EGFR-mutant Non-small Cell Lung Cancer Cells Research Articles

Vismodegib Potentiates Marine Antimicrobial Peptide Tilapia Piscidin 4-Induced Cytotoxicity in Human Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is a common cancer with several accepted treatments, such as chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, and immune checkpoint inhibitors. Nevertheless, NSCLC cells often become insensitive to these treatments, and therapeutic resistance is a major reason NSCLC still has a high mortality rate. The induction of therapeutic resistance in NSCLC often involves hedgehog, and suppression of hedgehog can increase NSCLC cell sensitivity to several conventional therapies. In our previous work, we demonstrated that the marine antimicrobial peptide tilapia piscidin 4 (TP4) exhibits potent anti-NSCLC activity in both EGFR-WT and EGFR-mutant NSCLC cells. Here, we sought to further explore whether hedgehog might influence the sensitivity of NSCLC cells to TP4. Our results showed that hedgehog was activated by TP4 in both WT and EGFR-mutant NSCLC cells and that pharmacological inhibition of hedgehog by vismodegib, a Food and Drug Administration-approved hedgehog inhibitor, potentiated TP4-induced cytotoxicity. Mechanistically, vismodegib acted by enhancing TP4-mediated increases in mitochondrial membrane potential and intracellular reactive oxygen species (ROS). MitoTempo, a specific mitochondrial ROS scavenger, abolished vismodegib/TP4 cytotoxicity. The combination of vismodegib with TP4 also reduced the levels of the antioxidant proteins catalase and superoxide dismutase, and it diminished the levels of chemoresistance-related proteins, Bcl-2 and p21. Thus, we conclude that hedgehog regulates the cytotoxic sensitivity of NSCLC cells to TP4 by protecting against mitochondrial dysfunction and suppressing oxidative stress. These findings suggest that combined treatment of vismodegib and TP4 may be a promising therapeutic strategy for NSCLC.

Multifunctional liposomal complex for detection of the acquired resistance to EGFR-TKIs and synergistic anticancer phototherapy

The emergence of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) poses formidable obstacles to non-small cell lung cancer (NSCLC) therapy. Real-time monitoring of the efficacy of EGFR-TKIs to avoid invalid treatment remains a great challenge. Herein, we rationally designed a EGFR-TKI-modified liposomal complex co-loaded with a iodine-containing contrast agent ioversol (IOV) and a photosensitizer indocyanine green (ICG), namely ELIG, for simultaneous EGFR-TKI-mediated computed tomography/fluorescence dual-modal imaging to monitor drug resistance during the treatment, and combined photodynamic therapy (PDT) and photothermal therapy (PTT) to reverse drug resistance. ELIG exhibited good physicochemical properties, favorable photothermal effects, and high singlet oxygen production. ELIG can protect ICG and IOV from rapid degradation and prolong the circulation time. In addition, ELIG had a great ability to recognize drug-sensitive EGFR-mutated NSCLC cells, and could monitor the changes in the intracellular amounts of the imaging agents to detect the drug resistance and predict the efficacy during the treatments. Additionally, the heavy atom effect of IOV can increase the PDT effect of ICG obviously. With 808 nm laser irradiation, ELIG showed satisfactory anticancer effects to reverse drug resistance by the synergism of PDT and PTT in vitro and in vivo. Hence, this work holds great promise for providing a new idea and an effective strategy for predicting and managing the efficacy of EGFR-TKIs.

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 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 946: Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration

