• Alzheimer’s disease
• Aβ
• EGFR
• EGFR inhibitor
• cancer
• learning and memory

• Apoptosis
• Cell cycle
• EGFR-TKI
• Lung adenocarcinoma
• Resistance
• STE029

Epidermal growth factor receptor (EGFR), the receptor for members of the epidermal growth factor family, regulates cell proliferation and signal transduction; moreover, EGFR is related to the inhibition of tumor cell proliferation, angiogenesis, invasion, metastasis, and apoptosis. Therefore, EGFR has become an important target for the treatment of cancer, including non-small cell lung cancer, head and neck cancer, breast cancer, glioma, cervical cancer, and bladder cancer. First- to third-generation EGFR inhibitors have shown considerable efficacy and have significantly improved disease prognosis. However, most patients develop drug resistance after treatment. The challenge of overcoming intrinsic and acquired resistance in primary and recurrent cancer mediated by EGFR mutations is thus driving the search for alternative strategies in the design of new therapeutic agents. In view of resistance to third-generation inhibitors, understanding the intricate mechanisms of resistance will offer insight for the development of more advanced targeted therapies. In this review, we discuss the molecular mechanisms of resistance to third-generation EGFR inhibitors and review recent strategies for overcoming resistance, new challenges, and future development directions.

• Cancer
• Drug resistance
• Epidermal growth factor receptor (EGFR)
• Inhibitors
• Structure–activity relationship
• Tyrosine kinase

Epidermal growth factor receptor (EGFR)-activating mutations are major oncogenic mechanisms in non-small cell lung cancer (NSCLC). Most patients with NSCLC with EGFR mutations benefit from targeted therapy with EGFR- tyrosine kinase inhibitors (TKIs). One of the main limitations of targeted therapy is that the tumor response is not durable, with the inevitable development of drug resistance. Previous studies demonstrated that the potential resistance mechanisms are diverse, including the presence of EGFR T790M, MET amplification, mesenchymal transformation, and anaplastic lymphoma kinase (ALK) rearrangement. The patient in our report was diagnosed with stage IA lung adenocarcinoma harboring the EGFR L858R mutation and underwent radical surgery. The patient received icotinib for 12 months after recurrence. Subsequent molecular analysis of the left pleural effusion indicated that LCLAT1-ALK fusion might be an underlying mechanism contributing to the acquired resistance to icotinib. Ensartinib was prescribed, but the lesion in the right lung continued to progress. Hence, a re-biopsy and molecular analysis of lesions in the right lung was performed to solve this problem. In contrast to the left pleural effusion, EGFR exon 20 T790M might have mediated the acquired resistance in lesions in the right lung of this patient. The combination of osimertinib and ensartinib has achieved a rapid partial response until now. The complexity and heterogeneity in our case may provide new insights into the resistance mechanisms of targeted therapy.

The aim of this study was to 1) investigate the effects of 27 CYP3A4 variants on the metabolism of osimertinib and 2) study the interactions between osimertinib and others as well as the underlying mechanism. A recombinant human CYP3A4 enzymatic incubation system was developed and employed to determine the kinetic profile of CYP3A4 variants. Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was applied to detect the concentration of the main metabolite, AZ5104. The results demonstrated that the relative clearance rates of CYP3A4.19, 10, 18, 5, 16, 14, 11, 2, 13, 12, 7, 8, and 17 in catalyzing osimertinib were significantly reduced to a minimum of 25.68% compared to CYP3A4.1, while those of CYP3A4.29, 32, 33, 28, 15, 34, and 3 were obviously enhanced, ranging from 114.14% to 284.52%. The activities of the remaining variants were almost equal to those of CYP3A4.1. In addition, 114 drugs were screened to determine the potential interaction with osimertinib based on the rat liver microsome (RLM) reaction system. Sixteen of them inhibited the production of AZ5104 to 20% or less, especially proton pump inhibitors, among which the IC₅₀ of rabeprazole was 6.49 ± 1.17 μM in RLM and 20.39 ± 2.32 μM in human liver microsome (HLM), with both following competitive and non-competitive mixed mechanism. In an in vivo study, Sprague–Dawley (SD) rats were randomly divided into groups, with six animals per group, receiving osimertinib with or without rabeprazole, omeprazole, and lansoprazole. We found that the AUC_(0–t), AUC_(0–∞), and C_max of osimertinib decreased significantly after co-administration with rabeprazole orally, but they increased remarkably when osimertinib was administered through intraperitoneal injection. Taken together, our data demonstrate that the genetic polymorphism and proton pump inhibitors remarkably influence the disposition of osimertinib, thereby providing basic data for the precise application of osimertinib.

