Epidermal Growth Factor Receptor TKI Resistance Research Articles

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.

Treatment Patterns and Adverse Event-Related Hospitalization Among Patients with Epidermal Growth Factor Receptor (EGFR)-Mutated Metastatic Non-small Cell Lung Cancer After Treatment with EGFR Tyrosine Kinase Inhibitor and Platinum-Based Chemotherapy Regimens.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR TKIs) are established first-line treatments among patients with metastatic non-small cell lung cancer harboring EGFR-sensitizing mutations. Upon EGFR TKI resistance, there are scant data supporting a standard of care in subsequent lines of therapy. We aimed to characterize real-world treatment patterns and adverse events associated with hospitalization in later lines of therapy. This retrospective analysis of administrative claims included adults with metastatic non-small cell lung cancer who initiated a next line of therapy (index line of therapy) following EGFR TKI and platinum-based chemotherapy discontinuation on/after 1 November, 2015. Treatment regimens and adverse event rates during the index line of therapy were described. Among 195 eligible patients (median age: 59 years; female: 60%), the five most common index line of therapy regimens were immune checkpoint inhibitor monotherapy (29%), EGFR TKI monotherapy (21%), platinum-based chemotherapy (19%), non-platinum-chemotherapy (13%), and EGFR TKI combinations (9%). The overall median (95% confidence interval) time to discontinuation of the index line of therapy was 2.8 (2.1-3.2) months. Common adverse events associated with hospitalizations included infection/sepsis, pneumonia/pneumonitis, and anemia (2.9, 2.8, and 2.0 per 100 person-months, respectively). Among EGFR TKI-resistant patients who discontinued platinum-based chemotherapy, the duration of the next line of therapy was short, treatment was highly variable, and re-treatment with EGFR TKIs and platinum-based regimens was common, suggesting a lack of standard of care in later lines. Adverse event rates associated with hospitalization were high, especially among platinum-treated patients. These results underscore the unmet need for new therapies in a later line of treatment to reduce the clinical burden among patients in this population.

Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells.

Epithelial-mesenchymal-transition (EMT) is an epigenetic-based mechanism contributing to the acquired treatment resistance against receptor tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR)-mutations. Delineating the exact epigenetic and gene-expression alterations in EMT-associated EGFR TKI-resistance (EMT-E-TKI-R) is vital for improved diagnosis and treatment of NSCLC patients. We characterized genome-wide changes in mRNA-expression, DNA-methylation and the histone-modification H3K36me3 in EGFR-mutated NSCLC HCC827 cells in result of acquired EMT-E-TKI-R. CRISPR/Cas9 was used to functional examine key findings from the omics analyses. Acquired EMT-E-TKI-R was analyzed with three omics approaches. RNA-sequencing identified 2,233 and 1,972 up- and down-regulated genes, respectively, and among these were established EMT-markers. DNA-methylation EPIC array analyses identified 14,163 and 7,999 hyper- and hypo-methylated, respectively, differential methylated positions of which several were present in EMT-markers. Finally, H3K36me3 chromatin immunoprecipitation (ChIP)-sequencing detected 2,873 and 3,836 genes with enrichment and depletion, respectively, and among these were established EMT-markers. Correlation analyses showed that EMT-E-TKI-R mRNA-expression changes correlated better with H3K36me3 changes than with DNA-methylation changes. Moreover, the omics data supported the involvement of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 signaling axis for acquired EMT-E-TKI-R. CRISPR/Cas9-mediated analyses corroborated the importance of ZEB1 in acquired EMT-E-TKI-R, MIR200C and MIR141 to be in an EMT-E-TKI-R-associated auto-regulatory loop with ZEB1, and FGFR1 to mediate cell survival in EMT-E-TKI-R. The current study describes the synchronous genome-wide changes in mRNA-expression, DNA-methylation, and H3K36me3 in NSCLC EMT-E-TKI-R. The omics approaches revealed potential novel diagnostic markers and treatment targets. Besides, the study consolidates the functional impact of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1-signaling axis in NSCLC EMT-E-TKI-R.