Abstract Introduction: Olaparib, an oral poly polymerase (PARP) inhibitor has received approval for treating malignancies displaying homologous recombination deficiency (HRD). Current data is insufficient in establishing the role of PARP inhibitors in non-small cell lung cancer (NSCLC) in the setting of third generation EGFR tyrosine kinase inhibitor (TKI) use. Preclinical investigation suggests heightened PARP inhibitor sensitivity of TKI-resistant EGFR mutant NSCLC cells compared to TKI-sensitive cells. Methods: We utilized The Cancer Genome Atlas (TCGA) to investigate the prevalence of HRD gene mutations in patients with NSCLC harboring EGFR mutations. We also present a case demonstrating a favorable response to the dual therapy of olaparib and osimertinib in NSCLC harboring EGFR, RAD50, and ARID1A mutations that progressed on osimertinib. Results: There were 2298 patients in the NSCLC cohort (MSKCC database). Among 687 patients with EGFR mutations, 24 patients (3.5%) exhibited ARID1A mutations, 8 patients (1.2%) had BRCA1 mutations, 11 patients (1.6%) had BRCA2 mutations, and 5 (0.7%) had RAD50 mutations. A 71-year-old female presented with stage IV adenocarcinoma of the lung. She had recurrent malignant pericardial effusion requiring pericardiocentesis for previous cardiac tamponade. Computed tomography (CT) of the chest revealed a 3cm irregular left hilar mass with bilateral mediastinal and hilar lymph nodes. Tissue next generation sequencing (NGS) at baseline identified EGFR exon19 deletion along with RAD50 R726H which is a HRD pathway gene. Circulating tumor DNA (ct-DNA) NGS (Guardant360) showed EGFR E746_A750del 4.8%. The patient achieved partial response to osimertinib for 28 months. Follow-up CT showed disease progression. Olaparib 200mg twice daily was added to osimertinib based on repeat ct-DNA NGS indicating a new ARID1A alteration (splice site SNV) along with the known BRCA1 mutation. Follow up scans currently have consistently demonstrated ongoing partial response for 19 months. Combining olaparib with osimertinib can be effective in an osimertinib-resistant NSCLC patient with EGFR mutation and HRD aberration. The favorable response could be attributed not only to pre-existing RAD50 mutation but also to acquired mutations in ARID1A HRD genes. Conclusion: HRD aberrations are uncommon in EGFR mutated lung cancer patients. Further investigation on the role of PARP inhibitor in EGFR mutated lung cancer is warranted. Citation Format: Jinah Kim, Horyun Choi, Liam Il-Young Chung, Young Kwang Chae. Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration [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 946.

Abstract 4746: PIM kinase inhibition synergizes with a MERTK inhibitor to treat osimertinib resistant EGFR-mutant non-small cell lung cancer

Abstract There is an unmet need for effective treatment in the setting of acquired osimertinib resistance in EGFR-mutant non-small cell lung cancer (NSCLC) patients and MERTK tyrosine kinase is a potential therapeutic target in this context. Treatment with the MERTK inhibitor MRX-2843 was sufficient to abrogate downstream PI3K-AKT and MAPK-ERK signaling, cell expansion, and colony formation in osimertinib-resistant EGFR-mutant NSCLC cell lines, suggesting dependence on MERTK signaling for tumor cell proliferation/survival. Further, two structurally distinct PIM kinase inhibitors, SGI-1776 and PIM447, provided synergistic inhibition of cell expansion and colony formation in osimertinib-resistant cell line cultures when combined with MRX-2843 treatment. Mechanistically, treatment with MRX-2843 in combination with either PIM kinase inhibitor decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Furthermore, knockdown of PIM kinases (PIM1, 2, 3) using nine sets of siRNAs led to various changes in expression of TAM receptor family members (TYRO3, AXL, MERTK), indicating a role for PIM kinases in regulation of TAM kinase expression. These studies suggest a novel treatment strategy for osimertinib-resistant EGFR-mutant NSCLC using a first-in-class MERTK kinase inhibitor that is currently under evaluation in multiple Phase I clinical trials. Citation Format: Dan Yan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. PIM kinase inhibition synergizes with a MERTK inhibitor to treat osimertinib resistant 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 4746.

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.

Tanshinone IIA reverses gefitinib resistance in EGFR-mutant lung cancer via inhibition of SREBP1-mediated lipogenesis.

Gefitinib resistance is an urgent problem to be solved in the treatment of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA) is one of the main active components of Salvia miltiorrhiza, which exhibits significant antitumor effects. The aim of this study is to explore the reversal effect of Tan IIA on gefitinib resistance in the epidermal growth factor receptor (EGFR)-mutant NSCLC and the underlying mechanism. CCK-8, colony formation assay, and flow cytometry were applied to detect the cytotoxicity, proliferation, and apoptosis, respectively. The changes in lipid profiles were measured by electrospray ionization-mass spectrometry (MS)/MS. Western blot, real-time q-PCR, and immunohistochemical were used to detect the protein and the corresponding mRNA levels. The in vivo antitumor effect was validated by the xenograft mouse model. Co-treatment of Tan IIA enhanced the sensitivity of resistant NSCLC cells to gefitinib. Mechanistically, Tan IIA could downregulate the expression of sterol regulatory element binding protein 1 (SREBP1) and its downstream target genes, causing changes in lipid profiles, thereby reversing the gefitinib-resistance in EGFR-mutant NSCLC cells in vitro and in vivo. Tan IIA improved gefitinib sensitivity via SREBP1-mediated lipogenesis. Tan IIA could be a potential candidate to enhance sensitivity for gefitinib-resistant NSCLC patients.

