wtEGFR Non-small Cell Lung Cancer 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.

Abstract 1914: Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression

Abstract Amivantamab (RYBREVANT™) is an FDA-approved, low-fucose IgG1 bispecific antibody targeting EGF and Met receptor tyrosine kinases, with proven clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Preclinical studies demonstrated that three context-dependent mechanisms of action (MOA) contribute to amivantamab efficacy: immune cell-mediated killing by antibody-dependent cellular cytotoxicity and trogocytosis, receptor internalization and degradation, and inhibition of ligand binding to both EGF and Met receptors. In tumors lacking known cancer-driver genetic aberrations, ligand binding to wildtype EGFR (EGFRWT) and/or MetWT may activate downstream signaling pathways, inducing pro-tumorigenic processes. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in EGFRWT NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers, and induces EGFR phosphorylation, promoting downstream signaling and increasing cell proliferation. Importantly, treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab, or an AREG-targeting antibody, inhibits ligand-induced signaling and cell/tumor proliferation/growth. Across 10 EGFRWT NSCLC patient-derived xenograft mouse models, amivantamab efficacy correlated with AREG RNA levels, suggesting AREG as a potential predictive marker for amivantamab activity in this population. Interestingly, in these xenograft models, amivantamab anti-tumor activity was independent of amivantamab's Fc engagement with immune cells, suggesting that, in this context, the amivantamab’s ligand-blocking function is sufficient for its efficacy. Juxtaposedly, we previously demonstrated that low fucose amivantamab, active Fc domain, was necessary for maximal anti-tumor activity against tumors with EGFR or Met activating mutations. Altogether, these results demonstrated amivantamab context-dependent MOA for efficacy. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab against anti-EGFRWT therapies. Citation Format: Ricardo Rivera-Soto, Benjamin Henley, Marian Pulgar, Stacey Lehman-Tenuto, Himanshu Gupta, Megan Weindorfer, Smruthi Vijayaraghavan, Tsun-Wen Yao, Sylvie Laquerre, Sheri Moores. Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression [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 1914.

Statin shapes inflamed tumor microenvironment and enhances immune checkpoint blockade in non–small cell lung cancer

Immune checkpoint blockade (ICB) therapy has achieved breakthroughs in the treatment of advanced non-small cell lung cancer (NSCLC). Nevertheless, the low response due to immuno-cold (i.e., tumors with limited tumor-infiltrating lymphocytes) tumor microenvironment (TME) largely limits the application of ICB therapy. Based on the glycolytic/cholesterol synthesis axis, a stratification framework for EGFR-WT NSCLC was developed to summarize the metabolic features of immuno-cold and immuno-hot tumors. The cholesterol subgroup displays the worst prognosis in immuno-cold NSCLC, with significant enrichment of the cholesterol gene signature, indicating that targeting cholesterol synthesis is essential for the therapy for immuno-cold NSCLC. Statin, the inhibitor for cholesterol synthesis, can suppress the aggressiveness of NSCLC in vitro and in vivo and can also drastically reverse the phenotype of immuno-cold to an inflamed phenotype in vivo. This change led to a higher response to ICB therapy. Moreover, both our in-house data and meta-analysis further support that statin can significantly enhance ICB efficacy. In terms of preliminary mechanisms, statin could transcriptionally inhibit PD-L1 expression and induce ferroptosis in NSCLC cells. Overall, we reveal the significance of cholesterol synthesis in NSCLC and demonstrate the improved therapeutic efficacy of ICB in combination with statin. These findings could provide a clinical insight to treat NSCLC patients with immuno-cold tumors.

Telisotuzumab vedotin (Teliso-V) monotherapy in patients (pts) with previously treated c-Met–overexpressing (OE) advanced non-small cell lung cancer (NSCLC).