Impact of KEAP1/NFE2L2/CUL3 mutations on duration of response to EGFR tyrosine kinase inhibitors in EGFR mutated non-small cell lung cancer

ObjectivesFor patients with Epidermal Growth Factor Receptor (EGFR)-mutated non-small cell lung cancer (NSCLC), frontline EGFR-tyrosine kinase inhibitor (TKI) therapy compared to chemotherapy improves outcomes. However, resistance to these agents uniformly develops. Recently, mutations in the KEAP1-NFE2L2 pathway have been implicated as a potential mechanism of acquired EGFR TKI resistance. Materials and methodsWe examined all patients with metastatic NSCLC with mutations in both EGFR and KEAP1/NFE2L2/CUL3 identified on next generation sequencing from 2015 - 2018. These patients were compared to a NSCLC control cohort with mutations in EGFR and wild type in KEAP1/NFE2L2/CUL3 matched on the basis of sex, smoking status, age and race. Time to treatment failure on EGFR TKI therapy and overall survival were examined. ResultsAmong 228 EGFR mutant NSCLCs, 17 (7%) also carried mutations in KEAP1, NFE2L2, or CUL3. The most common co-mutation in both the KEAP1/NFE2L2/CUL3 mutant and wild-type cohort was TP53. Patients with KEAP1/NFE2L2/CUL3 mutations had a shorter median time to treatment failure on EGFR TKI (4.7 months) compared with the wild-type matched cohort (13.0 months), p= 0.0014. There was no difference in overall survival. ConclusionFor NSCLC patients with mutations in EGFR, co-mutations in KEAP1/NFE2L2/CUL3 are associated with significantly decreased time to treatment failure. Our results suggest that these mutations represent a mechanism of intrinsic resistance to TKI treatment.

Long non-coding RNA LOC554202 promotes acquired gefitinib resistance in non-small cell lung cancer through upregulating miR-31 expression.

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation inevitably have a relapse due to the occurrence of acquired resistance, resulting in treatment failure. However, little is known about the mechanisms of acquired resistance of NSCLC patients. Here, we elucidated the expression pattern of LOC554202 and miR-31, and their biological functions and mechanisms in NSCLC with acquired EGFR TKI resistance to gefitinib. In the present study, we observed that LOC554202 and miR-31 promoted proliferation and clonogenic growth of gefitinib-resistant NSCLC cells in vitro. LOC554202 upregulated miR-31 expression and they both reduced sensitivity of NSCLC cells to gefitinib. In a xenograft mice model, we found that knockdown of miR-31 significantly repressed gefitinib-resistant NSCLC cells growth in vivo. Furthermore, both LOC554202 and miR-31 levels were significantly increased in NSCLC patients acquiring resistance to gefitinib, and the expression of LOC554202 was positively correlated with the expression of miR-31. By luciferase reporter assays, we identified RAS P21 Protein Activator 1 (RASA1) and Hypoxia Inducible Factor 1 Subunit Alpha Inhibitor (FIH-1) as direct targets of miR-31 in NSCLC cells. Mechanistically, miR-31 directly repressed RASA1 and FIH-1 expression, and thus, at least partially activated the RAF-MEK-ERK and PI3K-AKT signaling pathways in NSCLC with acquired resistance to gefitinib. In conclusion, these data will help us develop potential therapeutic targets for the diagnosis and treatment of acquired EGFR TKI resistance in EGFR-mutant NSCLC.

Effects and mechanisms of betulinic acid on improving EGFR TKI-resistance of lung cancer cells.

