NCI-H460 Cell Lines Research Articles

Abstract 2058: Compound library screening to identify modulators of alternative splicing in non-small cell lung cancer (NSCLC)

Abstract Alternative splicing is a molecular mechanism that allows a single gene to encode multiple proteins. It is a complex and highly controlled process used to regulate normal gene expression but is often dysregulated in cancer. Compounds that can modulate alternative splicing are currently being explored as a potential new class of therapeutic agent in cancer. This highlights the need for a deeper understanding of the splicing process, its regulation, and its impact. The discovery of novel and specific tool compounds that modulate splicing would therefore not only be beneficial in investigating the regulation and dysregulation of splicing in cancer but could also be exploited therapeutically. To identify novel regulators of splicing, we generated a cell-based split luciferase screening assay based on alternative splicing of MCL-1 (myeloid cell leukemia-1 protein) pre-mRNA. The MCL1 gene usually produces an mRNA encoding a ‘long’ variant (MCL1L) that is an anti-apoptotic protein often highly expressed in cancers. However, in some circumstances, for example following genetic knockdown of splicing factors, a pro-apoptotic ‘short’ variant (MCL1S) is expressed as a result of exon-skipping. Here we engineered the human NSCLC cell line NCI-H1299 to express a luminescent-tagged version of the MCL1S splice variant to monitor its induction upon spliceosome modulation. Using this engineered cell line, we screened an unannotated library of 12,000 compounds selected to have low molecular weights and favorable properties for cellular uptake. The screen had an average Z’ value of 0.89 over 45 microplates, indicating high assay robustness. Hits were identified as any compound with a luminescence of greater than the average + two standard deviations of the screening dataset. The compounds were validated by repetition in the screening assay, giving 34 candidate hits that were then triaged by qPCR assay to confirm splicing modulation of MCL1. One interesting hit was found to increase mRNA and cellular protein levels of MCL1S and induced altered splicing across the transcriptome that was distinct from the splicing profiles of a diverse panel of splicing modulators, including compounds targeting SF3B1, CLK, and RBM39. In addition, treatment with this hit compound resulted in accumulation of the splicing factor SC35 in cytoplasmic granules, an unusual phenotype not seen with known splicing modulators, that could explain the distinct splicing modulation we have observed. Deconvolution and further characterization of screening hits and potentially unique new mechanisms of action could improve current understanding of alternative splicing and its regulation in NSCLC. The discovery of novel compounds to expand our current toolset of splicing modulators will be key in building the foundation for future splicing targeted drug discovery. Citation Format: Rachel Cooley, Marissa V. Powers, Adam G. Bond, Patrizia Jensen, Gary Newton, Andrea Scarpino, Juliane Braun, Christina Esdar, Paul A. Clarke. Compound library screening to identify modulators of alternative splicing in non-small cell lung cancer (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 2058.

Abstract 4668: Characterization of fourth-generation EGFR inhibitors in binding experiments with C797S mutant EGFR and cell-based assays with osimertinib-resistant non-small cell lung cancer cell lines

Abstract Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that inhibits the EGFR kinase domain by irreversible binding of the cysteine-797 residue (C797). Osimertinib was originally designed to target the T790M mutation, which is clinically the most frequently observed resistance mechanism to treatment with first- or second-generation EGFR inhibitors. Osimertinib has a high selectivity for the classical EGFR driver mutations (exon 19 deletion and L858R) compared to wild-type EGFR. Due to this favorable selectivity profile, osimertinib has become the standard first-line therapy for non-small cell lung cancer (NSCLC). Common resistance mechanisms following osimertinib treatment are MET oncogene amplification, and cysteine-to-serine substitution at position 797 (C797S) of EGFR, which precludes binding of osimertinib. Fourth-generation EGFR inhibitors and combinations with other targeted agents are potential strategies to overcome osimertinib resistance or to increase its efficacy. Three fourth-generation EGFR inhibitors were profiled in biochemical and cell-based assays: BDTX-1535, BLU-945, and JBJ-09-063. The binding kinetics for wild-type and C797S mutant EGFR were determined in surface plasmon resonance binding experiments on a Biacore 1S+ (Cytiva). The effect on osimertinib-resistant cells, generated using two different approaches, was studied in cell viability assays. In the first model, subclones of the NSCLC cell line PC-9, harboring an exon 19 deletion activating EGFR mutation, were selected for osimertinib resistance by in vitro drug selection [1]. In the second model, the EGFR C797S mutation was introduced into the NSCLC cell line NCI-H1975, which expresses both the L858R activating mutation and the T790M resistance mutation, by gene-editing with CRISPR-Cas9. Synergies with fourth-generation EGFR inhibitors were determined by profiling combinations in a 6-by-6 matrix, and quantifying synergistic effects using the Excess over Bliss score. We will present a detailed comparison of the binding kinetics of the three fourth-generation EGFR inhibitors on wild-type and C797S-mutant EGFR, and their ability to compete with osimertinib for binding to the kinase domain of EGFR. The different fourth-generation inhibitors show various selectivities for the various EGFR mutants and differences in their ability to spare wild-type EGFR. Profiling results of the three fourth-generation EGFR inhibitors in the osimertinib-resistant cell lines and on a panel of more than one hundred human cancer cell lines will be presented. Reference: [1] Bertran-Alamillo et al. Nature Communications 10, 1812; 2019 Citation Format: Janneke J. Melis, Kirsten Kevenaar, Jeffrey J. Kooijman, Yvonne Grobben, Jacob Ytsma, Jordi Bertran-Alamillo, Miguel-Angel Molina-Vila, Nicole Willemsen-Seegers, Guido J. Zaman. Characterization of fourth-generation EGFR inhibitors in binding experiments with C797S mutant EGFR and cell-based assays with osimertinib-resistant non-small cell lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4668.