9016 Background: Teliso-V is an antibody-drug conjugate composed of a c-Met antibody (ABT-700) and a microtubule inhibitor (monomethyl auristatin E). The phase 2 M14-239 trial (LUMINOSITY, NCT03539536) aims to identify the c-Met OE NSCLC populations best suited to Teliso-V (Stage 1) and expand selected groups for further evaluation of efficacy (Stage 2). In Stage 1, pts were enrolled into cohorts defined by histopathology (non-squamous [NSQ] or squamous [SQ]) and EGFR mutation status (mutant or wild type [WT]); NSQ cohorts were further divided in groups on the basis of c-Met expression (high or intermediate). Updated data from the fourth interim analysis (IA4) are presented. Methods: Pts had locally advanced/metastatic NSCLC, ≤2 prior lines of systemic therapy, ≤1 line of chemotherapy, and tumors that were c-Met OE by central immunohistochemistry (IHC; Ventana; Tucson, AZ). c-Met OE was defined for the NSQ cohort as ≥25% 3+ by IHC (high, ≥50% 3+; intermediate, 25 to <50% 3+) and for the SQ cohort as ≥75% 1+ by IHC. The planned enrollment was up to approximately 150 pts in Stage 1 and 160 pts in Stage 2. Teliso-V was dosed at 1.9 mg/kg IV Q2W. The primary endpoint is objective response rate (ORR) by independent central review. Secondary endpoints include duration of response (DOR). Results: As of data cutoff (27 May 2021), 136 pts were treated with Teliso-V; 122 were evaluable for ORR. ORR was 36.5% in the NSQ EGFR WT cohort (52.2% in c-Met high group and 24.1% in c-Met intermediate group), but was modest in the NSQ EGFR mutant and SQ cohorts. Efficacy data in groups/cohorts are in the Table. The most common any-grade adverse events (AEs) were peripheral sensory neuropathy (25.0%), nausea (22.1%), and hypoalbuminemia (20.6%). Grade 5 AEs considered possibly related to Teliso-V occurred in 2 pts (sudden death and pneumonitis in 1 pt each in the SQ cohort). Conclusions: Teliso-V demonstrated a promising ORR in pts with previously treated c-Met OE NSQ EGFR WT NSCLC; this cohort is currently expanding in Stage 2. ORR was modest in the cohorts of pts with c-Met OE NSQ EGFR mutant NSCLC and with c-Met OE SQ NSCLC; both cohorts have now met the protocol-specified stopping criteria and are no longer enrolling. The safety profile observed was consistent with IA3. Clinical trial information: NCT03539536. [Table: see text]

BEZ235 reduction of cisplatin resistance on wild-type EGFR non-small cell lung cancer cells

Cisplatin, as a first-line chemotherapy drug for advanced wild-type epidermal growth factor receptor (wtEGFR) non-small cell lung cancer (NSCLC), often loses effectiveness because of acquired drug resistance. We found that ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) of DNA repair kinases and signal transduction molecules, protein kinase B (AKT)/target mammalian target of rapamycin (mTOR), were significantly phosphorylated in cisplatin-resistant wtEGFR NSCLC cell lines (H358R and A549R) than in their parental cells. Also, BEZ235 (dual phosphatidylinositol-3-kinase (PI3K)/mTOR inhibitor) significantly decreased the phosphorylation levels of these kinases/proteins, as detected by Western blot analysis. In H358R and A549R cells, the results of indirect immunofluorescence, single-cell gel electrophoresis, flow cytometry, methylthiazolyldiphenyl-tetrazolium bromide, clone formation assay, and scratch healing experiment showed that BEZ235 enhanced cisplatin-induced DNA damage and cell apoptosis, and effectively inhibited cellular proliferation/migration when combined with cisplatin. The data indicated that the abnormal activation of ATM/ATR/DNA-PKcs kinases and AKT/mTOR pathway might induce wtEGFR NSCLC cell resistance to cisplatin. The effects of the combination of BEZ235 and cisplatin suggested that BEZ235 should be considered as a combination therapy for patients with cisplatin-resistant wtEGFR NSCLC.

Hedgehog Suppresses Paclitaxel Sensitivity by Regulating Akt-Mediated Phosphorylation of Bax in EGFR Wild-Type Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is one of the most common and deadly cancers worldwide. Among NSCLC patients, almost half have wild-type epidermal growth factor receptor (EGFR WT). The primary therapeutic option for these EGFR WT NSCLC patients is chemotherapy, while NSCLC patients with EGFR mutations have more diverse therapeutic options, including EGFR tyrosine kinase inhibitors. Moreover, NSCLC patients with EGFR WT have worse chemotherapy response than EGFR mutant NSCLC patients. Thus, an urgent need exists for novel therapeutic strategies to improve chemotherapy response in EGFR WT NSCLC patients. Hedgehog signaling is known to be highly active in NSCLC; however, its potential role in chemoresistance is not fully understood. In the present study, we found that paclitaxel (PTX) treatment induces hedgehog signaling in EGFR WT NSCLC cells, and inhibition of hedgehog signaling with GDC-0449 (Vismodegib) increases sensitivity to PTX-stimulated apoptosis. Furthermore, GDC-0449 potentiates PTX-induced reactive oxygen species and mitochondrial dysfunction. In contrast, a hedgehog agonist, Hh-Ag1.5, attenuates PTX-induced apoptosis. Mechanistic experiments revealed that hedgehog induces phosphorylation of Akt at Ser473. Akt then phosphorylates Bax at Ser184, which can switch its activity from pro-apoptosis to anti-apoptosis. Taken together, our findings suggest that inhibition of hedgehog signaling might be a promising therapeutic strategy to improve PTX response in EGFR WT NSCLC.