Abstract B099: Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach

Abstract The epidermal growth factor receptor (EGFR) is a crucial signaling protein involved in regulating cellular growth, survival, and proliferation. EGFR mutations, particularly in non-small cell lung cancer (NSCLC), have been extensively studied due to their role in driving tumor progression and resistance to conventional therapies. While EGFR-mutant tumors initially respond well to EGFR inhibitors, emergence of resistance remains a significant challenge. Ferroptosis is a recently discovered form of regulated cell death characterized by accumulation of lipid peroxides and oxidative stress. It has gained attention as a potential therapeutic strategy in combating drug resistance. However, the vulnerability of various EGFR-mutant and resistant NSCLC cells to ferroptosis induction remains poorly understood. To bridge the existing knowledge gap, we investigated the responsiveness of both EGFR-mutant and their resistant counterpart NSCLC cell lines to ferroptosis induction. Interestingly, NSCLC cell lines and patients’ tumors which harbor EGFR mutations are distinct from those carrying STK11 and KEAP1 mutations, which are known to contribute to resistance to ferroptosis. This observation prompted us to test whether ferroptosis inducers could effectively target this specific subset of NSCLC. In our experimental approach, we first utilized a panel of commercially available EGFR mutant cell lines (n=3), including one harboring T790M resistance mutation, which is a common mechanism of acquired resistance to first-generation inhibitors. We subjected the EGFR-mutant cell lines in vitro to a process of acquiring resistance against three generations of EGFR inhibitors (n=4). Subsequently, upon confirmation of resistance, we evaluated their susceptibility to our proprietary ferroptosis inducer. Exome sequencing data of EGFR-inhibitor resistant cell lines and their age-matched control cell lines revealed that the former acquired genetic alterations that have previously been found to confer resistance in EGFR-inhibitor resistant patients’ tumors, suggesting that our approach is valid for modeling relevant resistance mechanisms. Our results indicate that both EGFR-mutant and resistant cell lines exhibit sensitivity to ferroptosis induction. Furthermore, we observed that EGFR-mutant patient-derived cells, which are refractory to EGFR inhibitors of several generations, were strongly cytostatic to our ferroptosis inducer. These findings provide valuable insights into the interplay between EGFR mutations, drug resistance, and ferroptosis. Exploiting the vulnerability of EGFR-inhibitor resistant NSCLC cells to ferroptosis could offer novel therapeutic options for overcoming drug resistance in EGFR-driven cancers. Further investigations into the underlying molecular mechanisms of ferroptosis inducers in NSCLC are warranted for the development of effective therapies. Citation Format: Taronish Dubash, Maria Cristina Munteanu, Cristian Nunez, Shrouq Farah, Laurence Jadin, Branko Radetich, Francesco M Marincola, Darby Schmidt, Nadia Gurvich. Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach [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 B099.

Periostin secreted by cancer-associated fibroblasts promotes cancer progression and drug resistance in non-small cell lung cancer.

