Oncogenic Epidermal Growth Factor Receptor Mutation Research Articles

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

The budget impact of introducing mobocertinib for the postplatinum treatment of advanced non-small cell lung cancer harboring epidermal growth factor receptor exon 20 insertion mutations.

BACKGROUND: Lung cancer is a leading cause of cancer morbidity and death in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, and oncogenic mutations in the gene encoding the epidermal growth factor receptor (EGFR) are among its most common genetic causes. Although NSCLC tumors harboring more common oncogenic EGFR mutations can be effectively treated with EGFR tyrosine kinase inhibitors (TKIs), those harboring EGFR exon 20 insertion mutations respond poorly to treatment with therapies approved for advanced NSCLC, including TKIs. Mobocertinib, a first-in-class potent, oral, irreversible TKI, is effective in this population. OBJECTIVE: To estimate the budget impact, for a US health plan with 10 million members, of introducing mobocertinib for the treatment of patients with locally advanced or metastatic NSCLC harboring EGFR exon 20 insertion mutations who have been previously treated with platinum-based chemotherapy. METHODS: A budget impact model was developed to compare 2 scenarios: a reference scenario in which 50% of patients received amivantamab and 50% received physician's choice/usual care therapy and an alternative scenario in which mobocertinib replaced the physician's choice/usual care option. The model had a 5-year time horizon in the base case. The model included epidemiologic inputs to estimate the size of the treatment-eligible population; clinical inputs to estimate treatment duration and efficacy, as well as adverse event frequency; and cost inputs for treatment acquisition and administration, management of adverse events, monitoring, and terminal care. The duration and cost of subsequent therapies were also considered. Budget impact was reported as a total cost, as per-member per-year costs, and as per-member per-month (PMPM) costs. To assess the robustness of model estimates and identify cost drivers, one-way sensitivity analyses and a range of scenario analyses were conducted. RESULTS: The model estimated an eligible treatment population of 55 patients (11 per year) over a 5-year time horizon. In the base case, the estimated budget impact of introducing mobocertinib was $5,615,808, or $0.01 PMPM. Model findings were robust to one-way sensitivity analyses and a range of sensitivity analyses; none of these analyses led to a PMPM budget impact of more than $0.06. Cost drivers included the percentage of eligible patients, the median duration of physician's choice/usual care therapy, patient weight, and the percentage of patients who undergo molecular testing. CONCLUSIONS: The estimated budget impact of mobocertinib is low, primarily because NSCLC harboring EGFR exon 20 insertion mutations is rare. DISCLOSURES: Dr Hernandez is an employee of Takeda Pharmaceuticals America, Inc. Dr Young was an employee of Takeda Pharmaceuticals America, Inc., at the time this study was conducted. This study and the editorial assistance were funded by Takeda Pharmaceuticals America, Inc.

Multiscale in silico knowledge-based model predicts tumor growth in advanced EGFR+ NSCLC patients on gefitinib.

