Second-generation Irreversible Tyrosine Kinase Inhibitors Research Articles

Rational design of EGFR dimerization-disrupting peptides: A new strategy to combat drug resistance in targeted lung cancer therapy

Although a variety of tyrosine kinase inhibitors (TKIs) have been developed to target human epidermal growth factor receptor (EGFR) for lung cancer therapy, many patients treated with first-line small-molecule TKIs are clinically observed to eventually establish drug-resistant mutations T790 M and C797S around kinase active site, which play a primary role in development of acquired drug resistance to first-generation reversible and second-generation irreversible TKIs, respectively. Here, instead of developing small-molecule drugs to directly target the active site, we attempt to derive self-inhibitory peptides (SIPs) from the EGFR:EGFR asymmetric dimerization interface, where is separated from kinase active site and has a relatively low conservation as compared to the active site. It is found that the dimerization is a typical peptide-mediated protein interaction, where the first EGFR N-lobe adopts an N-terminal binding sequence (nBS) to interact with the dimerization interface of second EGFR C-lobe. A core binding sequence (nCBS, 676NQALLRILKE68) is identified in the nBS region as hotspot segment, which is then rationally optimized to generate a number of SIPs. Consequently, three designed SIPs are determined as promising candidates; they have high affinity to EGFR kinase domain, strong competitive potency with native nBS peptide for the dimerization interface, and effective cytotoxicity on human lung cancer cell lines. It is also demonstrated that these peptides are insensitive to drug-resistant EGFR T790 M/C797S mutation at molecular and cellular levels, and exhibit a good selectivity for EGFR over HER2 at molecular level.

Validated LC-MS/MS assay for quantification of the newly approved tyrosine kinase inhibitor, dacomitinib, and application to investigating its metabolic stability.

Dacomitinib (DMB) is a second-generation irreversible tyrosine kinase inhibitor (TKI) that is claimed to overcome the disadvantages of the resistance reported for first-line epidermal growth factor receptor (EGFR) TKIs. Towards the end of 2018, the US Food and Drug Administration approved DMB in the form of VIZIMPRO tablets. In the current study, a validated LC-MS/MS assay was established for DMB quantification in rat liver microsomes (RLMs) with application to the drug metabolic stability assessment. Chromatographic resolution of DMB and lapatinib (internal standard) was achieved using an isocratic mobile phase and a reversed-phase C18 column. The linearity of the established LC-MS/MS assay ranged from 2 to 500 ng/mL with r2 ≥ 0.9999. The limit of detection (LOD) and limit of quantification (LOQ) were 0.35 and 1.1 ng/mL, respectively. The precision and accuracy (both intra-day and inter-day) were 0.84–3.58% and 92.2–100.32%, respectively. The metabolic stability of DMB in the RLM matrix was estimated by calculating two parameters, in vitro t1/2 (0.97 mL/min/kg) and intrinsic clearance (157.5 min). Such values infer that DMB would be excreted very slowly from the human body, which might lead to possible bioaccumulation. To the best of our knowledge, this is the first method for DMB analysis in RLMs with metabolic stability estimation.

Molecular Correlates of In Vitro Responses to Dacomitinib and Afatinib in Bladder Cancer.

Background:The HER family of proteins (EGFR, HER2, HER3 and HER4) have long been thought to be therapeutic targets for bladder cancer, but previous clinical trials targeting these proteins have been disappointing. Second generation agents may be more effective.Objective:The aim of this study was to evaluate responses to two second-generation irreversible tyrosine kinase inhibitors, dacomitinib and afatinib, in bladder cancer cell lines.Methods:Cell lines were characterized by targeted next generation DNA sequencing, RNA sequencing, western blotting and flow cytometry. Cell survival responses to dacomitinib or afatinib were determined using (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) or [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosylfate (PMS) cell survival assays.Results:Only two cell lines of 12 tested were sensitive to afatinib. Sensitivity to afatinib was significantly associated with mutation in either HER2 or HER3 (p < 0.05). The two cell lines sensitive to afatinib were also responsive to dacomitinib ralong with an additional 4 other cell lines out of 16 tested. No characteristic was associated with dacomitinib sensitivity. Molecular profiling demonstrated that only two genes were high in both afatinib and dacomitinib sensitive cells. Further rhigher expression of RAS pathway genes was noted for dacomitinib responsive cells.Conclusions:This study confirms that cell line screening can be useful in pre-clinical evaluation of targeted small molecule inhibitors and suggests that compounds with similar structure(s) and target(s) may have distinct sensitivity profiles. Further rcombinational targeting of additional molecularly relevant pathways may be important in enhancing responses to HER targeted agents in bladder cancer.

Current Role of Dacomitinib in Head and Neck Cancer

ABSTRACTIntroduction: Recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) has a dismal prognosis. With the emergence of monoclonal antibodies and tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR), several drugs were developed and tested in HNSCC. To date, the monoclonal antibody cetuximab is the only approved therapy for curative and recurrent/metastatic patients. Other EGFR-targeting drugs either failed in the clinical trials or are still in the early phases of drug development and research.Areas covered: In this article, previously published data and ongoing studies regarding dacomitinib, a second-generation irreversible TKI, for the treatment of HNSCC are presented and discussed.Expert opinion: The current body of evidence is not mature enough to indicate the use of dacomitinib for the treatment of HNSCC in curative or in recurrent/metastatic settings. Phase II data suggest the potential of improved outcome in selected recurrent/metastatic HNSCC based on several biomarkers, which need to be evaluated in randomized phase III trials. Meanwhile, an ongoing phase I study is investigating dacomitinib’s optimal dosing combined with and without cisplatin in the curative concomitant chemoradiotherapy setting.

