Non-small Cell Lung Cancer Cells Research Articles

Abstract 3279: MUC1 is necessary for the inflammatory memory response to osimertinib resistance in NSCLC cells

Abstract Emergence of resistance is an invariable outcome in cancer progression that may involve memory of cancer cells to treatment. The oncogenic MUC1-C protein confers pleotropic resistance of cancer cells to diverse cytotoxic and targeted agents by unclear mechanisms. We demonstrate that MUC1-C regulates the interferon (IFN) type I/II pathways in H1975 non-small cell lung cancer (NSCLC) cells selected for osimertinib resistance (H1975-OR). Targeting MUC1-C in H1975-OR cells suppressed STAT1 and inflammatory IFN signaling in association with reversing the osimertinib-resistant phenotype. Studies of H1975-OR cells selected for growth in the absence of osimertinib demonstrate that revertant H1975-RT cells retain MUC1-C-dependent memory necessary for recalling osimertinib resistance. Mechanistically, osimertinib treatment of H1975-RT cells activates the MUC1 gene at a (i) promoter-like signature (PLS) by MUC1-C and STAT1, and (ii) proximal enhancer-like signature (pELS) by PBAF and JUN/AP-1. Targeting MUC1-C in osimertinib-treated H1975-RT and MGH170 NSCLC MET-amplified cells isolated from a patient refractory to osimertinib treatment suppressed induction of STAT1 and downstream IFN-stimulated genes (ISGs) encoding MX1, OAS1 and ISG15. Targeting MUC1-C, STAT1, PBRM1/PBAF and JUN/AP-1 further confirmed their dependencies for reemergence of osimertinib resistance in H1975-RT and MGH170 cells. In support of these results, high MUC1-C protein expression analyzed by IHC is associated with shorter progression free survival for patients treated with osimertinib. These findings and the demonstration that MUC1-C is upregulated in NSCLC cells from patients refractory to osimertinib indicate that MUC1-C drives osimertinib resistance in NSCLC cells by promoting an inflammatory memory response. Citation Format: Naoki Haratake, Atrayee Bhattacharya, Ayako Nakashoji, Hiroki Ozawa, Mototsugu Shimokawa, Donald Kufe. MUC1 is necessary for the inflammatory memory response to osimertinib resistance in NSCLC cells [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 3279.

Abstract 6266: Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells

Abstract The neurotensin receptor 1 (NTSR1) is a 418 amino acid type A G protein-coupled receptor which causes phosphatidylinositol turnover and proliferation of non-small cell lung cancer (NSCLC) cells (Moody et al., Peptides 2021; 137: 710480). Adding the agonist NTS to NSCLC cells increases transactivation of the receptor tyrosine kinases (RTK) EGFR, HER2 and HER3 (Moody et al., Biology 2023 12: 957). The RTK tyrosine phosphorylation and growth caused by NTS is impaired by SR48692, a small molecule NTSR1 antagonist. The effects of NTS were investigated on HER4 using NSCLC cells. By Western blot, cell lines NCI-H522 and H661 had high levels of NTSR1, HER 4 and neuregulin 1 (NRG1), a ligand for HER4 but not NTSR2, HER3 or NRG2. By RT-PCT, high levels of HER4 and its isoforms JM-a, CYT1 and CYT2 were present in NCI-H522 and H661 cells. Adding NTS (0.1 μM) or NRG1 (0.01 μg/ml) to NCI-H522 or H661 cells increased P-Tyr1284-HER4 and P-ERK 3-fold. Using immunoprecipitation techniques, adding NTS to NSCLC cells increased formation of HER4-HER2 heterodimers which leads to cancer proliferation (Lucas et al., Pharmacol Rev 2022; 74:18). The increase in PY1284-HER4 caused by NTS was impaired by SR48692, HER4 siRNA, NRG1 siRNA or ibrutinib (TKI). The NTSR1 regulation of HER4 transactivation is impaired by GM6001 (MMP inhibitor]), PP2 (Src inhibitor) or N-acetylcysteine (antioxidant). The clonal growth of NSCLC cells is increased by NTS or NRG1 but decreased by SR48692 or ibrutinib. The results indicate that NTS increases the formation of HER2/HER4 heterodimers leading to the proliferation of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramos-Alvarez, Robert T. Jensen. Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells [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 6266.

Abstract 4687: Primary study on the anticancer role of BAM15 in lung cancer cells

Abstract Background: Lung cancer remains the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancer cases. BAM15, a mitochondrial uncoupler, is a potential therapeutic agent for many diseases, including obesity, diabetes, nonalcoholic fatty liver disease, Sepsis and cancers. However, there is no report on the anti-tumor role of BAM15 in NSCLC. Methods: To investigate the direct anti-cancer effects of BAM15 in NSCLC, we examined its inhibitory role on cell proliferation of NSCLC cells using the Cell Counting Kit-8(CCK8), colony forming assays and EdU staining assays. The impact of BAM15 on the cell cycle and cell apoptosis of NSCLC cells was analyzed by flow cytometric. A xenotransplanted NSCLC model in mice were used to test the anticancer role of BAM15. Result: Using the CCK8 assay, we found that the inhibitory role of BAM15 on the proliferation and colony formation of NSCLC cells is dose-dependent. The half maximal inhibitory concentration (IC50) values for cell proliferation were 2.71, 4.74, and 9.50 μM for A549, PC9, and H1299 NSCLC cells, respectively. The EdU staining assay demonstrated that BAM15 treatment significantly inhibited DNA synthesis in NSCLC cells. Flow cytometric analysis revealed that BAM15 arrested cell cycle at G2 phase in NSCLC cells and caused a striking increase in the percentage of apoptotic cells in NSCLC cells. The xenotransplanted NSCLC experiment indicated that BAM15 could significantly suppressed the growth of NSCLC tumors compared with the control tumors. Conclusions: Our study demonstrates that BAM15 significantly inhibits cell proliferation and promotes apoptosis in NSCLC cells, indicating that BAM15 is a potential therapeutic candidate for the treatment of NSCLC. Citation Format: Mei-yin Zhang, Yue-Ning Wang, Yu-Feng Zhou, Shuo-Cheng Wang, Shi-Juan Mai, Hui-Yun Wang. Primary study on the anticancer role of BAM15 in lung cancer cells [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 4687.