A multifunctional nanotheranostic agent potentiates erlotinib to EGFR wild-type non-small cell lung cancer

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as Erlotinib, have demonstrated remarkable efficacy in the treatment of non-small cell lung cancer (NSCLC) patients with mutated EGFR. However, the efficacy of EGFR-TKIs in wild-type (wt) EGFR tumours has been shown to be marginal. Methods that can sensitize Erlotinib to EGFR wild-type NSCLC remain rare. Herein, we developed a multifunctional superparamagnetic nanotheranostic agent as a novel strategy to potentiate Erlotinib to EGFR-wt NSCLCs. Our results demonstrate that the nanoparticles can co-escort Erlotinib and a vascular epithermal growth factor (VEGF) inhibitor, Bevacizumab (Bev), to EGFR-wt tumours. The nanotheranostic agent exhibits remarkable effects as an inhibitor of EGFR-wt tumour growth. Moreover, Bev normalizes the tumour embedded vessels, further promoting the therapeutic efficacy of Erlotinib. In addition, the tumour engagement of the nanoparticles and the vascular normalization could be tracked by magnetic resonance imaging (MRI). Collectively, our study, for the first time, demonstrated that elaborated nanoparticles could be employed as a robust tool to potentiate Erlotinib to EGFR-wt NSCLC, paving the way for imaging-guided nanotheranostics for refractory NSCLCs expressing EGFR wild-type genes.

Bioinformatics-Based Identification of HDAC Inhibitors as Potential Drugs to Target EGFR Wild-Type Non-Small-Cell Lung Cancer.

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) greatly benefit from EGFR-tyrosine kinase inhibitors (EGFR-TKIs) while the prognosis of patients who lack EGFR-sensitive mutations (EGFR wild type, EGFR-WT) remains poor due to a lack of effective therapeutic strategies. There is an urgent need to explore the key genes that affect the prognosis and develop potentially effective drugs in EGFR-WT NSCLC patients. In this study, we clustered functional modules related to the survival traits of EGFR-WT patients using weighted gene co-expression network analysis (WGCNA). We used these data to establish a two-gene prognostic signature based on the expression of CYP11B1 and DNALI1 by combining the least absolute shrinkage and selection operator (LASSO) algorithms and Cox proportional hazards regression analysis. Following the calculation of risk score (RS) based on the two-gene signature, patients with high RSs showed a worse prognosis. We further explored targeted drugs that could be effective in patients with a high RS by the connectivity map (CMap). Surprisingly, multiple HDAC inhibitors (HDACis) such as trichostatin A (TSA) and vorinostat (SAHA) that may have efficacy were identified. Also, we proved that HDACis could inhibit the proliferation and metastasis of NSCLC cells in vitro. Taken together, our study identified prognostic biomarkers for patients with EGFR-WT NSCLC and confirmed a novel potential role for HDACis in the clinical management of EGFR-WT patients.

Gefitinib sensitization of cisplatin-resistant wild-type EGFR non-small cell lung cancer cells.

PurposeThe usual first-line strategy of wild-type EGFR (wtEGFR) non-small cell lung cancer (NSCLC) remains cisplatin-based chemotherapy. However, cisplatin often loses effectiveness because most tumors acquire drug resistance over time. As EGFR is the most important pro-survival/proliferation signal receptor in NSCLC cells, we aimed at investigating whether cisplatin resistance is related to EGFR activation and further evaluating the combined effects of cisplatin/gefitinib (EGFR-tyrosine kinase inhibitor, EGFR-TKI) on cisplatin-resistant wtEGFR NSCLC cells.Materials and methodsEGFR activation was analysed in parental and cisplatin-resistant wtEGFR NSCLC cell lines (H358 and H358R, A549 and A549R). Cellular proliferation and apoptosis of H358R/A549R cells treated with cisplatin or gefitinib, alone or in combination were investigated, and the related effector protein was detected by western blot analysis. Anti-tumor effect of two drugs combined was evaluated in animal models of H358R xenografts in vivo.ResultsEGFR was significantly phosphorylated in cisplatin-resistant wtEGFR NSCLC cells H358R and A549R than their parental cells. In H358R and A549R cells, anti-proliferative ability of gefitinib was further improved, and gefitinib combined with cisplatin enhanced inhibition of cellular survive/proliferation, and promotion of apoptosis in vitro. The combined effects were also associated with the inhibition of EGFR downstream effector proteins. Similarly, in vivo, gefitinib and cisplatin in combination significantly inhibited tumor growth of H358R xenografts.Conclusion Abnormal activation of EGFR may induce wtEGFR NSCLC cell resistance to cisplatin. The combined effects of cisplatin/gefitinib suggest that gefitinib, as a combination therapy for patients with cisplatin-resistant wtEGFR NSCLC should be considered.