Abstract 5770: Inhibition of KRAS G12D mutant with small molecules

Abstract Background: RAS mutations are occurring at a high rate in colorectal cancer, lung cancer, and pancreatic cancer. RAS is family of small GTPase proteins with important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The The RAS gene is the most commonly found oncogene in human cancer. KRAS exhibits a highest mutation rate compared to those of HRAS and NRAS in various cancer types. Methods: Colorectal cell line LIM1215, SW48 and NSCLC cell line NCI-H1975, NCI-H838 were used for MTT assays. Apoptosis assay was performed using the annexin V-FITC apoptosis detection kit (BioBud, Cat. LS-02-100). Colony forming assay was performed with 6-well culture plates by seeding 500 cells/well. The expression levels of proteins involved in Q2a-induced signaling pathway were determined by Western blot. In vivo study was performed with tumor xenografts. HT-29 cells were implanted into the right flank of 5-week-old BALB/c nude mice. Tumor size was determined by measuring the diameter (width, length) of the tumor every two days with a digital caliper. All group mice sacrificed at day 19. Results: Patients with cancer involving KRAS mutations are resistant to therapeutics that target epidermal growth factor receptor and show poor outcomes. In addition, effective treatments that target G12D in KRAS have not been developed. Thus, therapeutic agents targeting the G12D mutation in KRAS are needed. In this study, we identified the Q2a compound, a quinazoline-based derivative that specifically acts on the KRAS G12D mutation. This compound induced cell apoptosis by reducing phosphorylation of AKT in the PI3K pathway downstream of KRAS. Q2a compound specifically inhibited the growth of KRAS G12D mutant cells. In addition, cells with the KRAS G12D mutation were more sensitive to Q2a compound compared to the sensitivity of wild-type cells and those with other KRAS mutations, resulting in apoptosis. Treatment with the Q2a compound also decreased C-RAF phosphorylation in the MAPK pathway, which is downstream of KRAS. In KRAS G12D-mutated colorectal cancer xenografts, Q2a compound inhibited tumor production without significant liver toxicity. Conclusion: Q2a compounds show potential as therapeutic agents that target KRAS G12D mutations in colorectal and pancreatic cancers. Kyewords: Anti-Cancer Drug, KRAS G12D Mutant, apoptosis, colon cancerJi Yoon Lee, Dae-Hee Lee*Department of Marine Bio Food Science, Gangneung-Wonju national University Citation Format: Dae-Hee Lee, Ji Yoon Lee. Inhibition of KRAS G12D mutant with small molecules [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 5770.

Abstract 1004: Establishing lung adenocarcinoma cell lines to mitigate lung cancer health disparities

Abstract Lung cancer remains the leading cause of cancer death worldwide and is a pronounced source of cancer health disparities. Black/African American (B/AA) individuals demonstrate the highest lung cancer death rates when compared to other ethnic/racial groups. B/AA males exhibit a 15% higher lung cancer death rate and a 12% higher lung cancer incidence when compared to White males. Lung adenocarcinoma (LUAD) is the most common histological subtype in all races including B/AA. To study lung cancer and its related disparities, essential tools such as lung cancer cell lines are important; they can be used for molecular studies of lung cancer development, progression, development of new therapies and pre-clinical drug testing. We reviewed the literature (Leon et al. 2023, Frontiers in Oncology, Vol. 13, p. 1187585, 2023) and identified only 30 unique B/AA cell lines out of ~800 cell lines available, with the majority of cell lines being from White and Asian patients. Moreover, only 6 B/AA cell lines were derived from patients with LUAD. We are in the process of developing additional LUAD cell lines from patients as well as alveolar epithelial cell lines that can be used to model LUAD development. In addition, we are generating multiple independent isogenic cell line variants from existing lung cancer cell line NCI-H23, derived from a B/AA male patient harboring a KRASG12C heterozygous mutation. We are using CRISPR/Cas-9-based genome editing to replace the KRASG12C mutation with other KRAS mutations found in B/AA patients, including KRASG12V, KRASG12D, KRASG12A, and KRASG13C. We will perform whole genome sequencing to exclude off target effects of the CRISPR engineering. We will use isogenic cell lines to test the efficacy of polyisoprenylated cysteinyl amide inhibitors (PCAIs) and other drugs on cell lines bearing the different KRAS mutations. PCAIs were developed to target cells carrying mutant KRAS. Our efforts will provide tools to limit cancer health disparities and serve as a foundation for future research. Supported by grants U54CA233396, U54CA233444, and U54CA233465 from the National Institutes of Health (NIH)/National Cancer Institute (NCI) and the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI. Citation Format: Sofia Lugo, Christopher Leon, Matthew Gladstone, Chunli Yan, Nazarius S. Lamango, Ite A. Offringa, Kweku Ofosu-Asante, Yong Huang, Justin K. Mensah-Mamfo. Establishing lung adenocarcinoma cell lines to mitigate lung cancer health disparities [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 1004.