e20565 Background: Tumor heterogeneity and treatment resistance remains a continued threat to patients with advanced non-small cell lung cancer (NSCLC), despite the emergence of targeted medicines, e.g. against epidermal growth factor receptor (EGFR) alterations. We developed an in silico EGFR mutated lung adenocarcinoma (EGFR+ LUAD) model to predict the effect of known oncogenic EGFR mutations (common EGFR mutations, EGFR exon 20 insertions). The model provides mechanistic representation of tumor progression, including response to gefitinib and captures tumor heterogeneity, patient age, gender, initial clinical disease stage, and smoking status. Methods: 5-step in silico model development: Model building: biology of EGFR+LUAD was characterized by extracting biological features and their functional relationships from > 300 published papers and translating them into ordinary differential equations (ODEs). Mutational burden, EGFR-downstream-pathways, tumor growth and heterogeneity, gefitinib-PK/PD, treatment-induced resistance and clinical outcome were incorporated into a knowledge-based model containing 27-97 variables, 108-258 parameters and 13-83 ODEs reflecting intra-tumor clonal heterogeneity in a mechanistic manner. Calibration: published spheroid, xenograft and clinical data were used for stepwise calibration to find the correct parameter values. Relevant virtual populations (VPOPs) matching real patients baseline characteristics were generated for model benchmarking and validation. Benchmarking against a published data-based model: coverage of experimental interquartile range (IQR) with simulated IQR (precision) assesses model fit with experimental data, coverage of simulated IQR with experimental IQR (overlap) assesses model fit with experimental variability. Validation: a VPOP with comparable baseline characteristics was simulated and results compared against a published patient dataset that wasn’t used in any step during calibration. Matching of prediction over clinical data was done using both coverage and bootstrapped log-rank metrics. Results: Our model provides comparable outputs to the data-based model without having access to the exact original data (our model: precision of 62%, overlap of 91% VS data-based model: precision of 72% and an overlap of 86%). Besides, as a validation criterion, our model also successfully reproduces the time to progression observed in an independent clinical trial (coverage > 99%, negative log-rank tests > 98%). Conclusions: We simulated tumor growth and treatment response in advanced EGFR+ LUAD patients and successfully validated results both against an existing data-based model and a published clinical data. Our model highlights the potential of in silico modeling to better understand complex diseases progression and support efficient development of innovative therapies.

A molecular mechanism for the generation of ligand-dependent differential outputs by the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

Impact of concurrent genomic alterations in epidermal growth factor receptor (EGFR)-mutated lung cancer.

Comprehensive characterization of the genomic landscape of epidermal growth factor receptor (EGFR)-mutated lung cancers have identified patterns of secondary mutations beyond the primary oncogenic EGFR mutation. These include concurrent pathogenic alterations affecting p53 (60–65%), RTKs (5–10%), PIK3CA/KRAS (3–23%), Wnt (5–10%), and cell cycle (7–25%) pathways as well as transcription factors such as MYC and NKX2-1 (10–15%). The majority of these co-occurring alterations were detected or enriched in samples collected from patients at resistance to tyrosine kinase inhibitor (TKI) treatment, indicating a potential functional role in driving resistance to therapy. Of note, these co-occurring tumor genomic alterations are not necessarily mutually exclusive, and evidence suggests that multiple clonal and sub-clonal cancer cell populations can co-exist and contribute to EGFR TKI resistance. Computational tools aimed to classify, track and predict the evolution of cancer clonal populations during therapy are being investigated in pre-clinical models to guide the selection of combination therapy switching strategies that may delay the development of treatment resistance. Here we review the most frequently identified tumor genomic alterations that co-occur with mutated EGFR and the evidence that these alterations effect responsiveness to EGFR TKI treatment.

Abstract 858: Oncogenic EGFR mutations as genomic biomarkers for cetuximab and panitumumab response in colorectal adenocarcinoma

Abstract Somatic mutations of Epidermal Growth Factor Receptor (EGFR) occur in ~3% of colorectal cancer patients. Here, through systematic functional screening of 21 recurrent EGFR mutations selected from public datasets, we show that 11 colon cancer-derived EGFR mutants; G63R, E114K, R165Q, R222C, S492R, P596L, K708R, E709K, G719S, G724S and L858R, are oncogenic, and able to transform cells in a ligand-independent manner. We demonstrate that cellular transformation by these mutants requires receptor dimerization. Importantly, the EGF-induced and constitutive oncogenic potential of these EGFR mutants are inhibited by cetuximab or panitumumab in vivo and in vitro. Taken together, we propose that a subset of EGFR mutations can serve as genomic predictors for response to anti-EGFR antibodies and that metastatic CRC patients with such mutations may benefit from these drugs as part of the first-line therapy. Citation Format: Nayoung Kim, Jeonghee Cho. Oncogenic EGFR mutations as genomic biomarkers for cetuximab and panitumumab response in colorectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 858.