Epidermal growth factor receptor (EGFR) mutations have been identified in approximately 55% of lung cancer patients in Taiwan. Gefitinib (Iressa) and Erlotinib (Tarceva) are the first-generation targeting drugs to patients with EGFR gene mutants a work by inhibiting tyrosine kinase activity. However, resistance in EGFR-mutated patients to first-generation tyrosine kinase inhibitor (TKI) therapy after 8-11 months of treatment has occurred. Betulinic acid (BetA) is a pentacyclic triterpenoid natural product derived from widespread plants. BetA has been reported to have a cytotoxic effect in several cancers. The purpose of this study is to investigate the effects and mechanisms of BetA on dampening EGFR TKI-resistance of lung cancer cells. Our study has demonstrated by MTT assay that combining BetA and an EGFR TKI increased the cytotoxicity against EGFR TKI-resistance lung cancer cells. Based on flow cytometry, combination treatments of BetA with an EGFR TKI enhanced Sub-G1 accumulation, induced apoptosis and induced mitochondrial membrane potential loss. Using western blotting, BetA and EGFR TKI combined treatments inhibited cell cycle related protein and triggered apoptosis- and autophagy- related protein expression. Taken together, our data suggests that a target therapy combining BetA with an EGFR TKI improves drug efficacy in EGFR TKI-resistant lung cancer cells.

Abstract LB-196: EGFR-mutant lung adenocarcinomas mutation profiles reveal ARID1A might be a novel tyrosine kinase resistance pathway

Abstract Lung cancer has the highest mortality across all cancers in the world. Epidermal growth factor receptor (EGFR) is commonly mutated in lung adenocarcinomas from Asian non-smoking females. These tumors usually respond to EGFR tyrosine kinase inhibitors (EGFR-TKI), but the outcomes vary in different patients. Though EGFR T790M, amplification of MET and other mechanisms have been shown to confer EGFR-TKI resistance, the molecular mechanisms in around 30% of patients are still unknown. To uncover novel candidate pathways of EGFR-TKI resistance, we analyzed the whole exome mutation profiles of pre-treatment, EGFR T790M-free lung adenocarcinomas with typical activating mutations (L858R, exon 19 deletions). Amongst 19 non-responding (NR) tumors without significant shrinkage after gefitinib or erlotinib treatment, 67 cancer-related genes showed coding region non-synonymous single nucleotide variations (SNV) or insertion-deletions (indels) but they were not detected in 22 responding tumors. Gene set enrichment analysis (GSEA) showed 14 of the 67 genes were involved in the EGFR and WNT signaling network (false discovery rate q=1.14 e-8 for EGFR and q=4.3 e-9 for WNT), including well-known TKI resistance-related genes PIK3CA, PTEN and NF1. Notably, ARID1A, which encodes a subunit of the chromatin remodeling complex, showed nonsense mutations in 2 NR but not responding tumors, which were validated by Sanger sequencing. More than 40% of ARID1A mutations in lung adenocarcinomas recorded in COSMIC were nonsense mutations or frameshift indels. Loss of function mutations of ARID1A have also been reported in various cancers, together suggesting ARID1A might be a tumor suppressor but its involvement in EGFR mutant lung cancers is unknown. To investigate the potential role of ARID1A, its expression was first studied using Kaplan-Meier plotter. Low expression of ARID1A is associated with significantly better overall survival than high expression level in lung adenocarcinoma patients (p=8.5 e-7). To test whether loss of ARID1A could cause TKI resistance, ARID1A was knocked down by shRNA in HCC827 lung cancer cell line harboring EGFR exon19 deletion. MTT cell viability assay using gefitinib showed increase of IC50. Western blot assay further showed ARID1A knockdown increased phosphorylation of AKT at Ser473. Together, the findings suggest loss of ARID1A could lead to EGFR bypass and promote activation of the downstream PI3K/AKT pathway, leading to EGFR TKI resistance. Overall, our findings suggest EGFR-TKI resistance mutations could be detected before TKI drug selection. Resistant gene mutations present in pre-treatment tumors could help to predict the treatment response. More specifically, our study identified a promising gene list involved in EGFR-TKI resistance, and suggested ARID1A loss could be a novel EGFR-TKI resistance mechanism. Citation Format: Xuyuan Gao, Hang Xu, James CM Ho, Oscar SH Chan, Feng Xu, Junwen Wang, Victor HF Lee, Vicky PC Tin, Zhijie Xiao, Siqi Wang, Judy WP Yam, Maria P Wong. EGFR-mutant lung adenocarcinomas mutation profiles reveal ARID1A might be a novel tyrosine kinase resistance pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-196.