Polyphyllin Ⅲ regulates EMT of lung cancer cells through GSK-3β/β-catenin pathway.

Some studies have found that the application of traditional Chinese medicine in the treatment of lung cancer has achieved satisfying results. Polyphyllin Ⅲ (PP Ⅲ) is a natural steroid saponin from P. polyphylla var. yunnanensis, and its analogs have played a wide role in anticancer research. This study aimed to investigate the effect of PP Ⅲ on the development of lung cancer and its molecular mechanism. A549 and NCI-H1299 cell lines were treated with PP Ⅲ in gradient concentration to detect the IC50 of the cells, and the optimal concentration was selected for subsequent experiments. The effects of PP III treatment on lung cancer were investigated in vitro and in vivo. In vitro experiments, it was found that the proliferation, invasion, migration, and colony formation ability of cancer cells were significantly reduced after PP III treatment, while accompanied by a large number of cell apoptosis. Further detection showed that N-cadherin was significantly decreased, E-cadherin was increased, and Snail and Twist were decreased in A549 cells and NCI-H1299 cells, respectively. In addition, GSK-3β expression was increased, while β-catenin expression was reduced with PP III treatment. In the mouse model, it was demonstrated that the volume of transplanted tumors was significantly reduced after PP Ⅲ treatment. PP Ⅲ has the capacity to inhibit the progression of lung cancer and regulate epithelial-mesenchymal transition through the GSK-3β/β-catenin pathway to suppress the malignant behavior of cancer cells. The application of PP Ⅲ is expected to be an effective method for the treatment of lung cancer.

Discovery of Pyroptosis-inducing Drugs and Antineoplastic Activity based on the ROS/ER Stress/Pyroptosis Axis.

Pyroptosis, a cell death process triggered by chemotherapy drugs, has emerged as a highly promising mechanism for combating tumors in recent years. As the lead of new drugs, natural products play an important role in the discovery of anticancer drugs. Compared to other natural products, the medicine food homologous natural products (MFHNP) exhibit a superior safety profile. Among a series of MFHNP molecular skeletons, this study found that only benzylideneacetophenone (1) could induce cancer cell pyroptosis. However, the anti-cancer activity of 1 remains to be improved. This study aimed to find a pyroptosis inducer with highly effective antitumor activity by modifying the chalcone structure. To examine the effect of the Michael receptor in compound 1 on the induction of pyroptosis, several analogs were synthesized by modifying the Michael acceptor. Subsequently, the anticancer activity was tested by MTT assay, and morphological indications of pyroptosis were observed in human lung carcinoma NCI-H460 and human ovarian cancer CP-70 cell lines. Furthermore, to improve the activity of the chalcone skeleton, the anticancer group 3,4,5- trimethoxyphenyl was incorporated into the phenyl ring. Subsequently, compounds 2-22 were designed, synthesized, and screened in human lung cancer cells (NCI-H460, H1975, and A549). Additionally, a quantitative structure-activity relationship (QSAR) model was established using the eXtreme Gradient Boosting (XGBoost) machine learning library to identify the pharmacophore. Furthermore, both in vitro and in vivo experiments were conducted to investigate the molecular mechanisms of pyroptosis induced by the active compound. α, β-unsaturated ketone was the functional group of the chalcone skeleton and played a pivotal role in inducing cancer cell pyroptosis. QSAR models showed that the regression coefficients (R2) were 0.992 (A549 cells), 0.990 (NCI-H460 cells), and 0.998 (H1975 cells). Among these compounds, compound 7 was selected to be the active compound. Moreover, compound 7 was found to induce pyroptosis in lung cancer cells by upregulating the expression of CHOP by increasing the ROS level. Furthermore, it effectively suppressed the growth of lung cancer xenograft tumors. Compound 7 exhibits antineoplastic activity by regulating the ROS/ER stress/pyroptosis axis and is a kind of promising pyroptosis inducer.

Lung cancer cells detection by a photoelectrochemical MoS2 biosensing chip.

This research aims to explore the potential application of this approach in the production of biosensor chips. The biosensor chip is utilized for the identification and examination of early-stage lung cancer cells. The findings of the optical microscope were corroborated by the field emission scanning electron microscopy, which provided further evidence that the growth of MoS2 is uniform and that there is minimal disruption in the electrode, hence minimizing the likelihood of an open circuit creation. Furthermore, the bilayer structure of the produced MoS2 has been validated through the utilization of Raman spectroscopy. A research investigation was undertaken to measure the photoelectric current generated by three various types of clinical samples containing lung cancer cells, specifically the CL1, NCI-H460, and NCI-H520 cell lines. The findings from the empirical analysis indicate that the coefficient of determination (R-Square) for the linear regression model was approximately 98%. Furthermore, the integration of a double-layer MoS2 film resulted in a significant improvement of 38% in the photocurrent, as observed in the device's performance.

Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence.

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence.

Ameliorative inhibition of sirtuin 6 by imidazole derivative triggers oxidative stress-mediated apoptosis associated with Nrf2/Keap1 signaling in non-small cell lung cancer cell lines.