MERTK Promotes Resistance to Irreversible EGFR Tyrosine Kinase Inhibitors in Non-small Cell Lung Cancers Expressing Wild-type EGFR Family Members.

Lung cancer is the leading cause of cancer-related death. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and over 60% express wild-type EGFR (wtEGFR); however, EGFR tyrosine kinase inhibitors (TKIs) have limited effect in most patients with wtEGFR tumors. We previously identified MERTK tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843, a novel MERTK-selective inhibitor with favorable properties for clinical translation. The goal of this study was to determine whether MERTK and EGFR inhibitor combination therapy could provide antitumor efficacy against wtEGFR NSCLC. An unbiased screen of 378 kinase inhibitors was conducted to identify synergistic interactions with MRX-2843 and biochemical and therapeutic effects were determined in vitro and in vivo. Numerous irreversible EGFR TKIs, including CO-1686 and osimertinib, synergized with MRX-2843 to inhibit wtEGFR NSCLC cell expansion, irrespective of driver oncogene status. CO-1686 and MRX-2843 combination therapy inhibited MERTK, wtEGFR, and ERBB2/ERBB3 and decreased downstream PI3K-AKT, MAPK-ERK, and AURORA kinase (AURK) signaling more effectively than single agents. Inhibition of PI3K, AKT or AURK, but not MEK, synergized with CO-1686 to inhibit tumor cell expansion, suggesting their roles as key redundant resistance pathways. Treatment with MRX-2843 and CO-1686 or osimertinib prevented xenograft growth while single agents had limited effect. Tumor growth inhibition was durable even after treatment with combination therapy was stopped. Our data support the application of MRX-2843 in combination with an irreversible EGFR TKI as a novel strategy for treatment of patients with wtEGFR NSCLC.

Metronomic vinorelbine is directly active on Non Small Cell Lung Cancer cells and sensitizes the EGFRL858R/T790M cells to reversible EGFR tyrosine kinase inhibitors

Metronomic vinorelbine (mVNR) has been described primarily as an antiangiogenic therapy, and no direct effects of mVNR on Non Small Cell Lung Cancer (NSCLC) cells has yet been demonstrated. The aims of this study were i) to establish the direct activity of mVNR on NSCLC cells either EGFR wt or EGFRL858R/T790M, and ii) to quantify the synergism of the combination with reversible EGFR tyrosine kinase inhibitors (TKIs), investigating the underlying mechanism of action. Proliferation assays were performed on A-549 (wt EGFRhigh), H-292 (EGFR-wt), H-358 (EGFR-wt), H-1975 (EGFRL858R/T790M) NSCLC cell lines exposed to mVNR, its active metabolite deacetyl-VNR (D-VNR), gefitinib and erlotinib for 144 h treatments. The synergism between mVNR and EGFR TKIs was determined by the combination index (CI) in EGFR-wt and H-1975 NSCLC cells. Cyclin-D1 and ABCG2 genes expression and protein levels were measured by RT-PCR and ELISA assays, as well as the phosphorylation of ERK1/2 and Akt. Intracellular concentrations of EGFR TKIs and VNR were investigated with a mass spectrometry system. mVNR, and its active metabolite D-VNR, were extremely active on NSCLC cells, in particular on H-1975 (IC50 = 13.56 ± 2.77 pM), resistant to TKIs. mVNR inhibited the phosphorylation of ERK1/2 and Akt and significantly decreased the expression of both cyclin-D1 and ABCG2 m-RNA and protein. The simultaneous combination of VNR and reversible EGFR TKIs showed a strong synergism on EGFR-wt NSCLC cells and on H-1975 cells (e.g. CI = 0.501 for 50% of affected cells), increasing the intracellular concentrations of EGFR TKIs (e.g. +50.5% vs. gefitinib alone). In conclusions, mVNR has direct effects on NSCLC cells and sensitizes resistant cells to EGFR TKIs, increasing their intracellular concentrations.