Abstract 1976: Targeting metabolism in non-small cell lung cancer (NSCLC) with the novel multitarget small molecule PI3K-CDK4/6-CDK9 inhibitor LCI139

Abstract Introduction: NSCLC is a heterogenous disease estimated to cause the majority of all 2023 United States cancer-related deaths. There is no broadly applicable targeted therapy to treat lung cancer and those FDA-approved drugs target patient-specific biomarker mutations such as EGFR, ALK, ROS1, etc. Targeting multiple oncogenic pathways to induce synthetic lethality is an attractive strategy for anticancer therapeutic development. Here, we describe the effects of novel multitarget small molecule PI3K-CDK4/6-CDK9 inhibitor LCI139 on a panel of human NSCLC cells of mixed histologic and mutational backgrounds. Methods: LCI139 specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and KINOMEscan. LCI139 (0.001-10μM) was screened against NCI-H1975, NCI-H1703, NCI-H1373, NCI-H1781 and NCI-H226 human cell lines; IC50 values determined by CellTiter-Glo/WST-8 Assay and apoptotic rate by flow cytometry (Annexin V/7-AAD). Metabolomic profile of representative LCI139 sensitive (LCI139S) and resistant (LCI139R) NSCLC cells was evaluated by western blot. Results: LCI139 is a highly potent inhibitor of PI3K-CDK4/6-CDK9: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). NCI-H1703, NCI-H226, NCI-H1373 and NCI-H1975 were highly sensitive to LCI139 (0.68, 3.2, 35.27 and 342.4nM IC50), while NCI-H1781 remained insensitive up to 10μM. Confirming those findings, LCI139 promoted robust apoptotic cell death in NCI-H1703, NCI-H226, NCI-H1373 and NCI-H1975 but not NCI-H1781. LCI139S H1703 cells displayed increased bioenergetic reliance on oxidative phosphorylation (OXPHOS), compared with the LCI139R H1781 cells. In the LCI139S H1703 cells, LCI139 treatment decreased expression of all mitochondrial complexes (I-IV) and glycolytic enzymes (HK2, GAPDH, LDHA). Conversely, LCI139 treatment of LCI139R H1781 cells only inhibited expression of mitochondrial complexes II and III, but increased glucose uptake (Glut1) and retained expression of other glycolytic enzymes. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor LCI139 is nanomolar potent against several NSCLC cells lines of mixed histologic and mutational backgrounds. NSCLC cells that are most reliant on OXPHOS to generate ATP, a classic metabolic adaptation to chemotherapy, appear to be most sensitive to LCI139. Taken together, these data support further drug characterization and mechanistic dissection to assess LCI139’s potential clinical utility in treating advanced stage and recurrent NSCLC. Citation Format: Cody C. McHale, Anna V. Ivanina Foureau, Hailey L. Dryden, Krishnaiah Maddeboina, Dhananjaya Pal, Bharath Yada, Page Mangum Arditti, Fei Guo, Kathryn F. Mileham, David M. Foureau, Donald L. Durden. Targeting metabolism in non-small cell lung cancer (NSCLC) with the novel multitarget small molecule PI3K-CDK4/6-CDK9 inhibitor LCI139 [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 1976.

Abstract 1797: Investigating the significance of OXPHOS activity in non-small cell lung cancer brain metastases

Abstract Background: Brain metastasis is a common and devastating occurrence for patients with non-small cell lung cancer (NSCLC). NSCLC patients with brain metastases have a poor median overall survival of 2-3 months, highlighting the urgency for novel treatment strategies. One approach to understanding and potentially targeting metastasis is via cancer metabolism. During the metastatic cascade, cancer cells must survive and adapt to the differential nutrient environments they encounter, including the brain. The blood brain barrier limits the availability of many metabolites, leading metastatic cells to alter their nutrient use to survive. RNA sequencing analyses of NSCLC brain metastases suggest that increased oxidative phosphorylation (OXPHOS) may be one such metabolic adaptation in the brain. The aim of this study was to determine the necessity of OXPHOS activity as a metabolic adaptation for NSCLC cells to proliferate in the unique metabolic environment of the brain. Materials and Methods: NSCLC cells were cultured in media mimicking the metabolic environments of the brain and blood. Expression of mitochondrial markers were evaluated by immunoblot in cells cultured across media conditions. The effects of the OXPHOS inhibitor IACS-010759 were assessed by cell growth as well as stable isotope tracing with [U-13C] glucose. Patient derived xenografts (PDXs) were derived from a cohort of patients with NSCLC who underwent stable isotope tracing with [U-13C] glucose. Mice bearing subcutaneously implanted PDXs were treated daily with 5mg/kg IACS-010759 and metastatic burden was measured by flow cytometry and histology. Results: Tracing studies in patients revealed that high enrichment in OXPHOS-related metabolites in the primary tumor correlated with worse patient outcomes. Compared with other media conditions, cells cultured in media replicating the metabolic environment of the brain have increased expression of mitochondrial proteins, increased 13C glucose labeling in the TCA cycle, and a larger reduction in proliferation with IACS-010759 treatment. Using our PDX models, inhibition of OXPHOS with IACS-010759 had little effect on subcutaneous tumor growth but reduced metastasis in the lung and the brain. Conclusions: OXPHOS activity may be enhanced in NSCLC brain metastases due to the unique metabolic environment of the brain. This elevated OXPHOS activity may be necessary for NSCLC brain metastasis growth, as OXPHOS inhibition reduced metastasis in the brain. NSCLC cells may undergo mitochondrial biogenesis to support survival in this unique metabolic environment, and this may represent an area of therapeutic vulnerability for brain metastases. Citation Format: Nia G. Hammond, Massar Alsamraae, Mayher Kaur, Rachel Zuckerman, Brianna Chang, Robert Cameron, Aaron Dyas, Brandon Faubert. Investigating the significance of OXPHOS activity in non-small cell lung cancer brain metastases [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 1797.

