan outside the submitted work. Dr. Ochi has received personal fees from Eli Lilly Japan K.K., Taiho Pharmaceutical Co. ltd., Chugai Pharmaceutical, Pfizer Japan Inc., Ono pharmaceutical, MSD, Bristol-Myers Squibb, Boehringer Ingelheim, and Nippon Kayaku outside the submitted work. Dr. Fukazawa has received personal fees from Boehringer-Ingelheim Japan outside the submitted work. Dr. Kiura has received grants and personal fees from Taiho Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Ono Pharmaceutical, Chugai Pharmaceutical, Nippon Kayaku Co., Ltd., Teijin Pharma Ltd., Shionogi Pharma Co., Ltd., Novartis Pharma, Takeda Pharmaceutical Co., Ltd., Kyorin Pharmaceutical Co., Ltd., Merck Biopharma Co., Ltd., MSD, and Pfizer Japan Inc. and personal fees from Eli Lilly Japan, AstraZeneca, Daiichi-Sankyo Pharmaceutical, Bristol-Myers Squibb, and Nipro Pharma Co. outside the submitted work. The other authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lung cancer is the leading cause of cancer-related deaths, in part due to its late diagnosis. Increased epidermal growth factor receptor (EGFR) expression in cancer cells is associated with a poor prognosis, and EGFR tyrosine kinase inhibitors are widely used in cancer treatment. This study aimed to clarify the relationship between EGFR expression on T cells and cancer prognosis in patients with non-small cell lung cancer (NSCLC). Forty patients with NSCLC and 40 healthy volunteers were included in this study. Peripheral CD4 + T helper (Th1, Th2, Th9, Th17, Th1Th17, follicular and peripheral Th) and cytotoxic T lymphocyte (CD8 + follicular and peripheral T) subsets were identified with flow cytometry according to their chemokine receptors. EGFR expression on T lymphocytes in relation to overall survival (OS) was investigated in patients with NSCLC. The patients [mean age (min-max) = 64.03 (45-83); 20 stage I-III and 20 stage IV] had increased EGFR expression on CD3 + T, CD4 + Th, Th1, Th2, and Th17 cells compared to the controls (p < 0.05). High EGFR expression on CD3 + T, CD4 + Th, Th1, and Th2 cells was associated with poor OS. Also, PD-1 expression on lymphocytes, CD3 + T, and Th cells was increased in patients with NSCLC compared to controls. The high expression of EGFR and PD-1 on Th cells and the reduced percentage of lymphocytes and Th cells, especially in stage IV patients with NSCLC, revealed that increased EGFR activity may trigger apoptosis of Th cells and promote the development of metastases, while high EGFR expression on CD3 + T, CD4 + Th, Th1, and Th2 cells may be an independent poor prognostic marker in NSCLC., (© 2024 The Scandinavian Foundation for Immunology.)

Background: The mutations of oncogenic epidermal growth factor receptor (EGFR) is an important cause of lung adenocarcinoma (LUAD) malignance. It has been knowm that metabolic reprogramming is an important hallmark of malignant tumors, and purine metabolism is a key metabolic pathway for tumor progression and drug resistance, but its relationship with the EGFR-mutant LUAD is unclear., Methods: Metabolic reprogramming was studied through capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolic profiling analysis. Cell proliferation in vitro was evaluated by EdU staining and cell cycle assay. Tumorigenicity in vivo was tested by subcutaneous tumor formation experiment in nude mice. The binding of hypoxia-inducible factor-1 alpha (HIF-1α) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was detected by DNA pull‑down assay and Chromatin immunoprecipitation (ChIP) assays. HIF-1α, HPRT1, DNA damage and cell apoptosis related genes were examined by western blot. In addition, RNA sequencing, mass spectrometry and bioinformatics analysis were performed., Results: We found that mutated EGFR (muEGFR) upregulates HPRT1 to promote purine metabolism and tumorigenesis of EGFR-mutant LUAD. Mechanistically, muEGFR increases HIF-1α expression through protein stability. Meanwhile, up-regulated HIF-1α bound to the promoter of HPRT1 and transcriptionally activates HPRT1 expression, enhancing purine metabolism to maintain rapid tumor cell proliferation in EGFR-mutant LUAD. Further, gefitinib inhibited the synthesis of purine nucleotides, and HPRT1 inhibition increased the sensitivity of gefitinib to EGFR-mutant LUAD., Conclusions: Our study reveals that muEGFR-HIF-1α-HPRT1 axis plays a key role in EGFR-mutant LUAD and provides a new strategy-inhibiting purine metabolism for treating EGFR-mutant LUAD., (© 2024. The Author(s).)