• National Key Research and Development Program of China
• National Natural Science Foundation of China

BIX01294 (BIX), an inhibitor of the G9a histone methyltransferase, has been reported to have antitumor activity against a variety of cancers. However, the molecular mechanisms underlying its anticancer effects, particularly those against lung cancer, remain unclear. Here, we report that BIX induces apoptotic cell death in EGFR-mutant non-small cell lung cancer (NSCLC) cells but not in their wild-type counterparts. Treatment with BIX resulted in a significant reduction in the EGFR level and inhibition of EGFR signaling only in EGFR-mutant NSCLC cells, leading to apoptosis. BIX also inhibited mitochondrial metabolic function and decreased the cellular energy levels that are critical for maintaining the EGFR level. Furthermore, BIX transcriptionally downregulated the transcription of branched-chain α-keto acid dehydrogenase (BCKDHA), which is essential for fueling the tricarboxylic acid (TCA) cycle. Interestingly, this BCKDHA downregulation was due to inhibition of Jumanji-domain histone demethylases but not the G9a histone methyltransferase. We observed that KDM3A, a Jumonji histone demethylase, epigenetically regulates BCKDHA expression by binding to the BCKDHA gene promoter. BIX exposure also led to a significant decrease in the EGFR level, causing apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Taken together, our current data suggest that BIX triggers apoptosis only in EGFR-mutant NSCLC cells via inhibition of BCKDHA-mediated mitochondrial metabolic function.

A drug known as BIX that is effective against bladder and breast cancers may also be effective in fighting non-small cell lung cancer (NSCLC). Although advances have been made in treatment of NSCLC, one of the most effective drugs targets a protein called EGFR, and EGFR gene mutations that lead to acquired drug resistance are common. Jaekyoung Son at the University of Ulsan, Seoul, South Korea, and colleagues investigated whether BIX is effective against NSCLC and attempted to elucidate its mechanism of action. The researchers found that BIX caused death of NSCLC cells, especially those with mutations in the EGFR gene. Further investigation showed that BIX was effective even against drug-resistant NSCLC cells, by acting via a different metabolic pathway. BIX shows promise as an alternative therapy for lung cancer, to overcome drug resistance.

• EMT
• NFATc1
• NSCLC
• microRNA-338
• proliferation

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib are effective for treating patients with EGFR mutant lung cancer; however, drug resistance inevitably emerges. Approaches to combine immunotherapies and targeted therapies to overcome or delay drug resistance have been hindered by limited knowledge of the effect of erlotinib on tumor-infiltrating immune cells.

Using mouse models, we studied the immunological profile of mutant EGFR-driven lung tumors before and after erlotinib treatment.

We found that erlotinib triggered the recruitment of inflammatory T cells into the lungs and increased maturation of alveolar macrophages. Interestingly, this phenotype could be recapitulated by tumor regression mediated by deprivation of the EGFR oncogene indicating that tumor regression alone was sufficient for these immunostimulatory effects. We also found that further efforts to boost the function and abundance of inflammatory cells, by combining erlotinib treatment with anti-PD-1 and/or a CD40 agonist, did not improve survival in an EGFR-driven mouse model.

Our findings lay the foundation for understanding the effects of TKIs on the tumor microenvironment and highlight the importance of investigating targeted and immuno-therapy combination strategies to treat EGFR mutant lung cancer.

The online version of this article (10.1186/s40425-019-0643-8) contains supplementary material, which is available to authorized users.