Abstract 861: Precise excision of an oncogenic allele of tumor by Adenovirus-based CRISPR/Cas9 system induces tumor regression

Abstract Approximately 15% of non-small cell lung cancer patients possesses several mutations in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. To this end, we demonstrate that an adenovirus-mediated co-delivery of Cas9 and a guide RNA targeting oncogenic EGFR mutation commonly found in lung cancer can selectively remove oncogenic allele while being inactive against wild-type EGFR allele. An EGFR mutant harboring a single-nucleotide missense mutation (CTG to CGG) was targeted as this mutation leads to generation of protospacer-adjacent motif sequence recognized by the Cas9 protein of S. pyogenes. The CRISPR/Cas9-mediated excision of mutant EGFR allele with high indel rate led to potent lung cancer cell killing. Importantly, intratumoral administration of Ad-based CRISPR/Cas9 system led to potent tumor growth inhibition and complete tumor regressions in number of mice harboring EGFR mutant xenograft tumors. Collectively, our findings show that precise and controlled excision of oncogenic mutation by Ad-mediated expression of CRISPR/Cas9 system offers a powerful genomic surgical strategy to excise oncogenic mutations to treat cancers. Note: This abstract was not presented at the meeting. Citation Format: Taeyoung Koo, A-Rum Yoon, Sangsu Bae, Jin-Soo Kim, Chae-Ok Yun. Precise excision of an oncogenic allele of tumor by Adenovirus-based CRISPR/Cas9 system induces tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 861.

Abstract 362: Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma

Abstract Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated Interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-β (TGF-β) signaling play crucial regulatory roles in cancer-associated inflammation. Here we show that Stat3 represses Smad3 in cooperation with c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827 cells resistant. IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance. By contrast, TGF-β upregulated gefitinib sensitivity. We found that IL-6 signaling via phosphorylated Stat3 repressed, whereas TGF-β upregulated the expression of Smad3 in HCC827 cells. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad-induced transcription of the Smad3 gene. Smad3 induced apoptosis by upregulating pro-apoptotic genes such as Caspase 3 and downregulating anti-apoptotic genes such as Bcl2. Our results suggest that preventing IL-6/Stat3-induced loss of Smad3 can be a therapeutic strategy to prevent gefitinib resistance in NSCLCs with gefitinib-sensitive EGFR mutation. Citation Format: Yojiro Makino, Jeong-Hwan Yoon, Eunjin Bae, Mitsuyasu Kato, Keiji Miyazawa, Tatsuo Ohira, Norihiko Ikeda, Masahiko Kuroda, Mizuko Mamura. Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma [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 362. doi:10.1158/1538-7445.AM2017-362

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma

Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-β (TGF-β) signaling pathways play crucial regulatory roles in cancer-associated inflammation. However, mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-β signaling, c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827 cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance as repressing transcription of Smad3, whereas TGF-β enhanced gefitinib sensitivity as activating transcription of Smad3 in HCC827 cells with gefitinib-sensitizing EGFR mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4-induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our results suggest that derepression of Smad3 can be a therapeutic strategy to prevent gefitinib-resistance in NSCLCs with gefitinib-sensitizing EGFR mutation.

Abstract 2931: Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation

Abstract Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic alterations responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the resistant clones and RNAi-based systematic synthetic lethal screening revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, we detected increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs refractory patients by immunohistochemistry. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs in a subset of lung cancer patients and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients. Citation Format: Nayoung Kim, Ahye Cho, Hideo Watanabe, Yoon-La Choi, Meraj Aziz, Michelle Kassner, Je-Gun Joung, Angela KJ Park, Joshua Francis, Joon Seol Bae, Soo-min Ahn, Kyoung-Mee Kim, Joon-Oh Park, Woong-Yang Park, Myung-Ju Ahn, Keunchil Park, Hongwei Holly Yin, Jeonghee Cho. Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation. [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 2931.