Background: Redox homeostasis is the vital regulatory system with respect to antioxidative response and detoxification. The imbalance of redox homeostasis causes oxidative stress. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, also called Nfe2l2)/Kelchlike ECH-associated protein 1 (Keap1) signaling is the major regulator of redox homeostasis. Nrf2/Keap1 signaling is reported to be involved in cancer cell growth and survival. A high level of Nrf2 in cancers is associated with poor prognosis, resistance to therapeutics, and rapid proliferation, framing Nrf2 as an interesting target in cancer biology. Sirtuins (SIRT1-7) are class III histone deacetylases with NAD + dependent deacetylase activity that have a remarkable impact on antioxidant and redox signaling (ARS) linked with Nrf2 deacetylation thereby increasing its transcription by epigenetic modifications which has been identified as a crucial event in cancer progression under the influence of oxidative stress in various transformed cells. SIRT6 plays an important role in the cytoprotective effect of multiple diseases, including cancer. This study aimed to inhibit SIRT6 using an imidazole derivative, Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate, to assess its impact on Nrf2/Keap1 signaling in A549 and NCI-H460 cell lines. Method: Half maximal inhibitory concentration (IC50) of Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate was fixed by cell viability assay. The changes in the gene expression of important regulators involved in this study were examined using quantitative real-time PCR (qRT-PCR) and protein expression changes were confirmed by Western blotting. The changes in the antioxidant molecules are determined by biochemical assays. Further, morphological studies were performed to observe the generation of reactive oxygen species, mitochondrial damage, and apoptosis. Results: We inhibited SIRT6 using Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate and demonstrated that SIRT6 inhibition impacts the modulation of antioxidant and redox signaling. The level of antioxidant enzymes and percentage of reactive oxygen species scavenging activity were depleted. The morphological studies showed ROS generation, mitochondrial damage, nuclear damage, and apoptosis. The molecular examination of apoptotic factors confirmed apoptotic cell death. Further, molecular studies confirmed the changes in Nrf2 and Keap1 expression during SIRT6 inhibition. Conclusion: The overall study suggests that SIRT6 inhibition by imidazole derivative disrupts Nrf2/Keap1 signaling leading to oxidative stress and apoptosis induction.

Chemical composition, antibacterial, anticancer, and anti-a-glucosidase activities of essential oils from Alpinia nelumboides

The chemical composition and biological properties of essential oils extracted from the rhizomes and leaves of Alpinia nelumboides were examined in this study. The oils were isolated via hydrodistillation using a Clevenger apparatus and analyzed with GC-FID/MS. Antibacterial activity against antibiotic-resistant bacterial strains was evaluated using the agar microdilution method. Anticancer and α-glucosidase inhibitory activities were assessed through in vitro assays. Eucalyptol was identified as the major component, accounting for 71.23% and 32.82% of the rhizome and leaves oils, respectively. Both rhizome and leaf extracts exhibited antibacterial activity against several antibiotic-resistant bacterial strains, with minimum inhibitory concentration values ranging from 8 to 16 mg/ ml. The rhizome essential oil demonstrated anticancer properties against NCI-H460 (IC50 = 98.28 ± 5.22 μg/ml) and HepG2 (IC50 = 189.87 ± 3.16 μg/ml) cell lines. Additionally, it showed α-glucosidase inhibitory activity (IC50 = 2.42 mg/ml). The essential oils obtained from A. nelumboides possess promising biological activities, including antibacterial, anticancer, and α-glucosidase inhibitory properties. These findings suggest their potential use as healthcare pharmaceuticals in the future.

Development of a cell adhesion-based prognostic model for multiple myeloma: Insights into chemotherapy response and potential reversal of adhesion effects.

Multiple myeloma (MM) is a hematologic malignancy notorious for its high relapse rate and development of drug resistance, in which cell adhesion-mediated drug resistance plays a critical role. This study integrated four RNA sequencing datasets (CoMMpass, GSE136337, GSE9782, and GSE2658) and focused on analyzing 1706 adhesion-related genes. Rigorous univariate Cox regression analysis identified 18 key prognosis-related genes, including KIF14, TROAP, FLNA, MSN, LGALS1, PECAM1, and ALCAM, which demonstrated the strongest associations with poor overall survival (OS) in MM patients. To comprehensively evaluate the impact of cell adhesion on MM prognosis, an adhesion-related risk score (ARRS) model was constructed using Lasso Cox regression analysis. The ARRS model emerged as an independent prognostic factor for predicting OS. Furthermore, our findings revealed that a heightened cell adhesion effect correlated with tumor resistance to DNA-damaging drugs, protein kinase inhibitors, and drugs targeting the PI3K/Akt/mTOR signaling pathway. Nevertheless, we identified promising drug candidates, such as tirofiban, pirenzepine, erlotinib, and bosutinib, which exhibit potential in reversing this resistance. In vitro, experiments employing NCIH929, RPMI8226, and AMO1 cell lines confirmed that MM cell lines with high ARRS exhibited poor sensitivity to the aforementioned candidate drugs. By employing siRNA-mediated knockdown of the key ARRS model gene KIF14, we observed suppressed proliferation of NCIH929 cells, along with decreased adhesion to BMSCs and fibronectin. This study presents compelling evidence establishing cell adhesion as a significant prognostic factor in MM. Additionally, potential molecular mechanisms underlying adhesion-related resistance are proposed, along with viable strategies to overcome such resistance. These findings provide a solid scientific foundation for facilitating clinically stratified treatment of MM.