Tumor-Targeted Nanoparticles Deliver a Vitamin D-Based Drug Payload for the Treatment of EGFR Tyrosine Kinase Inhibitor-Resistant Lung Cancer.

Mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor ( EGFR) gene drives the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR TKIs), including erlotinib and afatinib, are initially effective in treating EGFR mutant nonsmall cell lung cancer (NSCLC). However, drug resistance quickly develops due to several mechanisms, including induction of the epithelial-mesenchymal transition (EMT). No effective therapies are currently available for patients who develop EMT-associated EGFR TKI resistance. 1,25-Dihydroxyvitamin D3 (1,25D3) promotes epithelial differentiation and inhibits growth of NSCLC cells. 1,25D3 thus represents a promising agent for the treatment of EMT-associated EGFR TKI resistance. However, 1,25D3 induces the expression of 24-hydroxylase (24OHase), which decreases 1,25D3 activity. CTA091, a potent and selective 24OHase inhibitor, has been developed to attenuate this adverse effect. CTA091 also suppresses renal 24OHase activity and so may promote hypercalcemia. To exploit favorable effects of 1,25D3 plus CTA091 in tumor cells while avoiding problematic systemic effects of 24OHase inhibition, we developed EGFR-targeted, liposomal nanoparticles (EGFR-LP) to offer tumor-targeted co-delivery of 1,25D3 and CTA091. We then established an EMT-associated model of EGFR TKI resistance, and showed that such nanoparticles improved cellular uptake of 1,25D3 and CTA091, drove pro-epithelial signaling by upregulating E-cadherin ( CDH1), and significantly inhibited the growth of EGFR TKI resistant cells. Our results demonstrated that the delivery of vitamin D-based drug payloads via tumor-targeted EGFR-LP has promise as a new therapy for EFGR TKI resistant lung cancer. Future studies will focus on in vivo evaluation of biological activity, therapeutic benefits, and systemic toxicity prior to clinical translation.

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR–tyrosine kinase inhibitors (EGFR–TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR–TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure–activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

Transcriptome analysis of EGFR tyrosine kinase inhibitors resistance associated long noncoding RNA in non-small cell lung cancer.

The non-small cell lung cancer (NSCLC) patients harbor mutations in the epidermal growth factor receptor (EGFR) can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib, and show improved progression-free survival. However, most of the patients who are initially responsive to EGFR TKIs with activating EGFR mutations eventually develop acquired resistance after long-term therapy, and are followed by disease progression. Recently, diverse mechanisms of acquired EGFR TKI resistance have been reported, but little is known about the role of long noncoding RNAs in EGFR TKIs resistance. To gain insight into the expression pattern and potential function of lncRNAs in NSCLC cells EGFR-TKI resistance, we analyzed expression patterns in EGFR-TKIs-resistant cell lines and compared it with their parental sensitive cell line by using gene profiling datasets from Gene Expression Omnibus (GEO). Then, the expression levels of five chose lncRNAs were validated in PC9-gefitinib resistant cells (PC9G) and sensitive cells by using real-time quantitative PCR (qPCR). Among of these five lncRNAs, CASC9 expression was upregulated in PC9G and knockdown of its expression could increase the sensitivity of PC9G cells to gefitinib, while EWAST1 (LINC00227) is downregulated in PC9G cells and overexpressed EWAST1 also lead to increased sensitivity of PC9G cells to gefitinib. As indicated by GO analysis, the CASC9 and EWAST1 co-expressed genes are involved in several important pathways including regulation of cell growth, regulation of cell apoptosis and Chromatin assembly. Taken together, dysregulated lncRNAs play critical roles in EGFR-TKIs resistant NSCLC cells and might be used as novel potential targets to reverse EGFR-TKI resistance for NSCLC patients.

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR.In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells.

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.