Cancer-associated fibroblasts (CAFs) are important components in the tumor microenvironment, and we sought to identify effective therapeutic targets in CAFs for non-small cell lung cancer (NSCLC). In this study, we established fibroblast cell lines from the cancerous and non-cancerous parts of surgical lung specimens from patients with NSCLC and evaluated the differences in behaviors towards NSCLC cells. RNA sequencing analysis was performed to investigate the differentially expressed genes between normal fibroblasts (NFs) and CAFs, and we identified that the expression of periostin (POSTN), which is known to be overexpressed in various solid tumors and promote cancer progression, was significantly higher in CAFs than in NFs. POSTN increased cell proliferation via NSCLC cells' ERK pathway activation and induced epithelial-mesenchymal transition (EMT), which improved migration in vitro. In addition, POSTN knockdown in CAFs suppressed these effects, and in vivo experiments demonstrated that the POSTN knockdown improved the sensitivity of EGFR-mutant NSCLC cells for osimertinib treatment. Collectively, our results showed that CAF-derived POSTN is involved in tumor growth, migration, EMT induction, and drug resistance in NSCLC. Targeting CAF-secreted POSTN could be a potential therapeutic strategy for NSCLC. KEY MESSAGES: • POSTN is significantly upregulated in CAFs compared to normal fibroblasts in NCSLC. • POSTN increases cell proliferation via activation of the NSCLC cells' ERK pathway. • POSTN induces EMT in NSCLC cells and improves the migration ability. • POSTN knockdown improves the sensitivity for osimertinib in EGFR-mutant NSCLC cells.

YAP mediates resistance to EGF-induced apoptosis in EGFR-mutated non-small cell lung cancer cells

Mechanisms underlying the growth and survival of non-small cell lung cancer (NSCLC) cells positive for activating mutations of the epidermal growth factor receptor gene (EGFR) have remained unclear. We here examined the functional relation between such mutant forms of EGFR and Yes-associated protein (YAP), a transcriptional coactivator of the Hippo signaling pathway that regulates cell proliferation and survival. Under the condition of serum deprivation, epidermal growth factor (EGF) induced activation of YAP in NSCLC cell lines positive for mutated EGFR but not in those wild type (WT) for EGFR. Similar EGF-induced activation of YAP was apparent in A549 lung cancer cells forcibly expressing mutant EGFR but not in those overexpressing the WT receptor. Furthermore, EGF induced apoptotic cell death in serum-deprived A549 cells overexpressing the WT form of EGFR but not in those expressing mutant EGFR, and knockdown of YAP rendered the latter cells sensitive to this effect of EGF. Our results thus suggest that activation of YAP mediates resistance of EGFR-mutated NSCLC cells to EGF-induced apoptosis and thereby contributes specifically to the survival of such cells.

Clinicopathological impact of VEGFR2 and VEGF-C in patients with EGFR-major mutant NSCLC receiving osimertinib.

Vascular endothelial growth factor (VEGF) has been identified as one of the resistant mechanisms to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). However, the relationship between the efficacy of osimertinib and protein expression of VEGF family members in patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR mutations remains unclear. A total of 76 patients with advanced NSCLC with EGFR major mutations (del19 or L858R) receiving first-line osimertinib were eligible as the osimertinib (Osi) group, whereas 43 patients receiving first- or second-generation EGFR-TKIs were compared with the control group. The expression of vascular endothelial growth factor receptor 2 (VEGFR2) and vascular endothelial growth factor C (VEGF-C) in the tumor specimens was analyzed using immunohistochemistry. VEGFR2 and VEGF-C were highly expressed in 65.8% and 51.3% of patients, respectively, in the Osi group, and 69.7% and 76.7%, respectively, in the control group. High VEGFR2 and VEGF-C levels were significantly associated with poor performance status (PS) and female sex, respectively. In the Osi group, patients with co-high expression of VEGFR2 and VEGF-C showed significantly worse progression-free survival (PFS) and overall survival (OS) than those without co-high expression. In del19, VEGFR2 was a significant predictor of PFS and OS and independent predictor of OS in multivariate analysis. In L858R, co-high expression of VEGFR2 and VEGF-C was identified as a significant predictor of PFS and OS and independent predictor of PFS. VEGFR2 and VEGF-C are highly expressed in EGFR-mutant NSCLC cells. Increased expression of VEGFR2 was identified as a significant prognostic factor in patients with EGFR del19 mutation who received osimertinib, whereas co-high expression of VEGFR2 and VEGF-C was a significant predictor for those with EGFR L858R mutation.