Abstract 1082: MerTK promotes resistance to irreversible EGFR TKIs by activation of the PI3K-AKT pathway in NSCLCs expressing wild-type EGFR

Abstract Lung cancer is the leading cause of cancer-related death with poor survival rates worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and 60% of these have overexpression of wild-type EGFR (wtEGFR), which portends a poor prognosis. In addition, in a recent clinical trial 6 of 12 lung cancer patients whose tumors acquired a resistance-conferring T790M EGFR mutation during treatment with an EGFR TKI reverted to wtEGFR after treatment with Rociletinib/CO-1686, an irreversible EGFR TKI that is selective for the T790M mutant. These data suggest that targeting wtEGFR may improve treatment outcomes; however, to date single agents that target wtEGFR have not been effective in large scale clinical trials and new ways to target wtEGFR in this context are needed. Our laboratory previously identified the MerTK receptor tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843 as a novel MerTK-selective small molecule tyrosine kinase inhibitor with favorable properties for clinical translation. Irrespective of driver oncogene status, treatment with a MerTK inhibitor yields potent anti-tumor effects in NSCLC cell culture models and blocks tumor growth in xenografts of the MerTK-dependent wtEGFR-expressing A549 cell line. In an attempt to more potently block A549 lung cancer cell proliferation, we screened a library of 378 kinase inhibitors that are in various stages of development and identified synergy between MRX-2843 and multiple irreversible EGFR TKIs, including CO-1686 and Osimertinib/AZD-9291. Further, we found that wtEGFR and MerTK were frequently co-expressed and co-immunoprecipitated with each other in NSCLC cancer cell lysates. Synergistic inhibition of cell expansion was observed in a spectrum of NSCLC cell lines with wtEGFR expression treated with the combination therapy, including H1299 (NRAS mutation), H157 (KRAS mutation), H3122 (ALK fusion), and Colo699 (FGFR1 overexpression). On a mechanistic level, combined treatment with 1μM CO-1686 and 100 nM MRX-2843 dramatically inhibited phosphorylation of both MerTK and EGFR and downstream signaling through the PI3K-AKT and MAPK-ERK pathways, while treatment with equivalent doses of either single agent did not efficiently inhibit MerTK, EGFR or downstream signaling. Additionally, CO-1686 mediated synergistic inhibition of A549 expansion in combination with a PI3K or AKT inhibitor, suggesting a role for PI3K-AKT activation downstream of MerTK contributing to the resistance of wtEGFR NSCLCs to irreversible EGFR TKIs. Taken together, our data provide rationale for a novel strategy for treatment of NSCLC with wtEGFR overexpression by combining MRX-2843 and an irreversible EGFR inhibitor. Citation Format: Dan Yan, Xiaodong Wang, Stephen V. Frye, Shelton H. Earp, Deborah DeRyckere, Douglas K. Graham. MerTK promotes resistance to irreversible EGFR TKIs by activation of the PI3K-AKT pathway in NSCLCs expressing wild-type EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1082. doi:10.1158/1538-7445.AM2017-1082

Early Change in FDG-PET Signal and Plasma Cell-Free DNA Level Predicts Erlotinib Response in EGFR Wild-Type NSCLC Patients

INTRODUCTION: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a treatment option in the second- or third-line palliative setting in EGFR wild-type (wt) non–small cell lung cancer (NSCLC) patients. However, response rates are low, and only approximately 25% will achieve disease control. Early prediction of treatment resistance could accelerate discontinuation of ineffective treatment and reduce unnecessary toxicity. In this study, we evaluated early changes on 18F-fluoro-D-glucose (F-18-FDG) positron emission tomography/computed tomography (PET/CT) and in total plasma cell-free DNA (cfDNA) as markers of erlotinib response in EGFR-wt patients. METHODS: F-18-FDG-PET/CT scans and blood samples were obtained prior to erlotinib initiation and were repeated after 1 week (PET/CT) and 1 to 4 weeks (blood sample) of treatment. Level of cfDNA was measured by droplet digital polymerase chain reaction. Percentage change (%∆) in SULpeak and total lesion glycolysis (TLG) on FDG-PET/CT and in plasma cfDNA was correlated to radiological response, progression-free survival (PFS), and overall survival (OS). RESULTS: Fifty patients were prospectively enrolled. A significant correlation was found between CT response and %∆TLG (P=.003). All patients with early metabolic progression showed radiological progression. Increased %∆TLG and %∆cfDNA were significantly correlated with shorter PFS (P=.002 and P=.004, respectively) and OS (P=.009 and P=.009, respectively). Multivariate analysis indicated %∆cfDNA to be the strongest predictor of OS. CONCLUSION: Early increase in TLG on F-18-FDG-PET/CT correlates with radiological progression, and shorter PFS and OS. Early increase in cfDNA predicts shorter PFS and OS. Both assessments are promising tools for early detection of nonresponders and reduced OS in TKI-treated EGFR-wt NSCLC patients.

Gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancers by accelerating EGFR turnover

Gallic acid is a common botanic phenolic compound, which is present in plants and foods worldwide. Gallic acid is implicated in various biological processes such as cell growth and apoptosis. Indeed, gallic acid has been shown to induce apoptosis in many cancer types. However, the molecular mechanisms of gallic acid-induced apoptosis in cancer, particularly lung cancer, are still unclear. Here, we report that gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancer (NSCLC) cells, but not in EGFR-WT NSCLC cells. Treatment with gallic acid resulted in a significant reduction in proliferation and induction of apoptosis, only in EGFR-mutant NSCLC cells. Interestingly, treatment with gallic acid led to a robust decrease in EGFR levels, which is critical for NSCLC survival. Treatment with gallic acid had no significant effect on transcription, but induced EGFR turnover. Indeed, treatment with a proteasome inhibitor dramatically reversed gallic acid-induced EGFR downregulation. Moreover, treatment with gallic acid induced EGFR turnover leading to apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Thus, these studies suggest that gallic acid can induce apoptosis in EGFR-dependent lung cancers that are dependent on EGFR for growth and survival via acceleration of EGFR turnover.

Abstract 4651: Docosahexaenoic acid-induced degradation of epidermal growth factor receptor through lysosome and ubiquitin/proteasomal system in human non-small cell lung cancer cells

Abstract Mutations in EGFR gene frequently occurr in lung cancer and their expressions are related to poor prognosis and drug resistance. Although docosahexaenoic acid (DHA) has anti-cancer activity, the underlying mechanism is not well known in human non-small cell lung cancer (NSCLC) yet. In this study, we show a molecular mechanism of DHA-induced degradation of EGFR in human NSCLC cells. To determine whether DHA modulates EGFR degradation, we used three NSCLC cell lines, which is A549 (EGFR WT), and two mutant cells (PC9 and H1975). We confirmed that the viability of three lung cancer cells was decreased by DHA in a dose- and time-dependent manner. DHA augmented EGFR degradation and its ubiquitin in all three cell lines. Indeed, DHA increased the levels of E3-ligase, c-cbl and endosome-related molecules (Rab5, EEA1, Rab7, and LAMP1). Chloroquin (CQ) effectively blocked the DHA-induced EGFR degradation. Moreover, knockdown of lysosome associated membrane protein (LAMP) by its siRNAs, which regulates lysosome fusion, partially prevented EGFR degradation induced by DHA, and co-localization of EGFR with lysosome in immunocytochemistry, suggesting that DHA-induced EGFR degradation is associated with lysosomal degradation. On the other hand, MG132 (proteasome inhibitor) also protected DHA-triggered EGFR degradation, demonstrating that proteasomes also contribute to EGFR degradation in response to DHA treatment. To confirm the effects of endogenous ω3-PUFAs on EGFR degradation, fat-1-stablyexpressing A549 (f-A549) and PC9 cells (f-PC9) were established (The cells express a C. elegansω3-desaturase converting ω6- to ω3-PUFAs endogenously). The level of EGFR was reduced in f-A549 and f-PC9 cells, indicating ω3-PUFAs diminish EGFR level. The cell growth was retarded and the tumorigenicity was inhibited in nude mice. In addition, EGFR levels were significantly decreased in tumor tissues from f-A549 and f-PC9 cells-bearing mice. Taken together, DHA may induce the degradation of EGFR through lysosome and ubiquitin/proteosomal system in EGFR WT and EGFR mutant NSCLC cells. Thus, these findings provide important preclinical evidence and molecular insight for utilization of ω3-PUFAs in the chemoprevention and treatment of human NSCLC.[This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932)] Citation Format: Soyeon Jeong, Kaipeng Jing, Soyeon Shin, Soyeon Kim, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Docosahexaenoic acid-induced degradation of epidermal growth factor receptor through lysosome and ubiquitin/proteasomal system in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4651.

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer.

Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype. Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non-small cell lung cancer (NSCLC) cell lines. Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy. This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer.

Cabozantinib (C), erlotinib (E) or the combination (E+C) as second- or third-line therapy in patients with EGFR wild-type (wt) non-small cell lung cancer (NSCLC): A randomized phase 2 trial of the ECOG-ACRIN Cancer Research Group (E1512).

8003 Background: Cabozantinib (C) is a small molecule inhibitor of multiple receptor tyrosine kinases, including MET, VEGFR2 & RET. MET is involved in tumor differentiation & VEGFR2 is a mediator of angiogenesis. Erlotinib (E) is FDA approved for the treatment of NSCLC. Methods: The primary objective of this randomized phase II study was to compare progression-free survival (PFS) of patients (pts) treated with E vs. C, & E vs E+C; each comparison had 91% power to detect a PFS hazard ratio (HR) of 0.5 with a 1-sided 0.10-level test stratified on prior number of therapies & ECOG PS. Secondary objectives included overall survival (OS), RECIST 1.1 response & CTCAE v4 toxicity. Pts were selected with previously treated (1-2 regimens) metastatic non-squamous EGFR wt NSCLC. Submission of archival tissue for central MET IHC testing was required. Oral daily dosing was: E-150 mg; C-60 mg; E+C-150 mg E, 40 mg C. Imaging was performed every 8 weeks. Pts optionally crossed over to E+C following progression on E or C. ...