Abstract IA36: Treatment strategies to overcome EGFR TKI resistance.

Abstract IA36: Treatment strategies to overcome EGFR TKI resistance.

Abstract 2450: Either pan class I PI3K inhibitor or PI3K alpha selective inhibitor sensitizes head and neck squamous cell carcinoma cells to Erlotinib, an EGFR TKI.

Abstract The epidermal growth factor receptor (EGFR) is overexpressed in 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. EGFR is therefore a potential therapeutic target for HNSCC. However, most HNSCC patients do not respond well to small molecular EGFR inhibitors. One of the possible mechanisms of EGFR TKI resistance is compensatory collateral activation of PI3K/Akt pathway. This study sought to explore whether NVP-BKM120, a selective pan class I PI3 kinase inhibitor, and NVP-BYL719, a novel PI3 kinase alpha selective inhibitor, may reverse the resistance of HNSCC cells toward erlotinib, an EGFR TKI. Cells were treated with the PI3K inhibitors, either BKM120 or BYL719, alone, erlotinib alone, or in combination, and viability was measured at 72 hours with MTT assay. Combination effect was evaluated by median effect analysis. Synergism, indicated by a combination index (CI) value <1, were seen in all five cell lines (SAS, HSC3, UMCC1, CA922 and FaDu) studied. By immunoblotting analysis, combination of Erlotinib and either PI3K inhibitor resulted in dowregulation of p-EGFR and p-AKT expression in SAS cells. When SAS and HSC3 cells were treated with erlotinib in combination with either BKM120 or BYL719, an additional increase in the subG1 population by flow cytometry analysis were observed, indicating an increase of apoptotic cell death. Our data suggest that additional PI3K blockade might be an effective strategy to overcome resistance of HNSCC cells toward EGFR TKIs, and the beneficial effect is similar between pan Class I PI3K inhibitor and PI3K alpha selective inhibitor. (The study was supported by the grant of DOH101-TD-B-111-001). Citation Format: Ming Gao, Yen-Shen Lu, Li-Ping Hsiao, Suz Wen Chen, Ann-Lii Cheng. Either pan class I PI3K inhibitor or PI3K alpha selective inhibitor sensitizes head and neck squamous cell carcinoma cells to Erlotinib, an EGFR TKI. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2013-2450

A dramatic lung cancer course in a patient with a rare EGFR germline mutation exon 21 V843I: Is EGFR TKI resistance predictable?

We report on the medical history of a Caucasian smoker woman diagnosed with a stage IV NSCLC adenocarcinoma, characterized by a rare epidermal growth factor receptor (EGFR) point mutation in exon 21 codon 843 (p.V843I/c.2527G>A/COSMIC ID 85894). This genetic alteration revealed to be germline, after its presence was demonstrated in chondroblasts from the bone biopsy. While it is the first description of germline V843I mutation without concomitant additional known EGFR activating mutation, we modeled the EGFR ATP catalytic domain in complex with ATP, gefitinib and erlotinib using computer-aided approaches to estimate possible changes in affinity upon the V843I mutation.

Mechanisms of Resistance to Epidermal Growth Factor Receptor Inhibitors and Novel Therapeutic Strategies to Overcome Resistance in NSCLC Patients

The epidermal growth factor receptor (EGFR) is a well-characterized oncogene that is frequently activated by somatic kinase domain mutations in non-small cell lung cancer (NSCLC). EGFR TKIs are effective therapies for NSCLC patients whose tumors harbor an EGFR activating mutation. However, EGFR TKI treatment is not curative in patients because of both primary and secondary treatment resistance. Studies over the last decade have identified mechanisms that drive primary and secondary resistance to EGFR TKI treatment. The elucidation of mechanisms of resistance to EGFR TKI treatment provides a basis for the development of therapeutic strategies to overcome resistance and enhance outcomes in NSCLC patients. In this paper, we summarize the mechanisms of resistance to EGFR TKIs that have been identified to date and discusses potential therapeutic strategies to overcome EGFR TKI resistance in NSCLC patients.