LC-ESI QToF MS Non-Targeted Screening of Latex Extracts of Euphorbia seguieriana ssp. seguieriana Necker and Euphorbia cyparissias and Determination of Their Potential Anticancer Activity.

Euphorbia seguieriana ssp. seguieriana Necker (ES) and Euphorbia cyparissias (EC) with a habitat in the Deliblato Sands were the subject of this examination. The latexes of these so far insufficiently investigated species of the Euphorbia genus are used in traditional medicine for the treatment of wounds and warts on the skin. To determine their chemical composition, non-targeted screening of the latexes' chloroform extracts was performed using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry employing an electrospray ionization source (LC-ESI QTOF MS). The analysis of the obtained results showed that the latexes of ES and EC represent rich sources of diterpenes, tentatively identified as jatrophanes, ingenanes, tiglianes, myrsinanes, premyrsinanes, and others. Examination of the anticancer activity of the ES and EC latex extracts showed that both extracts significantly inhibited the growth of the non-small cell lung carcinoma NCI-H460 and glioblastoma U87 cell lines as well as of their corresponding multi-drug resistant (MDR) cell lines, NCI-H460/R and U87-TxR. The obtained results also revealed that the ES and EC extracts inhibited the function of P-glycoprotein (P-gp) in MDR cancer cells, whose overexpression is one of the main mechanisms underlying MDR.

Abstract C175: The potential effect of KRAS inhibitors on tumor microenvironments

Abstract KRAS is one of the most common driver oncogenes among various cancer types. Researches aiming to develop KRAS inhibitors has been surging recently and the first clinical KRAS inhibitor sotorasib was approved for non-small cell lung cancer in the US and Japan. As KRAS activity has been reported to have certain effects on tumor microenvironments, KRAS inhibitor could potentially induce collateral outcomes on tumor microenvironment. Therefore, I have analyzed the potential influences of resistance to KRAS inhibitors on the tumor microenvironment. At first, I have established pancreatic cancer cell line MIA-Paca2 that acquired resistance to sotorasib, MIA/AMG-R, by continuously exposing them to sotorasib (1 µmol/L). An RNA sequencing analysis were performed with MIA/AMG-R and revealed that a gene set related to hypoxia were upregulated. When non-small cell lung cancer cell line NCI-H358 cells were exposed to sotorasib (1 µmol/L) for 4 hrs, the protein levels of HIF-1A decreased while that in MIA Paca-2 cells were unaffected. To evaluate at which steps of protein synthesis from gene transcription to through protein degradation of HIF-1A, NCI-H358 cells were treated with transcription inhibitor actinomycin D, translation inhibitor cycloheximide or proteasome inhibitor MG-132. The treatment of transcription inhibitor actinomycin D on NCI-H358 cells for 6 hrs did not lead to the decrease of HIF-1A protein level however, translation inhibitor cycloheximide induced drastic decrease in HIF-1A protein. Although exposure of NCI-H358 cells to MG-132 increased the expression level of HIF-1A, the combinational treatment of sotorasib and MG-132 still led to slight decrease in HIF-1A protein level. Also, MG-132 failed to recover the protein expression level decreased by cycloheximide treatment. In total, the cellular expression level of HIF-1A is possibly dependent on translation activity and KRAS inhibitor potentially affect the HIF-1A expression through the regulation of its translation. Conclusively, KRAS inhibitors could potentially be used to reprogram the hypoxic tumor microenvironments besides its primary effect against the downstream signaling of KRAS. Citation Format: Noritaka Tanaka, Takeharu Sakamoto. The potential effect of KRAS inhibitors on tumor microenvironments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C175.

A Scalable, Off-the-Shelf, and Cost-Effective NK Cell Manufacturing Platform for Developing NK Cell-Based Immunotherapy