Despite targeted therapies like epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), non-small cell lung cancer (NSCLC) remains a clinical challenge due to drug resistance hampering their efficacy. Here, we designed an "AND" logic gate-based supramolecular therapeutic platform (HA-BPY-GEF-NPs) for the treatment of EGFR-TKI resistant NSCLC. This system integrates both internal and external stimuli-responsive mechanisms that need to be activated in a preset sequence, enabling it to precisely control drug release behavior for enhancing therapeutic precision. By programming the system to respond to sequential near-infrared (NIR) irradiation and enzyme (cathepsin B) inputs, the release of gefitinib is effectively confined to the tumor region. Moreover, the NIR irradiation induces reactive oxygen species production, suppressing tumor growth and inhibiting bypass signaling pathways. The designed drug delivery system offers a highly controlled and targeted therapeutic approach, effectively inhibiting tumor growth, suppressing bypass signaling pathways, and overcoming EGFR-TKI resistance, thus offering a potential solution for maximizing therapeutic benefits.

Background: A growing body of evidence indicates that histone variants play an oncogenic role in cancer progression. However, the role and mechanism of histone variant H2AZ1 in lung cancer remain poorly understood. In this study, we aim to identify novel functions and molecular mechanisms of H2AZ1 in lung cancer., Methods: We analyzed H2AZ1 expression in lung adenocarcinoma using several RNA-seq and microarray datasets. Immunohistochemistry staining for H2AZ1 was performed on two sets of lung cancer tissue microarrays. To study the function of H2AZ1, we conducted assays for cell proliferation, colony formation, invasion, and migration. We employed CUT&Tag-seq, ATAC-seq, RNA-seq, and Western blotting to explore the regulatory patterns and potential mechanisms of H2AZ1 in lung adenocarcinoma., Results: Our findings reveal that H2AZ1 is highly expressed in lung cancer and high levels of H2AZ1 mRNA are associated with poor patient survival. Silencing H2AZ1 impaired cell proliferation, colony formation, migration, and invasion. Mechanistically, our CUT&Tag-seq, ATAC-seq, and RNA-seq results showed that H2AZ1 is primarily deposited around TSS and affects multiple oncogenic signaling pathways. Importantly, we uncovered that H2AZ1 may drive lung cancer progression through the RELA-HIF1A-EGFR signaling pathway., Conclusion: H2AZ1 plays an oncogenic role via several cancer-related pathways, including the RELA-HIF1A-EGFR axis in lung cancer. Intervention targeting H2AZ1 and its related signaling genes may have translational potential for precision therapy., (© 2024. The Author(s).)