Abstract 3114: Targeting MET in preclinical models to support the clinical development of Volitinib in NSCLC

Abstract MET is a transmembrane tyrosine kinase receptor that is deregulated (gene amplification, mutation and over-expression) across multiple cancer types. Signaling through MET is normally activated through interactions with its specific ligand, hepatocyte growth factor (HGF). Aberrant MET/HGF activation can stimulate tumor growth, promote angiogenesis, induce metastasis and may contribute to resistance mechanisms in several tumor types. Several non-selective MET inhibitors have entered clinical development; results have been mixed based on potency, selectivity, and/or patient selection. Volitinib is a potent (IC50 4 nM) and selective (>650 fold selectivity over 265 kinases), small molecule inhibitor of MET. Recent evaluation of Volitinib across a panel of cancer cell lines demonstrated selectivity for MET-driven disease, with MET amplified cell lines being most sensitive (IC50s of 1nM) and also suggesting limited off target activity. Volitinib resistant cell lines with MET amplification were identified and used to better understand the relationship of concurrent mutations with response. In addition to cancer cell line selectivity, we are analyzing preclinical models of NSCLC (non small cell lung cancer) that are representative of key patient segments, namely MET amplification and over expression. In newly diagnosed NSCLC adenocarcinomas, focal MET amplification events represent ∼3% of the population while over expression of MET (without gene amplification) is observed in the majority of patients. In preclinical models, focal Met amplification in EBC-1 and NCI-H1993 caused significant tumor growth inhibition, confirming sensitivity to Volitinib. Given the prevalence of over expression in NSCLC, however, we sought to build a platform of evidence for the therapeutic use of Volitinib in preclinical models lacking amplification of the MET gene. Using a patient-derived xenograft model (PDX) of EGFR WT, KRAS WT and metastatic NSCLC disease (HLXF-036LN), we demonstrate that Volitinib induces tumor regression as monotherapy and has added therapeutic benefit when used in combination with taxotere. In addition, we show robust efficacy effects for additional preclinical models, LG0567, LG0645 and Calu-3, for either Volitinib alone or in combination with taxotere. In parallel pharmacodynamic studies, we demonstrate that Volitinib inhibits p-MET and downstream signaling in each model. Together, using an integrated platform of molecular characterization and MET FISH and IHC scores, corresponding antitumor responses to Volitinib are being evaluated for several patient segments. We are using these studies to inform the design of patient selection criteria for upcoming clinical trials in NSCLC. Citation Format: Celina D'Cruz, Melanie Frigault, Ammar Adam, Minhui Shen, Garry Beran, Evan Barry, Paul Gavine, Yongxin Ren, Shiming Fan, Feng Zhou, Weiguo Qing, Mike Zinda, Weiguo Su, Edwin Clark. Targeting MET in preclinical models to support the clinical development of Volitinib in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2014-3114

Abstract B276: A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells.