Natural killer (NK) cells are innate lymphocytes that play a key role in the control of infection and malignancy. NK cells are potent killers of target cells and act in an MHC-independent manner, enabling their application as allogenic therapy for cancer. Human iPSCs are pluripotent with the ability to self-renew, thus providing an inexhaustible source for deriving NK cells. With a two-tiered cell bank system, we can manufacture NK cells with more homogeneity between batches. In principle, one single iPSC clone-derived master cell bank and working cell banks can support the life cycle of an affordable, off-the-shelf NK cell product. We developed a robust stepwise protocol for large-scale generation of high-purity NK cells from human iPSCs without the cell isolation procedure. First, human iPSCs formed embryoid bodies and then were differentiated into highly purified (>98.0%) CD34 + CD43 + hematopoietic stem and progenitor cells (HSPCs) using a combination of cytokines and small molecles such as BMP-4, VEGF, BFGF, SCF, FLT-3 and ascorbic acid. During this period, we observed up to an 180-fold cell increase in the number of HSPCs compared with human iPSCs. Second, these HSPCs were differentiated into NK cells with FLT3, SCF, IL-3, IL-7, and IL-15 etc., with cell number increased approximately 9000-fold compared with iPSCs. In the final step, iPSC-derived NK cells were further expanded up to 8000-fold which enable us to produce 1E12 NK cells from less than 1 million iPSCs. After expansion, iNK cells became more homogenous and exhibited a more mature phenotype. More than 99.0% of cells expressed both CD45 and CD56, and more than 70% expressed CD16. The expression of activating and co-stimulatory receptors (NKG2D, NKp46,NKp44, NKp30, CD244 and CD226) of NK cells was also high in these iNK cells. Intrestingly, the cells exhibited robust anti-tumor efficacy against K562 (chronic myelogenous leukemia), RPMI 8226 (multiple myeloma). Furthermore, the cryopreserved iNK cells demonstrated dramatic cytotoxicity to U-87 MG cells (malignant gliomas) in 2D culture and the tumor organoid, indicating that iNK cells derived from our protocol can be used as off-the-shelf therapy for both hematological and solid tumors. We also observed that these iNK cells had variable cytotoxicity in other tumors. For example, our iNK were not obviously cytotoxic to the NCI-H929 cell line, derived from a plasmacytoma myeloma patient and expressing BCMA at a high level. To improve the specific cytotoxicity of NK cells to NCI-H929 cells, we transduced a anti-BCMA CAR to the donor iPSC. Following differentiation, the CAR-transduced iNK cells showed approximately 6-fold cytotoxicity compared with WT iNK. (Figure 1) Collectively, our serum-free and strom cell-free NK cell generation produre from human iPSCs provides a scalable, consistent, off-the-shelf and cost-effective NK/CAR-NK cell manufacturing platform for clinical application and NK cell-based immunotherapies.

Unmodified Polystyrene Nanoparticles Induce Inflammatory and Oxidative Stress Responses in Human Lung Epithelial Cells

Polystyrene (PS) is one of the commercially used polymers considered as a pollutant in both aquatic and terrestrial environments. Moreover, PS-NPs are used in nanosensors and nanocarrier for the drug. Thus, it is crucial to determine the potential toxicological effect of PS-NPs to cells. Here we used varying concentrations of unmodified PS-NPs of 60±12 nm in size and analyzed their cyto-toxic effect and stress and toxicity gene expressions in NCI-H292 cells. NCI-H292 cells were ex-posed to minimum of 5 µg/mL and maximum of 50 µg/mL of PS-NPs; 50 µg/mL of PS-NPs showed about 50% cell viability. Moreover, unmodified PS-NPs and induce expressions of stress-related genes, such as those encoding interleukin 6 (IL 6), colony-stimulating factor 2 (CSF 2), C-X-C motif chemokine 10 (CXCL 10) responsible for inflammation and heme oxygenase 1 (HMOX 1) responsible for oxidative stress in NCI-H292 cell lines. Our findings suggest that PS-NPs are considered as a potential toxicological compound to the human cell line.

Deciphering the Role of Fatty Acid-Metabolizing CYP4F11 in Lung Cancer and Its Potential As a Drug Target.

Lung cancer is the leading cause of cancer deaths worldwide. We found that the cytochrome P450 isoform CYP4F11 is significantly overexpressed in patients with lung squamous cell carcinoma. CYP4F11 is a fatty acid ω-hydroxylase and catalyzes the production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE promotes cell proliferation and migration in cancer. Inhibition of 20-HETE-generating cytochrome P450 enzymes has been implicated as novel cancer therapy for more than a decade. However, the exact role of CYP4F11 and its potential as drug target for lung cancer therapy has not been established yet. Thus, we performed a transient knockdown of CYP4F11 in the lung cancer cell line NCI-H460. Knockdown of CYP4F11 significantly inhibits lung cancer cell proliferation and migration while the 20-HETE production is significantly reduced. For biochemical characterization of CYP4F11-inhibitor interactions, we generated recombinant human CYP4F11. Spectroscopic ligand binding assays were conducted to evaluate CYP4F11 binding to the unselective CYP4A/F inhibitor HET0016. HET0016 shows high affinity to recombinant CYP4F11 and inhibits CYP4F11-mediated 20-HETE production in vitro with a nanomolar IC 50 Cross evaluation of HET0016 in NCI-H460 cells shows that lung cancer cell proliferation is significantly reduced together with 20-HETE production. However, HET0016 also displays antiproliferative effects that are not 20-HETE mediated. Future studies aim to establish the role of CYP4F11 in lung cancer and the underlying mechanism and investigate the potential of CYP4F11 as a therapeutic target for lung cancer. SIGNIFICANCE STATEMENT: Lung cancer is a deadly cancer with limited treatment options. Cytochrome P450 4F11 (CYP4F11) is significantly upregulated in lung squamous cell carcinoma. Knockdown of CYP4F11 in a lung cancer cell line significantly attenuates cell proliferation and migration with reduced production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE). Studies with the unselective inhibitor HET0016 show a high inhibitory potency of CYP4F11-mediated 20-HETE production using recombinant enzyme. Overall, our studies demonstrate the potential of targeting CYP4F11 for new transformative lung cancer treatment.

In-silico and in-vitro functional validation of imidazole derivatives as potential sirtuin inhibitor.