Abstract 1333: Combining Afatinib and Cetuximab synergistically increases their cytotoxicity for EGFR T790M-harboring cells

Abstract Epidermal growth factor receptor (EGFR) is an important therapeutic target for non-small cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, are effective for patients with EGFR-activating mutations. However, these patients eventually develop resistance, usually through a secondary EGFR mutation, T790M: substitution of methionine for threonine at position 790 in the kinase domain. While second-generation irreversible TKIs have higher affinities for the double-mutant EGFRs, their therapeutic effect leaves room for improvement. Combining a second-generation TKI like afatinib with an anti-EGFR monoclonal antibody has been shown to improve clinical outcomes, although the mechanism is not fully understood. To investigate this mechanism, we used human erythroleukemia K562 cells, which are EGFR-negative, to establish cells with known EGFR mutations. Two activating EGFR mutations, Ex19del and L858R, were sensitive to first-generation TKIs. Acquisition of resistance to these TKIs was confirmed by adding the second EGFR mutation, T790M. Double-mutant EGFRs were moderately sensitive to afatinib, but were only minimally affected by the monoclonal anti-EGFR antibody cetuximab. Combining afatinib and cetuximab synergistically increased cytotoxity for K562 cells carrying double-mutant EGFRs. Apoptosis in these cells was preceded by induction of the pro-apoptotic protein BIM (Bcl2L11) and activation of Caspase-3 and PARP. Afatinib induced autophagy in these cells correlated with AKT and ERK1/2 signaling in the course of treatment. Accordingly, we found that cetuximab enhanced the afatinib-induced cytotoxicity through the autophagic cell death pathway. Our results indicate that two distinct mechanisms contribute to the synergistic effect exerted by afatinib and cetuximab against NSCLCs. Citation Format: Nobuyuki Tanaka, Zenta Watanuki, Tatsuro Fukuhara, Makoto Maemondo. Combining Afatinib and Cetuximab synergistically increases their cytotoxicity for EGFR T790M-harboring cells. [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 1333. doi:10.1158/1538-7445.AM2014-1333

Epidermal Growth Factor Receptor Inhibition in Mutation-Positive Non–Small-Cell Lung Cancer: Is Afatinib Better or Simply Newer?

In the last decade we have witnessed a major evolution in both molecular diagnostics and individualized therapy in the management of advanced non–small-cell lung cancer (NSCLC). The identification of epidermal growth factor receptor (EGFR) mutation and the centrality of EGFR tyrosine kinase inhibitors (TKIs) in patients whose tumors harbor these mutations have irrevocably altered how we view NSCLC. The more recent recognition of echinoderm microtubuleassociated protein-like 4/anaplastic lymphoma kinase and ROS1 translocations as molecular drivers, for which specific treatment exists, has altered our perspective still more. NSCLC is no longer considered a monolithic illness, but it is now readily recognized as a constellation of genotypic entities for which targeted therapy has an increasing role. In this regard, despite clinical, radiographic, and even histologic similarities from tumor to tumor, the genotypic footprint, one believes, should ultimately trump phenotype when it comes to therapeutic decision making. As part of this evolution, we are now observing the second wave of EGFR TKIs. In their seminal article, Sequist et al highlight the results of the LUX-Lung 3 trial, which compared afatinib, a secondgeneration irreversible TKI, to cisplatin and pemetrexed in the firstline treatment of treatment-naive patients with advanced adenocarcinoma of the lung and EGFR mutation. Afatinib is clearly superior with respect to response rate (RR) and progression-free survival (PFS), and these benefits are even more pronounced in the vast majority of patients whose tumors harbor the common activating mutations in exons 19 and 21 (Table 1). In this regard, the LUX-Lung 3 trial joins a number of other trials examining erlotinib and gefitinib in the identical therapeutic setting, each of which has consistently shown a statistically significant and clinically meaningful RR and PFS benefit compared with standard chemotherapy (Table 2). Although many would argue that the results of the LUX-Lung 3 trial were entirely expected and scarcely surprising, this landmark phase III effort comparing afatinib with standard chemotherapy in patients with NSCLC and EGFR mutation is distinguished by a number of factors. It is the largest trial to date in the first-line EGFR mutant setting to be published; it is the first randomized phase III trial to compare a second-generation irreversible EGFR TKI with activity against both human epidermal growth factor receptor 1 (HER1) and HER2 versus platinum-based cytotoxics; it employs a state-of-the-art comparator (pemetrexed and cisplatin); it fully integrates quality-oflife (QoL) evaluation into outcome analysis; it is the first global trial of this sort; and it is explicitly a registration trial. Among those trials that have been confined to patients with actionable EGFR mutations, this trial enrolled nearly double the number of participants, well over 300, making it far more robust and thereby tightening the CIs around the benefits already noted in similar studies. The global nature of this trial and its size make the findings a Table 1. Top-Line Results of the LUX-Lung 3 Trial