Background: CD155 is a transmembrane protein that inhibits antitumor immune response and represents a predictor of worse prognosis in non-small-cell lung cancer (NSCLC). However, it remains unexplored its association with clinical characteristics and genomic status of Latin American patients. This study characterizes the CD155 expression and its clinical implications in this population., Methods: Tissue biopsies from 86 patients with locally-advanced or metastatic NSCLC were assessed for CD155 protein expression, ALK rearrangements and EGFR mutations. Cutoff values for high CD155 expression (CD155 high ) were determined from receiver operating characteristic (ROC) curves according to 2-year survival. It was evaluated its association with clinicopathological features, median progression-free survival (mPFS) and overall survival (mOS)., Results: the cutoff score for CD155 high was 155 in the entire cohort and in patients without oncogenic alterations, and it was 110 in patients with oncogenic alterations. Eighty-four patients (97.7%) were CD155 positive, of which fifty-six (65.0%) had CD155 high . EGFR L858R mutation related to lower CD155 IHC score than exon 19 deletion. Individuals with CD155 high showed a shorter mOS (13.0 vs. 30.8 months; HR: 1.96 [95% CI, 1.15-3.35]; p = 0.014). Patients without oncogenic alterations having a CD155 high displayed shorter mPFS (1.6 vs. 6.4 months, HR: 2.09 [95% CI, 1.06-4.20]; p = 0.034) and mOS (2.9 vs. 23.1 months; HR: 1.27 [95% CI, 1.07- 4.42]; p = 0.032). Patients with oncogenic alterations having CD155 high only showed a trend to shorter mOS (26.3 vs. 52.0 months; HR: 2.39 [95% CI, 0.98-5.83]; p = 0.058)., Conclusion: CD155 high is a predictor of worse outcomes in patients with advanced NSCLC, predominantly among those without oncogenic alterations. CD155 could be a potential biomarker and a molecular target in patients with poor responses to current therapies., (© 2024. The Author(s).)

EGFR-TKIs have been used as frontline treatment in patients with advanced non-small cell lung cancer (NSCLC) suffering from the EGFR mutation. Gefitinib, the first-generation EGFR-TKI, has greatly improved survival rates in lung cancer patients, whereas acquired gefitinib resistance is still a critical issue that needs to be overcome. In our research, high expression levels of CIB2 were found in gefitinib-resistant lung cancer cells. CIB2 knockout rendered gefitinib-resistant cells more sensitive to gefitinib, and overexpression of CIB2 in parental cells was sufficient to induce more resistance to gefitinib. Inhibition of CIB2 in gefitinib-resistant lung cancer cells significantly induced cell apoptosis. To clarify the major molecular mechanism by which CIB2 increases gefitinib resistance, we demonstrated that raised CIB2 in lung cancer cells promoted epithelial-to-mesenchymal transition (EMT) through upregulation of ZEB1. Moreover, FOSL1 transcriptionally regulated CIB2 expression. Finally, CIB2 rendered tumors resistant to gefitinib treatment in vivo . Our results explored a new mechanism: upregulated CIB2 promoted EMT through ZEB1 to regulate gefitinib resistance, which could be a candidate therapeutic target for overcoming acquired resistance to EGFR-TKIs in NSCLC patients.

Non-small cell lung cancer (NSCLC) remains a significant challenge, as it is one of the leading causes of cancer-related deaths, and the development of resistance to anticancer therapy makes it difficult to treat. In this study, we investigated the anticancer mechanism of deoxybouvardin (DB), a cyclic hexapeptide, in gefitinib (GEF)-sensitive and -resistant NSCLC HCC827 cells. DB inhibited the viability and growth of HCC827 cells in a concentration- and time-dependent manner. In vitro kinase assay showed DB inhibited epidermal growth factor receptor (EGFR), mesenchymal-epithelial transition (MET), and AKT, and their phosphorylation was suppressed in HCC827 cells treated with DB. A molecular docking model suggested that DB interacts with these kinases in the ATP-binding pockets. DB induces ROS generation and cell cycle arrest. DB treatment of HCC827 cells leads to mitochondrial membrane depolarization. The induction of apoptosis through caspase activation was confirmed by Z-VAD-FMK treatment. Taken together, DB inhibited the growth of both GEF-sensitive and GEF-resistant NSCLC cells by targeting EGFR, MET, and AKT and inducing ROS generation and caspase activation. Further studies on DB can improve the treatment of chemotherapy-resistant NSCLC through the development of effective DB-based anticancer agents., (© 2024. The Author(s).)