Epigenetic enzymes can interact with a wide range of genes that actively participate in the progression or repression of a diseased condition, as they are involved in maintaining cellular homeostasis. Sirtuins are a family of Class III epigenetic modifying enzymes that regulate cellular processes by removing acetyl groups from proteins. They rely on NAD+ as a coenzyme in contrast to classical histone deacetylases (HDACs) (Class I, II, and IV) that depend on Zn+ for their activation, linking their function to cellular energy levels. There are seven mammalian sirtuin isoforms (Sirt1-7), each located in different subcellular compartments. Sirtuins have emerged as a promising target, given that inhibitors of natural and synthetic sources are highly warranted. Imidazole derivatives are often investigated as sirtuin regulators due to their ability to interact with the binding site and modulate their activity. Imidazole bestows many possible substitutions on its ring and neighboring atoms to design and synthesize derivatives with specific target selectivity and improved pharmacokinetic properties, optimizing drug development. Ligand preparation, protein preparation, molecular docking, molecular dynamics, density function theory (DFT) analysis, and absorption, distribution, metabolism, and excretion (ADME) analysis were performed to understand the interacting potential and effective stability of the ligand with the protein. RT-PCR and Western blot analyses were performed to understand the impact of ligands on the gene and protein expression of Class III HDAC enzymes. We evaluated the sirtuin inhibition activity of our in-house compound comprised of imidazole derivatives by docking the molecules with the protein data bank. ADME properties of all the compounds used in the study were evaluated, and it was found that all fall within the favorable range of being a potential drug. The molecule with the highest docking score was analyzed using DFT, and the specific compound was used to treat the non-small cell lung cancer (NSCLC) cell lines A549 and NCI-H460. The gene and protein expression data support the in-silico finding that the compound Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl) acetate has an inhibitory effect on nuclear sirtuins. In conclusion, targeting sirtuins is an emerging strategy to combat carcinogenesis. In this study, we establish that Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl) acetate possesses a strong inhibitory effect on nuclear sirtuins in NSCLC cell lines.

Therapeutic potential of Lianhua Qingke in airway mucus hypersecretion of acute exacerbation of chronic obstructive pulmonary disease

BackgroundLianhua Qingke (LHQK) is an effective traditional Chinese medicine used for treating acute tracheobronchitis. In this study, we evaluated the effectiveness of LHQK in managing airway mucus hypersecretion in the acute exacerbation of chronic obstructive pulmonary disease (AECOPD).MethodsThe AECOPD model was established by subjecting male Wistar rats to 12 weeks of cigarette smoke (CS) exposure (80 cigarettes/day, 5 days/week for 12 weeks) and intratracheal lipopolysaccharide (LPS) exposure (200 μg, on days 1, 14, and 84). The rats were divided into six groups: control (room air exposure), model (CS + LPS exposure), LHQK (LHQK-L, LHQK-M, and LHQK-H), and a positive control group (Ambroxol). H&E staining, and AB-PAS staining were used to evaluate lung tissue pathology, inflammatory responses, and goblet cell hyperplasia. RT-qPCR, immunohistochemistry, immunofluorescence and ELISA were utilized to analyze the transcription, expression and secretion of proteins related to mucus production in vivo and in the human airway epithelial cell line NCI-H292 in vitro. To predict and screen the active ingredients of LHQK, network pharmacology analysis and NF-κB reporter system analysis were employed.ResultsLHQK treatment could ameliorate AECOPD-triggered pulmonary structure damage, inflammatory cell infiltration, and pro-inflammatory cytokine production. AB-PAS and immunofluorescence staining with CCSP and Muc5ac antibodies showed that LHQK reduced goblet cell hyperplasia, probably by inhibiting the transdifferentiation of Club cells into goblet cells. RT-qPCR and immunohistochemistry of Muc5ac and APQ5 showed that LHQK modulated mucus homeostasis by suppressing Muc5ac transcription and hypersecretion in vivo and in vitro, and maintaining the balance between Muc5ac and AQP5 expression. Network pharmacology analysis and NF-κB luciferase reporter system analysis provided insights into the active ingredients of LHQK that may help control airway mucus hypersecretion and regulate inflammation.ConclusionLHQK demonstrated therapeutic effects in AECOPD by reducing inflammation, suppressing goblet cell hyperplasia, preventing Club cell transdifferentiation, reducing Muc5ac hypersecretion, and modulating airway mucus homeostasis. These findings support the clinical use of LHQK as a potential treatment for AECOPD.

Lipid Handling Induces Ferroptosis and Conveys Sensitivity to Anti-BCMA Immunotherapy in Multiple Myeloma