A targeted enzyme approach to sensitization of tyrosine kinase inhibitor-resistant breast cancer cells

Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of potential use in patients with breast cancer. Unfortunately, in clinical studies, gefitinib is often ineffective indicating that resistance to EGFR inhibitors may be a common occurrence in cancer of the breast. EGFR has been shown to be overexpressed in breast cancer, and in particular remains hyperphosphorylated in cell lines such as MDA-MB-468 that are resistant to EGFR inhibitors. Here, we investigate the cause of this sustained phosphorylation and the molecular basis for the ineffectiveness of gefitinib. We show that reactive oxygen species (ROS), known to damage cellular macromolecules and to modulate signaling cascades in a variety of human diseases including cancers, appear to play a critical role in mediating EGFR TKI-resistance. Furthermore, elimination of these ROS through use of a cell-penetrating catalase derivative sensitizes the cells to gefitinib. These results suggest a new approach for the treatment of TKI-resistant breast cancer patients specifically, the targeting of ROS and attendant downstream oxidative stress and their effects on signaling cascades.

Randomized phase II trial of erlotinib (E) plus high-dose celecoxib (HD-C) or placebo (P) in advanced non-small cell lung cancer.

7518 Background: Cyclooxygenase-2 (COX-2)-dependent signalling represents a potential mechanism of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy in NSCLC. This is mediated in part through an EGFR-independent activation of MAPK/Erk by the COX-2 metabolite PGE2. In addition, PGE2 promotes downregulation of E-cadherin and epithelial to mesenchymal transition. We hypothesize that EGFR and COX-2 inhibition with E and HD-C will augment EGFR TKI efficacy by increasing tumor E-cadherin expression and blocking PGE2-mediated resistance to EGFR inhibition. Methods: Pts with stage IIIB/IV NSCLC who progressed following at least one line of therapy or refused standard chemotherapy were randomized to E (150mg/day)/HD-C (600mg/2x day) vs E/P in a 28-day cycle. Pts were stratified by smoking status and ECOG PS. The primary endpoint was PFS with 80% power to detect a 50% improvement; assessments were performed every 2 cycles. Secondary endpoints included response rate, OS and evaluation of molecular markers in tissue and serum to assess targeting COX-2-related pathways and evaluate EGFR TKI-resistance. All pts were required to have pre-treatment tissue available. Results: 107 pts were enrolled with comparable baseline characteristics in both arms. Disease control rate (DCR) was similar in both arms, and a trend toward improved PFS was seen in the E/HD-C arm with HR 0.81 (see Table). Analysis of those with EGFR wild type revealed a significantly increased PFS while those with EGFR mutation had similar PFS in both groups. Safety analysis showed similar toxicity in both arms. Additional biomarker correlations will be presented. Conclusions: The combination of E/C in metastatic NSCLC with HD-C is well tolerated and demonstrates significantly improved efficacy in EGFR wt population. This warrants further study into the COX-2-dependent mechanisms of primary resistance to EGFR TKI therapy. Supported by NIH 1P50 CA90388, K12 CA01727 and medical research funds from the Dept of Veterans’ Affairs. [Table: see text]

Circulating tumor cells and detection of EGFR mutation in cell-free tumor DNA in blood plasma in metastatic non-small cell lung cancer with EGFR mutation.