Abstract 3290: FTO mediates tumor growth and resistance to erlotinib in NSCLC

Abstract Lung cancer, among the most common cancer types, ranks first in cancer associated deaths. Nearly 85% of the lung cancer cases are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR), an established marker for NSCLC, is frequently overexpressed on the cell surface. Amplifying mutations often render these receptors activated in a ligand independent manner, therefore, therapies such as erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), target these activating mutations and inhibit tumor growth. Nevertheless, patients acquire resistance to EGFR TKI within 9-14 months of treatment. Recent studies show that fat mass and obesity- associated (FTO) gene, initially identified to increase the body mass index, plays a major role in cancer development and acquired therapy resistance in several cancer types. FTO demethylates m6A mRNA and modulates tumorigenesis, and drug resistance. In this study, we explored the functions of FTO in EGFR TKI resistance in NSCLC. To study the expression of FTO in erlotinib resistant cells, we used wildtype-EGFR NSCLC cell lines (H2170, H358) and mutant EGFR NSCLC cell lines (H1975, PC9) that were rendered resistant to erlotinib. Our RT-qPCR results demonstrated that FTO was upregulated by 1.7-3.0 fold in erlotinib resistant H2170, H358, PC9 and H1975 cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting, and by 1.7 to 2.6 fold in the nucleus of erlotinib resistant H1975, H2170 and PC9 cell lines by immunofluorescence. Furthermore, we investigated FTO inhibition with Dac51 (FTOi) on cell viability and cell migration properties. MTT cell viability assay showed a 9-17% decrease in cell viability when treated with a combination of Dac51 and erlotinib compared to Dac51 alone in erlotinib resistant H2170 and PC9 cells, respectively. The combinatory treatment showed an additive effect compared to the Dac51 and erlotinib treatments alone. Wound healing assay revealed an 18% open wound area after 48-hour treatment with both Dac51 and erlotinib compared to 0.1% after treatment with erlotinib alone in erlotinib resistant H2170 cells. To evaluate the effect of siRNA mediated FTO (siFTO) knockdown on cell viability and energy metabolism, MTT and luciferase assays were performed. MTT assay showed a 21-28% decrease in viability in erlotinib resistant H1975, H2170, PC9 cells, demonstrating increase in the erlotinib efficacy, as compared to the mock siRNA treatment. Luciferase assay showed an inverse relationship between the FTO and ATP, where the latter was upregulated by 1.3 to 3.4 folds in erlotinib resistant H1975, H2170 and PC9 cells, when treated with siFTO, suggesting its involvement in the energy requirements of the NSCLC cells. In conclusion, our study suggests FTO was upregulated in erlotinib resistant NSCLC cells on both the gene and protein levels. FTO could mediate resistance to EGFR TKI possibly through cell proliferation and migration and help with prognosis of NSCLC patients. Citation Format: Aayush Rastogi, Rong Qiu, Neelu Puri. FTO mediates tumor growth and resistance to erlotinib in 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 3290.

Abstract 1726: Targeted epigenomic control of MYC as a strategy to treat EGFR inhibitor-resistant NSCLC

Abstract EGFR tyrosine kinase inhibitors (EGFRi) have improved outcomes for non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. However, patients receiving EGFRi therapy frequently relapse with an EGFR T790M mutation. While osimertinib, a third generation EGFRi, blocks EGFR T790M activity, resistance eventually develops through various EGFR-dependent and EGFR-independent mechanisms, such as acquiring additional mutations in EGFR (e.g. C797S), activating bypass signaling pathways, or undergoing an epithelial to mesenchymal transition (EMT). MYC is a master transcription factor critical for mediating oncogenic signal transduction and has been implicated in EGFRi resistance, suggesting that MYC targeting may overcome multiple EGFRi resistance mechanisms. To evaluate this, we have developed a NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC epigenomic controller (NSCLC MYC-EC), designed to selectively target regulatory elements in MYC’s insulated genomic domain and downregulate MYC expression. We have previously shown that NSCLC MYC-EC effectively decreases MYC expression pre-transcriptionally and have demonstrated that MYC-EC combination with osimertinib synergistically reduces viability of EGFR mutant NSCLC cells. Here, we demonstrate NSCLC MYC-EC activity in models that have developed EGFRi resistance through EGFR-dependent and -independent mechanisms. To test the effect of NSCLC MYC-EC in NSCLC cells with T790M mutant EGFR, we treated H1975 (L858R, T790M) and PC9-T790M cells (del19, engineered T790M) with MYC-EC alone or in combination with osimertinib. MYC-EC combination with osimertinib enhanced MYC protein downregulation and synergistically reduced cell viability in both models. To evaluate the effect of MYC-EC in osimertinib-resistant cells, we engineered H1975 and PC9-T790M cells with an EGFR C797S mutation. While osimertinib treatment did not impact cell viability in these models, MYC-EC maintained activity, downregulating MYC levels and reducing cell viability. To investigate MYC-EC activity in cells with alternative resistance mechanisms, we generated osimertinib-resistant H1975 cells by stepwise dose escalation. Protein analysis showed highly reduced phospho-EGFR levels, suggesting an EGFR-independent mechanism of survival. RNA-sequencing analysis revealed that EMT pathway was highly activated in these resistant cells. Importantly, this model retained sensitivity to NSCLC MYC-EC treatment, demonstrating that selectively targeting MYC can treat osimertinib-resistant NSCLC cells with a mesenchymal phenotype. Together, these data demonstrate that pre-transcriptional downregulation of MYC through NSCLC MYC-EC treatment effectively inhibits viability of EGFRi-resistant NSCLC through both EGFR-dependent and -independent mechanisms and supports the development of NSCLC MYC-EC in EGFRi-resistant NSCLC. Citation Format: Eugine Lee, Padraich Flahardy, Cameron Vergato, Stephen Siecinski, Justin Chen, Charles O’Donnell, Graeme Hodgson, Defne Yarar, Thomas McCauley. Targeted epigenomic control of MYC as a strategy to treat EGFR inhibitor-resistant 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 1726.