BYL719 reverses gefitinib-resistance induced by PI3K/AKT activation in non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation often obtain de novo resistance or develop secondary resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs), which restricts the clinical benefit for the patients. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signal pathway is one of the most important mechanisms for the EGFR-TKIs resistance beyond T790M mutation. There are currently no drugs simultaneously targeting EGFR and PI3K signal pathways, and combination of these two pathway inhibitors may be a possible strategy to reverse theses resistances. To test whether this combinational strategy works, we investigated the therapeutic effects and mechanisms of combining BYL719, a PI3Kα inhibitor, with gefitinib, an EGFR-TKI inhibitor in EGFR-TKIs resistance NSCLC models induced by PI3K/AKT activation. Our results demonstrated that PIK3CA mutated cells showed increased growth rate and less sensitive or even resistant to gefitinib, associated with increased PI3K/AKT expression. The combination of BYL719 and gefitinib resulted in synergistic effect compared with the single agents alone in EGFR-mutated NSCLC cells with PI3K/AKT activation. The inhibition of AKT phosphorylation by BYL719 increased the antitumor efficacy of gefitinib in these cell lines. Moreover, the combined effect and mechanism of gefitinib and BYL719 were also confirmed in the NSCLC cells and patient-derived organoids under 3D culture condition, as well as in vivo. Taken together, the data indicate that PIK3CA mutation induces more aggressive growth and gefitinib resistance in NSCLC cells, and the combination treatment with gefitinib and BYL719 is a promising therapeutic approach to overcoming EGFR-TKIs resistance induced by PI3K/AKT activation.

Upregulation of long non‑coding RNA LINC00460 in EGFR‑mutant lung cancer indicates a poor prognosis in patients treated with osimertinib.

The long non-coding RNA (lncRNA) LINC00460 is involved in tumor growth, metastasis and drug resistance. The present study investigated the clinical significance of LINC00460 expression in patients with epidermal growth factor receptor (EGFR) mutation-positive lung cancer treated with osimertinib. Osimertinib-resistant cells we derived from EGFR-mutant non-small-cell lung cancer (NSCLC) cell lines, after which, small interfering RNA (siRNA)-mediated silencing and in vitro-transcribed (IVT), synthetic LINC00460 RNA transfection were used to investigate the effects of LINC00460 expression on acquired resistance to osimertinib. Reverse transcription-quantitative polymerase chain reaction was performed to evaluate LINC00460 expression in 54 samples (RNA extracted from the tumor tissues of 30 cases and cell-free RNA from 24 cases) obtained from patients with EGFR mutation-positive lung cancer who had received osimertinib as the initial treatment. The acquisition of osimertinib resistance increased the expression of LINC00460 in the EGFR-mutant NSCLC cell lines. By contrast, knockdown of LINC00460 in osimertinib-resistant cell lines increased their sensitivity to osimertinib, whereas treatment of NSCLC cells with IVT LINC00460 RNA decreased their sensitivity to osimertinib. The present study examined LINC00460 expression at the primary tumor site and demonstrated that compared with in the low-expression group (n=24), the high-expression group (n=6) had a significantly lower best overall response rate to osimertinib (16.6% vs. 60.0%; P=0.044), significantly shorter median progression-free survival (PFS; 224 days vs. 669 days; P=0.001) and significantly shorter median overall survival (724 days vs. not reached; P=0.011). Moreover, following osimertinib therapy, PFS was significantly shorter for patients with high LINC00460 expression in plasma cell-free RNA (n=12) than for those with low LINC00460 expression (n=12) (median PFS: 655 days vs. 210 days; P=0.020). In conclusion, the upregulation of LINC00460, the expression of which is implicated in osimertinib resistance, in the primary site and plasma of patients with EGFR mutation-positive lung cancer may be associated with a poor prognosis in those treated with osimertinib.

AX-0085, a novel AXL kinase inhibitor, and effects on osimertinib-resistance in non–small-cell lung cancer.