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI) approved for patients with EGFR T790M resistance mutations as first- or second-line treatment of EGFR-positive patients. Resistance to Osimertinib will inevitably develop, and the underlying mechanisms are largely unknown. In this study, we discovered that acquired resistance to Osimertinib is associated with abnormal DNA damage response (DDR) in lung adenocarcinoma cells. We discovered that the polycomb protein Lethal(3) Malignant Brain Tumor-Like Protein 1 (L3MBTL1) regulates chromatin structure, thereby contributing to DDR and Osimertinib resistance. EGFR oncogene inhibition reduced L3MBTL1 ubiquitination while stabilizing its expression in Osimertinib-resistant cells. L3MBTL1 reduction and treatment with Osimertinib significantly inhibited DDR and proliferation of Osimertinib-resistant lung cancer cells in vitro and in vivo. L3MBTL1 binds throughout the genome and plays an important role in EGFR-TKI resistance. It also competes with 53BP1 for H4K20Me2 and inhibits the development of drug resistance in Osimertinib-resistant lung cancer cells in vitro and in vivo. Our findings suggest that L3MBTL1 inhibition is a novel approach to overcoming EGFR-TKI-acquired resistance., (© 2024. The Author(s).)

Acquired resistance is inevitable in the treatment of non-small cell lung cancer (NSCLC) with osimertinib, and one of the primary mechanisms responsible for this resistance is the epithelial-mesenchymal transition (EMT). We identify upregulation of the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and functional inactivation of glycogen synthase kinase 3β (GSK3β) as drivers of EMT-associated osimertinib resistance. Upregulation of PIM1 promotes the growth, invasion, and resistance of osimertinib-resistant cells and is significantly correlated with EMT molecules expression. Functionally, PIM1 suppresses the ubiquitin-proteasome degradation of snail family transcriptional repressor 1 (SNAIL) and snail family transcriptional repressor 2 (SLUG) by deactivating GSK3β through phosphorylation. The stability and accumulation of SNAIL and SLUG facilitate EMT and encourage osimertinib resistance. Furthermore, treatment with PIM1 inhibitors prevents EMT progression and re-sensitizes osimertinib-resistant NSCLC cells to osimertinib. PIM1/GSK3β signaling is activated in clinical samples of osimertinib-resistant NSCLC, and dual epidermal growth factor receptor (EGFR)/PIM1 blockade synergistically reverse osimertinib-resistant NSCLC in vivo. These data identify PIM1 as a driver of EMT-associated osimertinib-resistant NSCLC cells and predict that PIM1 inhibitors and osimertinib combination therapy will provide clinical benefit in patients with EGFR-mutant NSCLC., (© 2024. The Author(s).)

Patients with lung adenocarcinoma who have never smoked (NSLA) and lack key driver mutations, such as those in the EGFR and ALK genes, face limited options for targeted therapies. They also tend to have poorer outcomes with immune checkpoint inhibitors than lung cancer patients who have a history of smoking. The proteogenomic profile of nonsmoking lung adenocarcinoma patients without these oncogenic driver mutations is poorly understood, which complicates the precise molecular classification of these cancers and highlights a significant area of unmet clinical need. This study analyzed the genome, transcriptome, and LC‒MS/MS-TMT-driven proteome data of tumors obtained from 99 Korean never-smoker lung adenocarcinoma patients. NSLA tumors without EGFR or ALK driver oncogenes were classified into four proteogenomic subgroups: proliferation, angiogenesis, immune, and metabolism subgroups. These 4 molecular subgroups were strongly associated with distinct clinical outcomes. The proliferation and angiogenesis subtypes were associated with a poorer prognosis, while the immune subtype was associated with the most favorable outcome, which was validated in an external lung cancer dataset. Genomic-wide impacts were analyzed, and significant correlations were found between copy number alterations and both the transcriptome and proteome for several genes, with enrichment in the ERBB, neurotrophin, insulin, and MAPK signaling pathways. Proteogenomic analyses suggested several targetable genes and proteins, including CDKs and ATR, as potential therapeutic targets in the proliferation subgroup. Upregulated cytokines, such as CCL5 and CXCL13, in the immune subgroup may serve as potential targets for combination immunotherapy. Our comprehensive proteogenomic analysis revealed the molecular subtypes of EGFR- and ALK-wild-type NSLA with significant unmet clinical needs., (© 2024. The Author(s).)