Lipid metabolic changes in cancer are attracting increased attention. BAFF/APRIL axis is a multifaceted immune regulator of adipose tissue function and an emerging target for anti-BCMA therapy in multiple myeloma (MM). However, lipid analyses are scarce in MM onset and response to treatment. First, we measured metabolites and proteins in sera obtained from matched bone marrow and peripheral blood of 17 newly diagnosed and 9 relapsed/refractory MM patients, by LC-MS/MS. String analysis identified crucial pathways including fibrinogen regulation, inflammatory response, structural integrity of lipoproteins and lipid transport, with a significant increase of apoliproteins APO-A1 (key component of HDL particle and cofactor for lecithin-cholesterol acyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters), A2,C2 (which hydrolyzes triglycerides to provide free fatty acids for cells), C3, components of HDL and LDL, confirming a deranged lipid handling in RR-MM patients. Second, we studied the lipid metabolic changes upon anti-BCMA exposure with Elranatamab (EL), an investigational, off-the-shelf, humanized BCMA-CD3-directed bispecific antibody engineered to elicit potent T-cell-mediated anti-MM activity. Combining in vitro assays, FACS, qRT-PCR, immunofluorescence, Western Blot and RNA-seq we evaluated the response to EL treatment in 3 human myeloma cell lines U266, NCI-H929 and OPM2 (3 cell lines chosen for their different basal bioenergetics profile). MM cells were co-incubated with various concentrations of EL in presence of CD3+T cells isolated from healthy donors in 96- well U-bottom plates for 4 h and 24 h at an E:T ratio of 1:5 and 1:10, then stained with anti-CD138-PE antibody . CD138 + cells were detected by flow cytometry and counted as surviving MM cells. BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) and BODIPY 493/503 for Neutral Lipid Droplets Staining for FACS and immunofluorescence were used to quantify lipid peroxidation and cytoplasmatic LDs on surviving CD138 + cells. Despite the BCMA target was equally expressed on U266, NCI-H929 and OPM2, the sensitivity to EL was different, inducing cell cycle arrest in the G2/M phase in U266 and NCI-H929 cell lines but not in OPM2, in E:T ratio independent-manner. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. Oil-Red-O staining revealed an increased lipid content after EL exposure in all tested MM cell lines, with OPM2 the richest in basal lipids content. To dissect the contribution of lipophagy versus lipolysis we performed an autophagy flux analysis by western blot and RNA-seq essay, showing that lipolysis was the emerging response to EL treatment. In EL-sensitive U266 we found an increase of lipid peroxides, hallmark of ferroptosis, a form of cell death due to missed control of membrane lipid peroxidation, a phenomenon negligible in EL-resistant OPM2. Lipidomics demonstrated higher levels of monounsaturated fatty acids (MUFA) than polyunsaturated fatty acids (PUFA) in OPM2, associated to an increased expression of Stearoyl CoA desaturase 1 (SCD1), the enzyme that catalyzes the rate-limiting step in the production of MUFA. While the PUFA oxidation is pro-ferroptotic, MUFA activation promotes a ferroptosis-resistant cell state. Indeed, a ferroptosis-primed resistant phenotype in EL-sensitive U266 was associated to downregulation of lipid peroxides scavengers GPX4 and SLC7A11 and up-regulation of pro-ferroptotic acyl-CoA synthetase long-chain family member 4 (ACSL4). Exogenous MUFA (via supplementation with palmitic acid 125 µM conjugated to BSA 1:6) alleviated ferroptosis induction in U266 making them resistant to EL, a phenotype associated to overexpression of CD36, the main free fatty acids (FFA) transporter. Conversely, in OPM2, inducing iron-dependent lipid peroxidation through RSL3 (3 µM) treatment rescued EL resistance, resulting in a time-dependent increase in cytosolic lipid peroxidation beginning at 6 hours, and overt cell death via ferroptosis at 18 hours. Taken together, our findings suggest that MUFA/PUFA ratio drives ferroptosis and mediates EL sensitivity, addressing lipid handling as a novel means to tailor immunotherapy in RRMM.

Applying HDACis to increase SSTR2 expression and radiolabeled DOTA-TATE uptake: from cells to mice

AimsThe aim of our study was to determine the effect of histone deacetylase (HDAC) inhibitors (HDACis) on somatostatin type-2 receptor (SSTR2) expression and [111In]In-/[177Lu]Lu-DOTA-TATE uptake in vitro and in vivo. Materials and methodsThe human cell lines NCI-H69 (small-cell lung carcinoma) and BON-1 (pancreatic neuroendocrine tumor) were treated with HDACis (i.e. entinostat, mocetinostat (MOC), LMK-235, CI-994 or panobinostat (PAN)), and SSTR2 mRNA expression levels and [111In]In-DOTA-TATE uptake were measured. Furthermore, vehicle- and HDACi-treated NCI-H69 and BON-1 tumor-bearing mice were injected with radiolabeled DOTA-TATE followed by biodistribution studies. Additionally, SSTR2 and HDAC mRNA expression of xenografts, and of NCI-H69, BON-1, NCI-H727 (human pulmonary carcinoid) and GOT1 (human midgut neuroendocrine tumor) cells were determined. Key findingsHDACi treatment resulted in the desired effects in vitro. However, no significant increase in tumoral DOTA-TATE uptake was observed after HDACi treatment in NCI-H69 tumor-bearing animals, whereas tumoral SSTR2 mRNA and/or protein expression levels were significantly upregulated after treatment with MOC, CI-994 and PAN, i.e. a maximum of 2.1- and 1.3-fold, respectively. Analysis of PAN-treated BON-1 xenografts solely demonstrated increased SSTR2 mRNA expression levels. Comparison of HDACs and SSTR2 expression in BON-1 and NCI-H69 xenografts showed a significantly higher expression of 6/11 HDACs in BON-1 xenografts. Of these HDACs, a significant inverse correlation was found between HDAC3 and SSTR2 expression (Pearson r = −0.92) in the studied cell lines. SignificanceTo conclude, tumoral uptake levels of radiolabeled DOTA-TATE were not enhanced after HDACi treatment in vivo, but, depending on the applied inhibitor, increased SSTR2 expression levels were observed.