• EGFR
• Immunotherapies
• Lung cancer
• Mouse models
• Targeted therapies

• EGFR T790M
• Non-small cell lung cancer (NSCLC)
• acquired resistance
• osimertinib

FHND004 is a newly synthesized epidermal growth factor receptor (EGFR) inhibitor for the treatment of non-small cell lung cancer (NSCLC). The aim of the present study was to investigate the impacts of FHND004 on human ether-à-go-go-related gene (hERG) K⁺ channels and the molecular mechanisms underlying of its action. Whole-cell patch clamp recording was performed on wild type (WT), mutant hERG channels heterologously expressed in human embryonic kidney (HEK) 293 cells or I_Kr endogenously expressed in HL-1 cells, respectively. FHND004 inhibited hERG K⁺ currents in a concentration-dependent manner with IC₅₀ values of 8.46 ± 0.33 μM in HEK293 cells and 7.52 ± 1.27 μM in HL-1 cells, respectively. However, the inhibitory potency of FHND004 on hERG channels was significantly less than its precursor AZD9291. FHND004-induced inhibition was state-dependent with a preference within open state, but did not alter other kinetics including activation, inactivation, and recovery from inactivation or deactivation. In addition, FHND004 exhibited more potent inhibitory effects on WT/A422T and WT/H562P-hERG, two known long QT syndrome (LQTS) associated KCNH2 mutations, than WT alone. Mutations of the residues at pore regions (F656C, Y652A, S624A, and F557L) in hERG channels attenuated block effects of FHND004. Taken together, our results demonstrate the evidence that FHND004 is a less potent hERG blocker than its precursor AZD9291. There is, however, a need for caution in the potential use of FHND004 for treating NSCLC patients, especially in those with other concurrent triggering factors.

• EGFR inhibitor
• FHND004
• cardiac safety
• human ether-à-go-go-related gene
• inhibitory effect

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy.

We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays.

Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay.

Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

• NGS
• NSCLC
• T790M
• cfDNA
• liquid biopsy

To identify the somatic mutated genes for optimal targets of non-small-cell lung cancer after resistance to osimertinib treatment.

Study patients all had advanced lung adenocarcinoma and acquired resistance to osimertinib as a second- or third-line treatment. These patients had harboring EGFR T790M mutation before osimertinib treatment, which was confirmed by Amplification Refractory Mutation System (ARMS) PCR or Next-Generation Sequencing (NGS). After resistance to osimertinib treatment, tumor tissue was collected by core needle biopsy. DNA was extracted from 15 × 5 um sliced section of formalin-fixed paraffin-embedded (FFPE) material and NGS was done. The genetic changes were analyzed.

A total of 9 Chinese patients were studied, 5 females and 4 males, age 51–89 years. After progression with osimertinib treatment, core needle biopsy was performed and next-generation sequencing was performed. Nine patients had harboring 62 point mutations, 2 altered gene copies, 2 amplifications, and 1 EML4-ALK gene fusion. No MET or HER2 amplification was found in this cohort study. Nine patients still maintained initial EGFR 19 del or L858R activating mutations, while 7 of them kept EGFR T790M mutations. Among the 7 patients, 5 had secondary EGFR C797S and/or C797G mutations, which all happened in the same allele with T790M mutation. All patients were treated with targets therapies, chemotherapy, or best supportive care (BSC) in accordance with NGS genetic results and patients' performance status; 7 of them are still alive and 2 of them died of disease progression at last follow-up.

EGFR C797S/G mutation and the same one presented on the same allele with EGFR T790M mutation were the most common mutation feature and played a key role in resistance to osimertinib in Chinese patients with NSCLC. Tumor cells losing T790M mutation and maintaining EGFR activating mutation might benefit from first-generation EGFR-TKI treatment.

• 3rd-generation
• EGFR-TKI
• IGF1R
• NSCLC
• resistance

• Blood-brain barrier
• Brain metastases
• Chemotherapy
• Immunotherapy
• Lung cancer
• Radiation
• Targeted therapy