Abstract 5868: Upregulation of HER3 and MET in non-small cell lung cancer cells resistant to EGFR tyrosine kinase inhibitor

Abstract Introduction: Prior research demonstrates the importance of studying epidermal growth factor receptor (EGFR) activating mutations for better understanding the biology of non-small cell lung cancer (NSCLC), which comprises the majority (~80%) of lung cancers. EGFR-targeted tyrosine kinase inhibitors (TKIs) have been used in the clinic to treat NSCLC with EGFR activating mutations. First and second-generation EGFR-TKIs are effective in NSCLC patients with the most common EGFR mutations (exon 19 deletion (Del19) and exon 21-point mutation (L858R)). A third generation EGFR-TKI, osimertinib (AZD9291), has been developed to target the T790M resistance mutation, but patients are starting to show resistance to this line of therapy as well. Our study aims to elucidate the underlying mechanism(s) of EGFR-TKI resistance in NSCLC cells. Methods: NSCLC cell lines, including PC-9 and HCC827, which have the Del19 EGFR activating mutation, were utilized for experiments. From the parental cell lines, several cell lines resistant to the EGFR-TKIs were previously established. Cell proliferation (MTS) assays were used to confirm the resistance phenotype of the established cell lines. Western blot assays and RT-qPCR were utilized to determine changes in the expression level of several membrane receptors and their downstream effectors. Results: Our preliminary analysis showed that both human epidermal growth factor 3 (HER3) and mesenchymal-epithelial transition factor (MET) were upregulated in the EGFR-TKI-resistant cell lines as compared to their parental counterparts. Further studies revealed an increased level of phosphorylated STAT3 (p-STAT3) and ERK1/2 (p-ERK1/2) in the resistant cell lines when compared to the parental cell lines, suggesting the possible involvement of HER3 and/or MET in the activation of downstream signaling pathways. Upregulation of HER3 and MET was found at both the protein and mRNA level. Conclusion: Based on our initial findings, membrane receptors, including HER3 and MET, could be working together to continue activating downstream signaling pathways to evade the effects of osimertinib, resulting in resistance. Additional work is underway to define the role of HER3 and MET in the NSCLC cells resistant to EGFR-TKIs. Citation Format: Margaret Larsen, Hui Lyu, CongCong Tan, Yakun Wu, Bolin Liu. Upregulation of HER3 and MET in non-small cell lung cancer cells resistant to EGFR tyrosine kinase inhibitor [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 5868.

Abstract 7581: Nicotine promotes the proliferation and metastasis of non-small cell lung cancer through c-Myc/EZH2 pathway

Abstract Objective: Lung cancer, particularly non-small cell lung cancer (NSCLC), continues to be a predominant contributor to global cancer-related mortality, with smoking emerging as a significant risk factor. Nicotine, which is one of the main chemicals from the tobacco smoke, is identified as carcinogen for lung cancer, especially for squamous carcinoma (LUSC). This study aims to investigates the intricate molecular mechanisms by which nicotine facilitates proliferation and migration in NSCLC through the c-Myc/EZH2 signaling pathway. Methods: Clinical tissue specimens were procured from surgical patients with lung cancer for immunohistochemical staining. Additionally, public databases were scrutinized to compare EZH2 expression between smokers and non-smokers. NSCLC cell lines underwent treatment with nicotine in a time- and concentration-dependent manner, and the impact on EZH2 was corroborated through Western Blot, Real-time PCR, and immunofluorescence staining. Proliferation and migration assays, encompassing CCK8 experiments, colony formation assays, EdU staining, and Transwell assays, were conducted to discern the effects of nicotine on NSCLC. Experimental techniques such as si-RNA and CHIP were deployed to investigate changes in c-Myc-mediated regulation of EZH2, elucidating the intricate role of the c-Myc/EZH2 pathway in nicotine-induced NSCLC proliferation and migration. Results: Smokers exhibited heightened EZH2 expression in tumor tissue compared to non-smokers. Nicotine treatment induced a concentration- and time-dependent upregulation of EZH2 in NSCLC cell lines. In 10 μM nicotine-treated NSCLC cells, concurrent treatment with 10 μM DZNep (EZH2 inhibitor) or si-EZH2 significantly augmented proliferation and migration. Moreover, the upregulation of EZH2 correlated with increased c-Myc expression, and siRNA-mediated c-Myc inhibition resulted in a corresponding reduction in EZH2 expression. Nicotine was identified as a promoter of NSCLC proliferation and migration through the c-Myc/EZH2 signaling pathway. This study establishes a robust association between nicotine and EZH2 in NSCLC cells, unveiling the novel regulatory axis of c-Myc/EZH2. Combined treatment with c-Myc and EZH2 inhibitors demonstrated efficacy in suppressing nicotine-induced promotion of NSCLC proliferation and migration. Conclusion: In summary, this study elucidates the mechanisms through which nicotine propels NSCLC development via the c-Myc/EZH2 pathway, providing a theoretical underpinning for future NSCLC treatment paradigms. The combined use of c-Myc and EZH2 inhibitors emerges as a promising therapeutic strategy, poised to inhibit nicotine-induced NSCLC proliferation and migration, offering enhanced treatment modalities for patients. Citation Format: Chen Ding, Hua Huang, Chen Chen, Yongwen Li, Hongyu Liu, Jun Chen. Nicotine promotes the proliferation and metastasis of non-small cell lung cancer through c-Myc/EZH2 pathway [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 7581.

Abstract 1958: EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs

Abstract RET fusions are an oncogenic driver mutation occurring in 1-2% non-small cell lung cancer (NSCLC). Currently, RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are approved for this patient population, demonstrating favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC. However, tumors eventually become refractory to selective RET inhibitor monotherapy. We sought to identify signaling pathways which mediate RET-independent acquired resistance to RET inhibitors and effective therapeutic combinations to overcome resistance. Using LC-2/ad (CCDC6-RET) NSCLC cells, we assessed the expression and activation of critical signaling pathways at a proteomic level following RET knockdown using reverse-phase protein array. RET knockdown resulted in rapidly increased adaptive upregulation of EGFR, HER2, MET, mTOR and MEK pathway signaling; observations were confirmed by Western Blotting. To determine whether EGFR or MET signaling facilitate RET inhibitor resistance, LC-2/ad and NCCE-TH1101 (KIF5B-RET) NSCLC cells were treated with increasing concentrations of selpercatinib or pralsetinib with or without ligands for these receptors, EGF or HGF respectively, and after five days cell viability was measured by CellTiter Glo. EGFR and MET signaling promoted RET TKI resistance in both cell lines. Next, to evaluate whether blockade of EGFR or MET signaling could enhance the activity of RET inhibitors, we treated LC-2/ad and NCCE-TH1101 cells with RET TKIs alone or in combination with the EGFR TKI erlotinib or poziotinib, or the MET inhibitor SU11274. We observed an additive effect with EGFR but not MET inhibitors when combined with RET TKIs. Western blot analysis revealed that EGFR signaling could reactivate mTOR and MAPK signaling after RET TKI treatment. Therefore, we next tested whether inhibition of these downstream pathways might enhance RET TKI sensitivity. We observed that treatment of LC-2/ad and NCCE-TH1101 cells with an mTOR inhibitor (everolimus) or a MEK inhibitor (trametinib) in combination with RET TKIs increased the anti-tumor activity or RET inhibitors. Finally, we established cells with acquired resistance to selpercatinib and pralsetinib. RET TKI resistant cells did not harbor secondary RET fusion mutations but underwent epithelial to mesenchymal transition and exhibited pan-RET inhibitor resistance. Moreover, RET TKI resistant cells showed increased cell surface expression of EGFR and MET by flow cytometry. RET TKI resistant cells were sensitive to mTOR or MEK inhibitors in combination with RET TKIs. Collectively, our findings indicate that in RET-fusion positive NSCLC tumor cells, activation of bypass pathways including the EGFR, MAPK, mTOR pathways may facilitate RET inhibitor resistance and that blockade of bypass pathways may enhance the efficacy of selective RET inhibitors and overcome acquired RET TKI resistance. Citation Format: Tomohiro Takehara, Monique B. Nilsson, Ximeng Liu, Hibiki Udagawa, Naoto Morikawa, Alissa Poteete, Junqin He, Xiaoxing Yu, Thiru Arumugam, Koichi Goto, John V. Heymach. EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs [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 1958.