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumours initially respond well to EGFR targeted monotherapy (e.g. gefitinib), but the benefit is usually limited due to the emergence of drug resistance. A major challenge in the field is to identify the most promising combination approaches that inhibit or prevent this resistance and then to prioritise these options for clinical testing in order to produce more durable patient benefits. Our aim in this study was to identify compounds that selectively and rapidly induced cell death but only when combined with gefitinib in EGFR M+ cells. Methods: We developed a high-throughput 96-well live-cell phenotypic assay (based on an acute apoptosis end point) to assess the combination potential of probe inhibitors targeting kinases (n=402), as well as compounds that are approved or in clinical development for the treatment of cancer (n=326). Screens were carried out using EGFR M+ (PC-9, HCC-827, HCC-2935, H1975) and EGFR WT (A549, H1993) NSCLC cells, and a non-tumor immortalized lung epithelial cell line (BEAS2B). Cells were treated with gefitinib (0-1 µM) and test compounds (0-10 µM) for 18 h. Hoechst 33258 (25 µM) was added and 30 min later cells with condensed nuclei and a high nuclear intensity were scored as apoptotic, and the percentage of apoptotic cells was calculated. SYTOX green DNA staining confirmed that apoptotic cells were scored positive using our simple Hoechst staining method. Results: The screening assay was robust and reproducible with Pearson correlation coefficients ranging from 0.66-0.92 when comparing inter-assay apoptosis scores. Using the compound library we identified 21 positive hits (Z-score≥10) that selectively induced apoptosis in the presence of gefitinib in EGFR M+ cells, but not in EGFR WT cells. Positive hits included compounds in four major target categories; BCL inhibitors (n=3), anti-mitotic agents (n=11), HDAC inhibitors (n=4), and AKT/PI3K inhibitors (n=5). Several of these targets have been suggested in the literature using different methodologies to our own, thereby validating our overall screening approach. Importantly, we have also identified several previously unreported combinations that induced marked apoptosis within 18 h of gefitinib treatment, and which we are currently evaluating in in vivo studies. In addition, we observed that combinations of EGFR TKIs with gefitinib were antagonistic in EGFR M+ cells. Finally, in the probe inhibitor library set, we have identified 20 diverse chemical structures that are strongly positive in our combination assay, suggesting new targets for selective killing of EGFR M+ NSCLC cells. Unexpectedly, screening the chemical probe library also identified 7 compounds that selectively induced cell death in combination with gefitinib in EGFR wild-type KRAS mutant A549 cells. Conclusion: We have developed a robust and reproducible high-throughput live cell apoptosis assay to screen compound libraries in combination with gefitinib in NSCLC cells. We have identified several novel combinations that are highly selective for EGFR M+ cells in the presence of gefitinib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B276. Citation Format: Sivan M. Bokobza, Aoife M. Devery, Anika M. Weber, Anderson J. Ryan. A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B276.

Abstract A263: Combination of quinacrine and erlotinib overcomes resistance to erlotinib in non-small cell lung cancer.

Abstract The epidermal growth factor receptor (EGFR) inhibitor erlotinib is highly effective in non-small cell lung cancer (NSCLC) patients whose tumors express EGFR mutations, e.g., EGFR-L858R or EGFR exon 19 del. However, the efficacy of erlotinib is limited by either primary or secondary resistance, i.e., in patients whose tumors express wild-type EGFR (wtEGFR), or in initially sensitive patients whose tumors develop the second-site mutation EGFR-L858R/T790M or Met amplification after 10-14 months of erlotinib treatment. To improve treatment with erlotinib, we combined it with quinacrine in several erlotinib-resistant NSCLC cells. Quinacrine is a 9-aminoacridine derivative that suppresses nuclear factor-kappa B (NF-κB) by causing chromatin trapping of the FACT (facilitates chromatin transcription) complex. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (Met amplification) cells, the combination of erlotinib and quinacrine at 5 to 1 or 10 to 1 fixed ratios was highly synergistic, as quantified by the Chou-Talalay combination indices [ED50: 0.61 (0.42-0.81); ED75: 0.53 (0.40-0.67); ED90: 0.63 (0.54-0.71)]. Addition of quinacrine to erlotinib treatment inhibited colony formation and induced significant cell cycle arrest and apoptosis in A549 and H1975 cells. Upon quinacrine treatment, the FACT subunit SSRP1 disappeared from the soluble protein fraction, and NF-κB-dependent luciferase activity was decreased. Knockdown of SSRP1 decreased cell viability and colony formation, and sensitized the cells to erlotinib treatment. Moreover, microarray analysis of A549 and H1975 cells treated with erlotinib only, quinacrine only, or both, showed that quinacrine or combination treatment significantly affected genes involved in cell cycle progression or nucleotide metabolism, and many of these genes contain binding sites for transcription factors that regulate SSRP1-enriched genes. These data support a Phase I/II clinical trial (NCT01839955) that is just beginning, combining erlotinib and quinacrine in wtEGFR NSCLC patients after first-line chemotherapy. To assess synergistic effects in this clinical trial, we identified genes from the microarray analysis that were more significantly suppressed by combination treatment than by either single treatment. We further narrowed them down to those whose increased expression is associated with poorer survival in lung adenocarcinoma patients (HR ranges from 1.58-2.92) as potential biomarkers of drug synergy for the clinical trial. These genes, most of which are involved in various cell cycle processes, included BIRC5, DLGAP5, FOSL1, FOXM1, HIST1H2AK, HIST1H2BM, KIFC1, MKI67, PBK, PLK1, TOP2A, and ZWILCH. We conclude that the combination of quinacrine and erlotinib helps overcome erlotinib resistance in NSCLC, potentially by targeting FACT and suppressing cell cycle progression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A263. Citation Format: Josephine Kam Tai Dermawan, Katerina Gurova, John Pink, Afshin Dowlati, Sarmishtha De, Goutham Narla, Neelesh Sharma, George Stark. Combination of quinacrine and erlotinib overcomes resistance to erlotinib in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A263.