7595 Background: The purpose of this study was to assess the correlation between the numbers of circulating tumor cells (CTCs), prognosis and the positivity of blood plasma EGFR mutation in cell-free tumor DNA in metastatic non-small cell lung cancer with positive epidermal growth factor receptor (EGFR) gene mutation in the primary site. Methods: Of the 20 patients with primary lung cancer who showed EGFR mutaion in the primary site, CTCs were counted using CellSearch system (Veridex) after the appearance of EGFR TKI resistance. Furthermore, cell-free tumor DNA was extracted from blood plasma samples of the 20 patients by QIAamp Circulating Nucleic Acid Kit (Qiagen), and was analyzed for EGFR mutation using cycleave real-time PCR assay. Results: Patient characteristics included 5 males and 15 females with a mean age of 64.1 yrs (43 to 82), performance status of 0 in 5, 1 in 11, and 2 in 4, respectively. Pathological classification of lung cancer were adenocarcinoma in 19, squamous cell carcinoma in 1, clinical stage were stage IV in 15 and recurrence after surgical resection in 5 with distant metastases, respectively. The localization of EGFR mutation in the primary site were exon 18 G719C in 1, exon 18 G725A + exon 19 deletion in 2, exon 19 deletion in 7, exon21 L858R in 9, exon 18 G791S + exon 21 L858R in 1→ exon 18 G719S + exon 21 L858R in 1, respectively. CTCs were detected in 7 out of 20 cases (35%). Numbers of CTCs ( per 7.5 ml) were 1 in 3 cases, 3 in 1 case, 8 in 1 case, 14 in 1 case, and 24 in 1 case, respectively. Negative CTCs group showed a significant longer survival than that of positive CTCs group (³a1 / 7.5 ml) from the times of detecting CTCs (MST: not reached vs. 3.8 months, p < 0.05). EGFR mutation in tumor cell-free DNA in blood plasma were detected in 6 out of 20 cases (30%). The lacalization of detected EGFR mutation were same as their primary site, without any new resistance mutation. Positive detective rates of EGFR mutation in blood plasma were 71.1% (5 out of 7 cases) in positive CTCs group and 7.7% (1 out of 13 cases) in negative CTCs group (p < 0.05). Conclusions: The numbers of CTCs were correlated not only prognosis but also the positivity of EGFR mutation in cell-free tumor DNA in blood plasma.

Abstract 278: Regulation of constitutive Met phosphorylation in breast cancer by EGFR

Abstract Breast cancer remains a major health problem, despite advances in detection, diagnosis, and treatment. Four genomic subtypes of breast cancer have been identified: luminal A and B, HER2 positive, and basal type breast cancers. Three of these subtypes help to define the proper targeted therapeutic as breast cancers of luminal subtypes tend to be estrogen receptor positive and treatable with tamoxifen while breast cancers of HER2 positive subtype are treatable with trastuzumab. The fourth subtype, the basal type breast cancers, has a poor prognosis and has yet to have a molecular target identified. One molecule highly expressed in basal type breast cancers is epidermal growth factor receptor (EGFR). However, targeting EGFR kinase activity with small molecule kinase inhibitors has yet to show efficacy in the treatment of breast cancers. Recently, a panel of EGFR expressing breast cancer cell lines was characterized as sensitive or resistant to EGFR kinase inhibitors. One mechanism of resistance to EGFR kinase inhibitors was found to be the constitutive activation of Met, another molecule associated with basal type breast cancers. Here, the mechanism of constitutive Met activation in EGFR tyrosine kinase inhibitor resistant breast cancer cells is explored. Previously, constitutive Met activation was found to associate with acquired EGFR inhibitor resistance in lung cancer via genetic amplification of Met. In the EGFR TKI resistant breast cancer cells with constitutive Met phosphorylation, Met was not amplified or significantly overexpressed. In addition, constitutive Met phosphorylation was found to occur independent of ligand (hepatocyte growth factor (HGF)) stimulation, as HGF was not expressed in the breast cancer cell lines and inhibiting HGF association with Met via an inhibitory antibody failed to reduce Met phosphorylation. These data suggest a ligand-independent mechanism for Met constitutive phosphorylation. Interestingly, ligand-independent Met activation can be mediated by Met association with other membrane molecules including integrins, tetraspanins and receptor tyrosine kinases, including EGFR. Co-immunoprecipitation studies demonstrated that EGFR and Met co-associate and that this association occurs independent of EGFR or Met kinase activities. In addition, Met phosphorylation was reduced with EGFR TKIs as well as with downregulation of EGFR expression. Taken together, these data suggest that one potential mechanism of EGFR TKI resistance in breast cancer cells is the association and transphosphorylation of Met by EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 278.