Abstract 383: Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment

Abstract Background: Mitophagy is a pivotal cellular process crucial for maintaining homeostasis in hypoxic conditions, selectively eliminating dysfunctional mitochondria through autophagy. This process is implicated in various diseases, including cancer. Non-Small Cell Lung Cancer (NSCLC), known for its prevalence and aggressiveness, often encounters hypoxic microenvironments. Mic19, a core protein of the mitochondrial cristae membrane, plays vital roles in maintaining mitochondrial architecture and function. However, its involvement in mitophagy in hypoxic NSCLC and the underlying molecular mechanisms remain inadequately explored. Methods: NSCLC cells were cultured under conditions with 1% O2 oxygen concentration to simulate a hypoxic microenvironment. Protein expression levels of Mic19 were analyzed using Western Blot and immunofluorescence (IF). Cellular proliferation and metastasis capability were assessed through CCK8 and transwell experiments. Immunoprecipitation experiments were conducted to validate the interaction between Mic19 and key mitophagy regulatory proteins. IF, confocal microscopy, ATP measurement, oxygen consumption rate (OCR), mitochondrial membrane potential, and transmission electron microscopy were employed to assess the formation of mitochondrial cristae and the level of mitophagy. Results: In hypoxic conditions, Mic19 exhibited up-regulation in NSCLC cells. Subsequent in vitro experiments confirmed Mic19's pivotal role in promoting the growth and metastasis of lung cancer cell lines. Through clinical data analysis, Mic19 emerged as an independent prognostic indicator for NSCLC. Inhibition of Mic19 expression exacerbated the disruption of mitochondrial morphology and structure in lung cancer cells, accompanied by the loss of mitochondrial cristae. Notably, Mic19 expression closely correlated with ATP and OCR production in tumor cells. Assessment of mitochondrial membrane potential and transmission electron microscopy revealed Mic19 as a critical downstream target of hypoxia, mediating mitophagy. Overall, these findings not only underscore the significance of Mic19 in regulating mitochondrial dynamics but also highlight its multifaceted role in tumor progression, positioning Mic19 as a promising therapeutic target and prognostic biomarker for NSCLC. Conclusion: This study provides novel insights into the mechanistic understanding of hypoxia-induced mitophagy in NSCLC, emphasizing the regulatory role of Mic19. Mic19 emerges as a potential therapeutic target for future interventions in NSCLC patients. Citation Format: Hua Huang, Chen Ding, Zhanrui Zhang, Di Wu, Chen Chen, Yongwen Li, Hongyu Liu, Jun Chen. Mic19 regulates mitophagy in non-small cell lung cancer under hypoxic microenvironment [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 383.

Abstract 4653: Unraveling the vulnerabilities of targeted therapy-tolerant persister cells in NSCLC

Abstract Genetic profiling and the development of targeted therapy has improved survival of many cancer patients, but resistance to targeted therapy remains a major barrier to long-term patient survival. To develop more pro-active strategies to improve clinical outcomes, our research focuses on drug-tolerant persister cells that constitute a reservoir of cancer cells that survive and adapt during the initial stage of therapy. These cells typically display therapy resistance in a reversible manner and set the stage for the subsequent emergence of cells with full acquired resistance. We utilized quantitative proteomics to characterize persister cells generated from EGFRmut, KRASmut, and ALKmut non-small cell lung cancer (NSCLC) cell lines after treatment with osimertinib, sotorasib, and lorlatinib, respectively. Integrating proteomics data with network analysis revealed a decrease in the signaling network associated with cell cycle regulation and DNA replication. Meanwhile, persister cells exhibited an enrichment in proteins linked to lysosomes, amino acids, and fatty acid metabolism. The data revealed a consistent metabolic shift in persister cells, with an increased dependence on oxidative phosphorylation (OXPHOS) for ATP generation. Small-scale drug screening was performed based on findings from proteomics and confirmed that persister cells are vulnerable to inhibitors of OXPHOS mitochondrial complexes. Combination treatment of OXPHOS inhibitors and each targeted therapy delayed the emergence of resistance. One complex V inhibitor, Gboxin, selectively inhibited the growth of persister cells. The therapeutic potential of complex V was further validated by CRISPR-based genetic knockdown studies. The metabolic transition in drug tolerant persister cells may serve as a critical adaptive resistance mechanism to provide a survival advantage under targeted therapy-induced stress. Understanding this metabolic shift may lead to new treatments that aim to prevent or delay the onset of acquired resistance in NSCLC. Citation Format: Donghwa Kim, Dmitry Kuchenov, Aubhishek Zaman, Neal Bennett, Emilio Ramos, Ken Nakamura, Arun P. Wiita, Sourav Bandyopadhyay, Trever Bivona. Unraveling the vulnerabilities of targeted therapy-tolerant persister cells in 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 4653.