Background/aim: Macrophages prevail in the microenvironment of several tumors, including non-small-cell lung cancer (NSCLC), where they secrete pro-tumorigenic factors that contribute to cancer progression. This study investigated the role of macrophages on the resistance of epidermal growth factor receptor (EGFR)-mutated NSCLC cells to tyrosine kinase inhibitors (TKIs)., Materials and Methods: EGFR-mutated cell lines PC-9 and HCC827 were cocultured with macrophages and treated with TKIs (erlotinib and gefitinib). The effects of the macrophage-conditioned medium (macrophage CM) on gefitinib resistance and cell migration were also evaluated., Results: Co-culture with macrophages significantly enhanced the resistance to erlotinib and gefitinib in PC-9 and HCC827 cells compared to that in cells cultured independently. Macrophage CM markedly induced gefitinib resistance in PC-9 cells, with maximum resistance observed at 50% CM concentration. This resistance persisted for up to 48 h post-CM removal. Macrophage CM inhibited gefitinib-induced apoptosis, as evidenced by the decreased expression of cleaved caspase-3, PARP, and BIM. Additionally, macrophage CM increased the migration ability of PC-9 cells, as shown by the wound healing and transwell migration assays. Studies have shown that TonEBP is crucial in cancer metastasis and drug resistance; we found that inhibition of TonEBP/NFAT5 expression reduced gefitinib resistance and migration in macrophage CM-induced PC-9 cells, indicating its role as mediator of these effects., Conclusion: Macrophages contribute to TKI resistance and enhance the migration of EGFR-mutated NSCLC cells through mechanisms involving TonEBP/NFAT5. Therefore, targeting TonEBP/NFAT5 represents a potential therapeutic strategy for overcoming macrophage-induced TKI resistance in NSCLC cells., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

There is an urgent need to fully understand the biology of third generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs), particularly osimertinib, and to develop mechanism-driven strategies to manage their acquired resistance. Transient receptor potential melastatin-2 (TRPM2) functions as an important regulator of Ca 2+ influx, but its role in mediating therapeutic efficacies of EGFR-TKIs and acquired resistance to EGFR-TKIs has been rarely studied. This study has demonstrated a previously undiscovered role of suppression of TRPM2 and subsequent inhibition of Ca 2+ influx and induction of ROS and DNA damage in mediating apoptosis induction and the therapeutic efficacy of osimertinib against EGFR mutant NSCLC. The rebound elevation represents a key mechanism accounting for the emergence of acquired resistance to osimertinib and other third generation EGFR-TKIs. Accordingly, targeting TRPM2 is a potentially promising strategy for overcoming and preventing acquired resistance to osimertinib, warranting further study in this direction including the development of cancer therapy-optimized TRPM2 inhibitors., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Ivonescimab ( ® ) is a first-in-class, humanized, tetravalent bispecific monoclonal antibody targeting programmed cell death protein 1 (PD-1) and vascular endothelial growth factor (VEGF)-A being developed by Akeso Biopharma for the treatment of non-small cell lung cancer (NSCLC) and other solid tumours, including breast cancer, liver cancer and gastric cancer. Ivonescimab simultaneously blocks the binding of PD-1 to its ligand (PD-L1), thereby relieving PD-1/PD-L1-mediated immunosuppression, and blocks the binding of VEGF-A to its receptor (VEGFR2), thus blocking tumour angiogenesis in the tumour microenvironment. In May 2024, ivonescimab, in combination with pemetrexed and carboplatin, received its first approval in China for the treatment of patients with EGFR-mutated locally advanced or metastatic non-squamous NSCLC who have progressed after tyrosine kinase inhibitor (TKI) therapy. Clinical studies of ivonescimab are underway in multiple countries worldwide. This article summarizes the milestones in the development of ivonescimab leading to this first approval for EGFR-mutated locally advanced or metastatic non-squamous NSCLC who have progressed after TKI therapy., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)