e15121 Background: Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) having high efficacy in treating patients with advanced non-small cell lung cancer (NSCLC) with EGFR activating mutations and EGFR T70M mutations. Although, osimertinib is a frontline anticancer agent for treating NSCLC, many patients inevitably develop osimertinib-resistance tumor recurrence. The activation of AXL was reported to be a major factor driving osimertinib-resistance in NSCLC. Thus, AXL is a promising therapeutic target for the treatment of osimertinib-resistant NSCLC, and we developed a new small-molecule AXL inhibitor, AX-0085. Methods: We established osimertinib-resistant cell lines from EGFR-mutated NSCLC cell lines, HCC827 and H1975, after high dose exposure and stepwise exposure to osimertinib in culture. We investigated the cell viability of AX-0085 and other kinase inhibitors, and the anti-tumor activity of AX-0085 on the proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) in osimertinib-resistant cells. Results: Compared to the parental cells, IC50 of resistant cell to osimertinib was significantly increased and osimertinib-resistant cells showed remarkably increased expression of AXL/pAXL and p-SRC as compared to the parental HCC827 cell. AX-0085 was completely blocked the AXL and SRC activation but EGFR inhibitors (gefitinib and osimertinib) did not inhibit the activation of AXL and SRC in the osimertinib-resistant cell line. AX-0085 (IC50: 4.4nM for AXL kinase in cell-based kinase assay) was one of the most potent AXL inhibitors and showed the strongest cytotoxicity against osimertinib-resistant cells compared to other kinase inhibitors. AX-0085 effectively down-regulated mRNA level of AXL and AXL regulating transcription factors in NSCLC cell lines such as HCC827 and H1975. AX-0085 treatment effectively inhibited the activation of AXL in acquired osimertinib-resistant cells and decreased the mRNA level of AXL and AXL regulating transcription factors. AX-0085 significantly inhibited the cell proliferation, apoptosis, migration, and EMT in acquired osimertinib-resistant cells. Conclusions: Our results showed that AX-0085 selectively inhibits the activation of AXL to overcome acquired resistance to osimertinib, suggesting an effective AXL-based therapy for patients with osimertinib-resistant tumors. Currently, AX-0085 is undergoing non-clinical trials.

Evaluating the efficacy and cardiotoxicity of EGFR-TKI AC0010 with a novel multifunctional biosensor

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatically improved the life expectancy of patients with NSCLC, concerns about TKI-induced cardiotoxicities have increased. AC0010, a novel third-generation TKI, was developed to overcome drug resistance induced by EGFR-T790M mutation. However, the cardiotoxicity of AC0010 remains unclear. To evaluate the efficacy and cardiotoxicity of AC0010, we designed a novel multifunctional biosensor by integrating microelectrodes (MEs) and interdigital electrodes (IDEs) to comprehensively evaluate cell viability, electrophysiological activity, and morphological changes (beating of cardiomyocytes). The multifunctional biosensor can monitor AC0010-induced NSCLC inhibition and cardiotoxicity in a quantitative, label-free, noninvasive, and real-time manner. AC0010 was found to significantly inhibit NCI-H1975 (EGFR-L858R/T790M mutation), while weak inhibition was found for A549 (wild-type EGFR). Negligible inhibition was found in the viabilities of HFF-1 (normal fibroblasts) and cardiomyocytes. With the multifunctional biosensor, we found that 10 μM AC0010 significantly affected the extracellular field potential (EFP) and mechanical beating of cardiomyocytes. The amplitude of EFP continuously decreased after AC0010 treatment, while the interval decreased first and then increased. We analyzed the change in the systole time (ST) and diastole time (DT) within a beating interval and found that the DT and DT/beating interval rate decreased within 1 h after AC0010 treatment. This result probably indicated that the relaxation of cardiomyocytes was insufficient, which may further aggravate the dysfunction. Here, we found that AC0010 significantly inhibited EGFR-mutant NSCLC cells and impaired cardiomyocyte function at low concentrations (10 μM). This is the first study in which the risk of AC0010-induced cardiotoxicity was evaluated. In addition, novel multifunctional biosensors can comprehensively evaluate the antitumor efficacy and cardiotoxicity of drugs and candidate compounds.

DMU-212 against EGFR-mutant non-small cell lung cancer via AMPK/PI3K/Erk signaling pathway

Although some important advances have been achieved in clinical and diagnosis in the past few years, the management of non-small cell lung cancer (NSCLC) is ultimately dissatisfactory due to the low overall cure and survival rates. Epidermal growth factor (EGFR) has been recognized as a carcinogenic driver and is a crucial pharmacological target for NSCLC. DMU-212, an analog of resveratrol, has been reported to have significant inhibitory effects on several types of cancer. However, the effect of DMU-212 on lung cancer remains unclear. Therefore, this study aims to determine the effects and underlying mechanism of DMU-212 on EGFR-mutant NSCLC cells. The data found that the cytotoxicity of DMU-212 on three EGFR-mutant NSCLC cell lines was significantly higher than that of normal lung epithelial cell. Further study showed that DMU-212 can regulate the expression of cell cycle-related proteins including p21 and cyclin B1 to induce G2/M phase arrest in both H1975 and PC9 cells. Moreover, treatment with DMU-212 significantly promoted the activation of AMPK and simultaneously down-regulated the expression of EGFR and the phosphorylation of PI3K, Akt and ERK. In conclusion, our study suggested that DMU-212 inhibited the growth of NSCLCs via targeting of AMPK and EGFR.

Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects of epidermal growth factor receptor inhibitor treatments

Precision cancer medicine has changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by the introduction of tyrosine kinase inhibitors (TKIs) towards mutated epidermal growth factor receptor (EGFR). However, as responses to EGFR-TKIs are heterogenous among NSCLC patients, there is a need for ways to early monitor changes in treatment response in a non-invasive way e.g., in patient's blood samples. Recently, extracellular vesicles (EVs) have been identified as a source of tumor biomarkers which could improve on non-invasive liquid biopsy-based diagnosis of cancer. However, the heterogeneity in EVs is high. Putative biomarker candidates may be hidden in the differential expression of membrane proteins in a subset of EVs hard to identify using bulk techniques. Using a fluorescence-based approach, we demonstrate that a single-EV technique can detect alterations in EV surface protein profiles. We analyzed EVs isolated from an EGFR-mutant NSCLC cell line, which is refractory to EGFR-TKIs erlotinib and responsive to osimertinib, before and after treatment with these drugs and after cisplatin chemotherapy. We studied expression level of five proteins; two tetraspanins (CD9, CD81), and three markers of interest in lung cancer (EGFR, programmed death-ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)). The data reveal alterations induced by the osimertinib treatment compared to the other two treatments. These include the growth of the PD-L1/HER2-positive EV population, with the largest increase in vesicles exclusively expressing one of the two proteins. The expression level per EV decreased for these markers. On the other hand, both the TKIs had a similar effect on the EGFR-positive EV population.

Abstract 3907: Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor

Abstract Resistance to targeted therapies is a major challenge in oncology. Disease progression is caused by multiple resistance mechanisms. Besides pre-existing and acquired genetic alternations, adaptive non-mutational reprogramming as well as modulation of phenotypic plasticity have emerged as drivers of disease progression. For example, in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) patients who were treated with osimertinib, ~50% of progressive disease could not be attributed to genetic mutations (Leonetti et al., BJC 2019). Similarly, in ~ 40% of patients suffering from KRAS G12C-mutated NSCLC and colorectal cancer (CRC), disease progressed under sotorasib treatment without identifiable acquired mutations (Zhao et al., Nature 2021). Thus, targeting non-genetic adaptive resistance mechanism such as drug-induced transcriptional reprogramming might be of great therapeutic benefit. Here we identified small molecules, that interfere with cancer drug-induced transcriptional escape mechanisms using a phenotypic screen based on an SRY-Box Transcription Factor 2 (SOX2) reporter system. The screen led to the development of TT125-802, a highly specific and potent, orally available small molecule inhibitor of the bromodomain of the transcriptional and epigenetic regulator CBP [cyclic adenosine monophosphate response element binding protein (CREB) binding protein] and its paralogue p300. TT125-802 dose-dependently prevented osimertinib resistance development in EGFR-mutated NSCLC cell lines HCC827 and HCC4006, as well as sotorasib resistance development in KRAS G12C-mutated NCI-H358 (NSCLC), SW837 and SNU-1411 (CRCs) as assessed by label-free long-term live microscopy assays. Data generated in mouse xenograft studies confirmed the ability of TT125-802 increasing response rates and prolonging the duration of response to osimertinib and sotorasib in vivo. In cells and tumours which were already resistant to osimertinib or sotorasib, TT125-802 could still delay cell or tumor growth. Complementary and longitudinal analysis of transcriptional changes using RNA sequencing in vitro and in vivo identified several early adaptive and late acquired resistance signatures that were reversed by TT125-802. A first-in-human study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Thomas Bohnacker, Dorothea Gruber, Sara Laudato, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3907.