Abstract 4005: HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC

Abstract Approximately 3% of non-small cell lung cancer (NSCLC) cases harbor ERBB2/HER2 mutations. Notably, the majority of actionable HER2 mutations in NSCLC are in-frame insertions in exon 20. Current HER2-targeting approaches, including antibody-drug conjugates (ADCs) such as trastuzumab deruxtecan, have shown promising response rates. To explore novel strategies for targeting HER2-positive NSCLC, we investigated chimeric antigen receptor (CAR)-based therapies to enhance the anti-tumor activity of immune effector cells. We engineered a series of HER2 CAR constructs utilizing scFvs derived from trastuzumab, pertuzumab, and FRP5, recognizing distinct domains of HER2. These scFvs were integrated into second-generation BB3z (with 4-1BB and CD3z intracellular domains) and third-generation 28BB3z (with CD28, 4-1BB, and CD3z intracellular domains) CAR backbones, which were subsequently transduced into activated T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. High transduction efficiencies were achieved, with over 80% in CAR-T cells and over 50% in CAR-NK cells. We next evaluated their activity against a panel of NSCLC cell lines expressing varying levels of HER2 in vitro using firefly luciferase assays. All HER2 CAR-T cells demonstrated more than 90% killing against H2170 (HER2 high) and H322 (HER2 medium) at an effector:target (E:T) ratio of 10:1, whereas HER2 CAR-T cells yielded a killing effect of 35% against A549 (HER2 low) cells. HER2-targeting CAR-NK cells also displayed potent cytotoxicity against HER2-expressing NSCLC cells. Moreover, we observed that treatment of HER2 mutant and HER2 amplified NSCLC cells with low doses of HER2 tyrosine kinase inhibitors (TKIs) such as poziotinib enhanced the expression of HER2 on the cell surface. This upregulation resulted in enhanced activation of HER2 CAR-T and CAR-NK cells in coculture experiments as measured by CD107a expression on effector cells. Additionally, treatment with the HER2 TKI poziotinib potentiated the killing activity mediated by HER2 CAR-T and CAR-NK cells. Using a HER2 YVMA duplication patient-derived xenograft (PDX) NSCLC model, we observed that the in vivo combination of poziotinib with HER2 CAR-NK cells exhibited superior anti-tumor activity as compared to poziotinib treatment alone. In conclusion, HER2 CAR-T and CAR-NK cells effectively target HER2-mutant and HER2-positive NSCLC, and their combination with HER2 TKIs enhances vulnerability to HER2-targeting strategies. Citation Format: Yan Yang, Monique B. Nilsson, Alissa Poteete, Junqin He, Qian Huang, John V. Heymach. HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against 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 4005.

Abstract 6972: Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer

Abstract High expression of programmed death-ligand 1(PD-L1) is associated with poor survival outcomes in epidermal growth factor receptor (EGFR) mutation non-small cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study we aimed to determine how increased PD-L1 affects the signal transduction of EGFR-mutant NSCLC cells. We first established PD-L1 overexpression EGFR mutant NSCLC cells, and discovered that PD-L1 overexpression cells had upregulated c-MET phosphorylation compared with parental cells using receptor tyrosine kinase array study. Subsequently, we demonstrated that knocking out PD-L1 (CD 274) in EGFR mutant NSCLC cells resulted in down-regulation of c-MET phosphorylation. Since protein tyrosine phosphatases (PTP) are known key regulators for c-MET phosphorylation and dephosphorylation, the activity of PTP was assessed. In PD-L1 overexpression EGFR mutant NSCLC cells, the PTP activity was decreased, whereas in PD-L1 knock-out cells, the PTP activity was increased. Additionally, the downregulation of c-MET phosphorylation in PD-L1 knock-out cells could be restored by adding PTP inhibitor (Na3VO4). The gene expression analysis showed that PTP expression was down-regulated in PD-L1 overexpression cells, indicating that PD-L1 upregulates c-MET phosphorylation by reducing PTP expression. We further established xenograft animal models using NSCLC cells with strong baseline c-MET phosphorylation to determine the effect of c-MET phosphorylation on osimertinib (a 3rd-generation EGFR-TKI) treatment. We found that tumor cells with strong c-MET phosphorylation were prone to develop MET amplification as acquired resistance under osimertinib, which could be overcome with combination of osimertinib and tepotinib, a c-MET inhibitor. Taken together, our results expand our understanding of PD-L1’s role beyond an immune checkpoint in EGFR mutant NSCLC. PD-L1-regulated MET alterations could be a crucial mechanism leading to poor treatment outcome of EGFR-TKIs in PD-L1 overexpression EGFR mutant NSCLC. Citation Format: Derek De-Rui Huang, Chia-Chi Hsu, Wei-Hsun Hsu, Bin-Chi Liao, James Chih-Hsin Yang. Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer [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 6972.

MicroRNA‑373‑3p inhibits the proliferation and invasion of non‑small‑cell lung cancer cells by targeting the GAB2/PI3K/AKT pathway.

MicroRNAs (miRNAs) were previously demonstrated to be involved in the pathogenesis of non-small-cell lung cancer (NSCLC); however, the roles of certain miRNAs in NSCLC remain to be elucidated. The present study aimed to investigate the functions of screened miRNAs in NSCLC and the potential mechanisms. First, expression profiles of miRNAs were downloaded from the Gene Expression Omnibus (dataset no. GSE29248) and the differentially expressed miRNAs were analyzed by bioinformatics methods. Reverse transcription-quantitative PCR was used to validate the differential expression of miR-373 in clinical samples. The association between miR-373 expression levels and clinicopathological characteristics was also investigated. To further examine how miR-373 mediates the emergence of NSCLC, western blot, Cell Counting Kit-8, cell invasion and wound-healing assays, as well as apoptosis detection and a luciferase assay were used. The results indicated significant downregulation of miR-373 in NSCLC tissues and its low expression was closely associated with the degree of differentiation, clinical stage and tumor size, and was indicative of an unfavorable prognosis for patients with NSCLC. A functional study indicated that overexpression of miR-373 inhibited the proliferation, promoted apoptosis, and suppressed invasion and migration of NSCLC cells. Bioinformatics prediction and functional assays suggested that Grb-associated binding protein 2 (GAB2) was a direct target of miR-373. In addition, GAB2 was found to be significantly upregulated in NSCLC tissues, and clinically, miR-373 was negatively associated with GAB2. Furthermore, overexpression of GAB2 blocked the tumor suppressive effects of miR-373 on NSCLC cells. Mechanistically, miR-373 mimics were able to reduce the expression of GAB2 and subsequently decrease the phosphorylation level of AKT and mTOR protein. The present results indicate that miR-373 exerts its anti-tumor effects in NSCLC cells by targeting the GAB2/PI3K/AKT pathway, suggesting that miR-373 may be a potential therapeutic target in NSCLC.