Efficacy of focal adhesion kinase inhibition in non-small cell lung cancer with oncogenically activated MAPK pathways.

Background:Focal adhesion kinase (FAK) is overexpressed in many types of tumours, including lung cancer. Y15, a small molecule which inhibits Y397 FAK autophosphorylation, decreases growth of human neuroblastoma, breast and pancreatic cancers. In this study, we investigated the in vitro and in vivo effects of Y15, and the underlying mechanism on non-small cell lung cancer cells.Methods:The cytotoxic effects of Y15 targeting FAK signalling were evaluated. Gene-knockdown experiments were performed to determine the anti-cancer mechanism. Xenografts with RAS or EGFR mutations were selected for in vivo Y15 treatment.Results:Y15 blocked autophosphorylation of FAK in a time- and dose-dependent manner. It caused dose-dependent decrease of lung cancer cell viability and clonogenicity. Y15 inhibited tumour growth of RAS-mutant (A549 with KRAS mutation and H1299 with NRAS mutation), as well as epidermal growth factor receptor (EGFR) mutant (H1650 and H1975) lung cancer xenografts. JNK activation is a mechanism underlying Y15-induced Bcl-2 and Mcl-1 downregulation. Moreover, knockdown of Bcl-2 or Bcl-xL potentiated the effects of Y15. The combination of various inhibitors of the Bcl-2 family of proteins with FAK inhibitors demonstrated synergy in multiple lung cancer cell lines in vitro.Conclusions:FAK inhibition demonstrated efficacy both in vitro and in vivo in lung cancers with either oncogenic RAS or EGFR mutations. In addition, FAK inhibition in combination with inhibitors of Bcl-2 family of anti-apoptotic proteins has synergistic activity in these MAPK-activated non-small cell lung cancer cell line models.

Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation.

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.

Clinical impact of the feasibility of epidermal growth factor receptor (EGFR) mutations detection in cytological samples of patients (pt) with non-small cell lung cancer (NSCLC) treated with erlotinib (E) at first line.

e18051 Background: EGFR mutations have been reported to be a predictor of efficacy of new drugs in NSCLC first line treatment. Recently, two phase III trials have shown the efficacy of gefinitib and erlotinib in this context, but the majority of pt were randomized after large biopsy samples. This study try to evaluate the efficacy and safety of E in untreated pt with advanced NSCLC and mutated EGFR (most of them detected in cytological specimens). Methods: Since 2007 we have screened 270 patients and we have found 40 patients with EGFR mutations (14%). 23 patients were treated with E at 150 mg per day at first line treatment. 13 of 23 pt were identify with cytological samples. Results: Median age was 72 years (33-90). Sex ratio (F/M): 74%/26%. 65% were non-smokers and 91% of them had ECOG 0-1. According with histology, 73.9% adenocarcinomas, 8.7% squamous cell carcinomas, 4.3% bronchioloalveolar carcinomas (13% non-specific). Oncogenic EGFR mutations detected were deletion of exon 19 (52.2%) and mutation of exon 21 (47.8%). Response has been evaluated in 18 p so far: complete response, 1 p; partial response, 11 p; stable disease, 3 p; progressive disease, 3 p; overall response rate, 67%; clinical benefit, 84%. In the cytological subgroup: overall response rate, 64%; clinical benefit, 82%. 11 patients continue under treatment with E. Progression free survival for all pt were 11 months, and 10 months for pt with mutations in EGFR gene detected in cytological specimens. Toxicity profile was predictable (mild to moderate rash, diarrhea and stomatitis). Conclusions: Our results suggest that detection of EGFR mutations in cytological samples is feasible and could have clinical impact when these pt are treated with tirosin-kinase inhibitors. A study comparing histological and cytological samples is on going.

TGF-β IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-beta-mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-beta-dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-beta are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR Mutations

BackgroundEpidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.Methods and FindingsTo study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.ConclusionsOur results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.