Epidermal growth factor receptor (EGFR) mutations (EGFRm⁺) occur in 10–35% of non-small-cell lung cancer (NSCLC) cases and confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs are standard treatments for NSCLC patients harboring EGFR exon 19 deletions or exon 21 L858R point mutations. Despite initial benefit, most patients develop drug resistance, posing a challenge to oncologists. The secondary T790M point mutation in EGFR exon 20 contributes to approximately 60% of resistance cases. Optimum strategies for overcoming acquired EGFR TKI resistance are not clearly defined, although current common practice is to switch to platinum-based chemotherapy following resistance onset. While the second-generation EGFR TKIs, including afatinib, dacomitinib, and neratinib, exhibit promising preclinical activity against T790M mutants, dose-limiting toxicities in patients have limited clinical success. However, third generation EGFR TKIs appear able to overcome this mutation. Other treatment options aimed at EGFR TKI resistance include use of an EGFR TKI beyond progression, and chemotherapy plus an EGFR TKI. This review focuses on improved anticancer agents and therapy options for NSCLC patients with acquired EGFR TKI resistance.

• chemotherapy
• drug resistance
• epidermal growth factor receptor
• non-small cell lung cancer
• tyrosine kinase inhibitors

• Brain metastases
• Disease-specific graded prognostic assessment
• EGFR mutation
• Leptomeningeal index
• Non-small cell lung cancer
• Prognostic index
• Stereotactic radiosurgery
• Whole brain radiotherapy

• drug therapy
• gefitinib
• non-small-cell lung cancer

In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans-activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies.

DOI:http://dx.doi.org/10.7554/eLife.21536.001

• activity
• cancer biology
• cell biology
• human
• in cell
• mouse
• profiling
• protein kinase
• receptor tyrosine kinase
• reporter

• carboplatin
• epidermal growth factor receptor gene mutation
• gefitinib
• non-small-cell lung cancer
• paclitaxel

Lung cancer, ~80%–85% of which is non-small-cell lung cancer (NSCLC), is the leading cause of cancer-related mortality worldwide. Sensitizing mutations in epidermal growth factor receptor (EGFR) gene (EGFRm⁺), such as exon 19 deletions and exon 21 L858R point mutations, are the most important drivers in NSCLC patients. In this respect, small-molecule EGFR tyrosine kinase inhibitors (TKIs) have been designed and developed, which launched the era of targeted, personalized and precise medicine for lung cancer. Patients with EGFRm⁺ could achieve good responses to the treatment with the first-generation EGFR TKIs, such as erlotinib and gefitinib. However, most patients develop acquired drug resistance mostly driven by the T790M mutation occurring within exon 20. Although the second-generation EGFR TKIs, such as afatinib, dacomitinib and neratinib, demonstrated promising activity against T790M in preclinical models, they have failed to overcome resistance in patients due to dose-limiting toxicity. Recently, the third-generation EGFR TKIs have shown to be effective against cell lines and murine models harboring T790M mutations while sparing wild-type EGFR, which represents a promising breakthrough approach in overcoming T790M-mediated resistance in NSCLC patients. This article provides a comprehensive review of the therapy revolution for NSCLC with three generations of EGFR TKIs.

• T790M mutation
• epidermal growth factor receptor
• lung cancer
• tyrosine kinase inhibitors

• ctcs
• egfr mutation
• nsclc

• Biomarkers, Tumor/genetics
• Carcinoma, Non-Small-Cell Lung/genetics
• ErbB Receptors/genetics
• Lung Neoplasms/genetics
• Mutation
• Neoplastic Cells, Circulating

• 肺肿瘤
• 化疗
• 靶向治疗
• lung neoplasms
• chemotherapy
• targeted therapy

• Antineoplastic Agents/therapeutic use
• Carcinoma, Squamous Cell/drug therapy
• Carcinoma, Squamous Cell/genetics
• ErbB Receptors/genetics
• Humans
• Lung Neoplasms/drug therapy
• Lung Neoplasms/genetics
• Mutation
• Protein Kinase Inhibitors/therapeutic use

• T790M mutation
• epidermal growth factor receptor
• non-small cell lung cancer
• osimertinib
• tyrosine kinase inhibitors

• AZD9291
• CO-1686
• EGFR mutations
• EGFR-TKIs
• T790M

• Combination drug therapy
• epidermal growth factor receptor (EGFR)
• non-small-cell lung cancer (NSCLC)

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).

METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.

RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.

CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.

• Epidermal growth factor receptor
• Tyrosine kinase inhibitor
• Gatekeeper mutation
• Non-small cell lung cancers