Abstract 7194: Targeting AXL and MCL-1 to eradicate lazertinib tolerance in EGFR-mutated NSCLC cells

Abstract Lazertinib, a novel third-generation EGFR-tyrosine kinase inhibitor, has shown marked efficacy in patients with EGFR-mutant lung cancer. However, after various periods of therapeutic intervention, most patients eventually acquire resistance to lazertinib. A potential therapeutic approach to address this clinical issue is to overcome the initial drug resistance mechanism. To determine the underlying adaptive lazertinib resistance mechanisms, we evaluated signal transduction, cell apoptosis, and drug sensitivity in mutant EGFR-expressing non-small cell lung cancer (NSCLC) cells treated with lazertinib. The siRNA screening assay demonstrated that AXL knockdown significantly inhibited 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 eventually led to the proliferation of residual cells and increased MCL-1 expression, which was mediated by the nuclear translocation of the transcription factor YAP. Cell-based assays showed that triple therapy with an MCL-1 or YAP inhibitor, in combination with EGFR-TKI lazertinib, an AXL inhibitor, significantly reduced cell viability and increased apoptosis. These results demonstrate that AXL and YAP/MCL-1 signals contribute to the adaptive lazertinib resistance in EGFR-mutant NSCLC cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be highly effective in eliminating lazertinib-resistant cells. Citation Format: Tadaaki Yamada, Yohei Matsui, Koichi Takayama. Targeting AXL and MCL-1 to eradicate lazertinib tolerance in EGFR-mutated NSCLC cells [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 7194.

Abstract 5843: Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer in the United States with a 5-year survival rate hovering between 20-30%. Mutations in EGFR can initially cause the tumors to respond to tyrosine kinase inhibitors (TKI). Osimertinib, a third generation TKI, is the preferred first or second line of treatment and despite being effective, recent reports indicate that patients develop resistance within 10-18 months of treatment. TKI resistance limits therapeutic options for patients and therefore, alternative strategies are being explored. In this context, Lysophosphatidic acid receptors (LPARs), a family of G-protein coupled receptors consisting of LPARs 1-6 expressed differentially in prostate, breast, ovarian, pancreatic, and brain cancers are being explored as potential targets for drug resistant cancers. Specifically, LPAR-1 has been shown to play a role in tumor cell clonogenicity, migration, and response to therapy in EGFR mutant NSCLC cells. Pharmacological inhibition or CRIPSR based deletion of LPAR-1 in EGFR mutant NSCLC cell lines HCC827 and NCI-H3255, increased TKI sensitivity by reducing clonogenic potential. Further, detailed pathway analysis in TKI sensitive and resistant EGFR mutant cell lines suggests that LPAR-1 expression influences drug sensitivity. Surprisingly, the role of LPAR-1 in TKI sensitivity is not well understood. In the present study, we have performed detailed investigation on the implications of LPAR-1 expression in the development of Osimertinib resistance. Methods: Osimertinib resistant EGFR mutant cell lines (OR) were generated using dose-escalation method and drug-tolerant persisters (DTPs) were generated using a short-term high exposure method. Gene and protein expressions were examined using qPCR and western blotting. Subcutaneous tumor cell derived xenografts were generated for in vivo studies. Results: LPAR-1 is downregulated in Osimertinib resistant NSCLC cells. Further, we also observe that LPAR-1 levels are lower in DTP cells, confirming a progressive loss of LPAR-1 with Osimertinib treatment. In vivo studies in cell derived xenografts correlated with the in vitro studies. Conclusion: Our studies indicate LPAR-1 downregulation correlates to Osimertinib resistance in EGFR mutant NSCLC cell lines. LPAR-1 downregulation has a key role in development of Osimertinib resistance. Future studies will focus on delineating LPAR-1 signaling pathways related to TKI resistance and investigating the effect of LPAR-1 overexpression using CRISPR technology. Citation Format: Varsha Srinivasan, Zhaohui Li, Raghuraman Kannan, Anandhi Upendran. Exploring the implications of LPAR-1 expression in TKI-resistant 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 5843.

Abstract 7599: Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer

Abstract Lung cancer is one of the five most common cancers in the world. Statistics indicate that approximately 80% to 90% of lung cancer cases fall under the classification of non-small cell lung cancer (NSCLC). For several decades, non-small cell lung cancer (NSCLC) has often been treated with chemotherapy drugs. However, lung cancer has high incidence and recurrence rates, mainly due to drug resistance and side effects during chemotherapy and radiotherapy. This is the hinder problem in the treatment of NSCLC, which highlight the necessity of developing new drugs or new treatments for NSCLC. Cisplatin is a chemotherapy drug that can treat about 50% of solid tumors, but the cisplatin-induced cytoprotective autophagy is the resistance to clinical treatment. It is urgent to elucidate the underlying molecular mechanisms regulating cisplatin resistance to improve its therapeutic value in lung cancer. Magnolol is a phenolic component isolated from the roots and bark of Magnolia officinalis and is used in traditional Chinese medicine. With a wide range of pharmacological activities, various studies have focused on the anti-cancer properties of magnolol in recent years. The effect of the drug is to keep cells in the G2/M phase, induce mitochondrial dysfunction and ROS production, affect the supply of cellular energy, and then lead to cell apoptosis. In this study, we investigated the ability of magnolol enhance cisplatin-induced cytotoxicity, showing dose dependence in NSCLC cell by MTT assay. We utilized the immunofluorescence staining and western blotting assay to confirm the downgrade expression of LC3B and the upregulate expression of p62 that define magnolol is able to suppress cisplatin-induced cytoprotective autophagy in NSCLC. We also detected the extrinsic/intrinsic apoptosis induction ability of magnolol combined with cisplatin in NSCLC. In the result, the cisplatin combined with magnolol have more significantly triggered the loss of mitochondrial membrane potential, and activate more protein cleaved caspase-3, -8 and -9 than monotherapy. We also used western blotting assay to confirm the expression of apoptosis inhibitory protein Bcl-2 and pro-apoptotic protein Bax to measure the degree of apoptosis. The migration and invasion effects of magnolol and cisplatin were identified by transwell invasion and wound healing on NSCLC. The magnolol combined with cisplatin extended the mouse survival rate and can inhibit tumor progression. These results suggest that combinational therapy of cisplatin and magnolol could be a promising treatment strategy for NSCLC. The potential regulation of magnolol on NSCLC is associated with triggering cisplatin-induced apoptosis and diminishing cisplatin-induce cytoprotective autophagy. Citation Format: Rong-Er Lu, Bo-Xun Chen, Fei-Ting Hsu, I-Tsang Chiang. Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer [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 7599.

Abstract 5907: Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-317 targeting NaPi2b

Abstract Introduction: NaPi2b is a multi-transmembrane type II sodium-dependent phosphate transporters that regulates phosphate homeostasis in normal physiological condition [1]. It is highly expressed in ovarian cancer (OC), non-small-cell lung cancer (NSCLC), and papillary thyroid cancer, with limited expression in normal tissue, making it a promising target for antibody-drug conjugates (ADCs) [2,3]. LM-317 is a novel NaPi2b targeted humanized monoclonal antibody LM-117 conjugated to the next-generation topoisomerase I inhibitor LDX2 via a cleavable linker, with antibody-drug ratio of 8. Here, we present the preclinical in vivo and in vitro results of LM-317. Results: LM-317 is highly specific cross-reactive with rhesus monkeys, cynomolgus monkeys, mouse, and rat NaPi2b. Concentration-dependent cell binding and internalization effects of LM-317 were observed in human NaPi2b-expressing cells, and OC and NSCLC cells expressing NaPi2b, with EC-50 of 0.92 - 9.7 nM and 1.51 - 4.76 nM, respectively. The cytotoxicity effect of LM-317 in NaPi2b -positive tumor cells was superior to LM-117-DXD, with IC-50 of 0.002 - 0.52nM. The in vivo studies showed that 1 mg/kg of LM-317 almost completely eradicated tumors in the OVCAR-3 xenograft mice model of OC, and similar antitumor activity was observed in the NCI-H175-xenograft mice model of NSCLC at the dose of 3 mg/kg. Single dose of 6 mg/kg of LM-317 showed sustained tumor eradication in the NSCLC xenograft -mouse model. LM-317 also exhibited potent tumor regression in the PDX model of OC and NSCLC. The tumor growth inhibition (TGI) value was 80%-100% in OC and NSCLC PDX models with different expression degree of NaPi2b after treatment with LM-317. The antitumor activity of LM-317 in cell line- and PDX models of OC and NSLCL was comparable to those of LM-117-DXD. Conclusion: The novel ADC LM-317 targeting NaPi2b showed robust antitumor activity in vitro, and in the OC and NSCLC cell line derived models and PDX models. LM-317 could potentially provide therapeutic benefit to treat NaPi2b-positive tumors including OC and NSCLC in the future. Reference: 1.S. Banerjee et al. Cancer Treatment Reviews 112 (2023) 1024892.Finstad CL, Lloyd KO, Federici MG, et al. Clin. Cancer Res. 1997;3(8):1433-14423.Zhang et al. Tumor Biology July 2017: 1-7. Citation Format: Wentao Huang, Zhifang Liu, Yifan Li, Heng Pan, Xia Qin, Da Fei, Runsheng Li. Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-317 targeting NaPi2b [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 5907.

Abstract 2803: Identification of microbiome and predictive immune biomarkers in patients with advanced non-small lung cancer treated with pembrolizumab with or without chemotherapy

Abstract Background: Recent studies have shown that gut microbiota modulates antitumor immunity, and affect clinical efficacy of immune checkpoint inhibitors, including anti-programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors. We aimed to explore the metagenomic profiles of previously untreated, treatment naïve, stage IV non-small cell lung cancer (NSCLC) treated with pembrolizumab, an anti-PD1 agent with or without platinum-based chemotherapy and identify distinct ecological niche between responder and non-responder groups. Methods: We performed metagenome sequencing, whole exome sequencing (WES), and RNA sequencing (RNA-seq) on 50 patients with stage IV NSCLC, including adenocarcinoma (N=31) and squamous cell carcinoma (N=22). We employed Dirichlet Multinomial Mixtures (DMM) community typing to cluster microbial community profiling data at the genus level and analyzed the changes in microbial composition and diversity between enterotype 1 (E1, N=27) and enterotype 2 (E2, N=23). Mutation landscape including genomic alterations in relation to immune response was analyzed between responders and non-responders using WES data. Differentially expressed genes (DEGs), Gene Set Enrichment Analysis (GSEA), and immune deconvolution analysis were carried out using RNA-seq data to analyze molecular profiling of two enterotypes between the responder (N=15) and non-responder (N=35) groups. Results: The two enterotypes exhibited distinct microbial abundances and alpha diversity indices. Abundances of members of Bacteroidaceae (P=0.011) and Oscillospiraceae (P<0.001) were higher in E2 members, and enrichment of Lactobacillaceae (P=0.006) and Bifidobacteriaceae (P=0.041) were higher in E1. Compared to E1, the number of observed bacterial species (P<0.001) and Shannon index (P<0.001) was significantly higher in E2, indicating a healthier gut environment. The proportion of responders was higher in E2 than in E1 cluster. Comparison between responders and non-responder groups within each enterotype revealed that Lactobacillaceae showed an association with the response in E2 cluster. Mutation landscape revealed TP53 mutation as the most common mutation, more frequently observed in the E2 (100%) than in the E1 (56%) cluster. Conclusion: We explored the association between the gut microbiome and clinical response to pembrolizumab with or without combination with chemotherapy and found that E2 cluster enriched with Lactobacillaceae is associated with treatment response. Further analysis with larger cohort is ongoing. Citation Format: Jii Bum (Joy) Lee, Su-Jin Choi, Gang-Taik Lee, Junwon Yang, Hyun-Seok Oh, Arim Min, Bo-Eun Kwon, Seong-san Kang, Sujeong Baek, Dong Kwon Kim, Jae Hwan Kim, Sun Min Lim, Min Hee Hong, Kyoung-Ho Pyo, Byoung Chul Cho. Identification of microbiome and predictive immune biomarkers in patients with advanced non-small lung cancer treated with pembrolizumab with or without chemotherapy [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 2803.