Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Erlotinib Research Articles

Design, synthesis and biological evaluation of novel heptamethine cyanine dye-erlotinib conjugates as antitumor agents

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) has been proved as a target for the treatment of non-small cell lung cancer (NSCLC) with specific gene mutations. However, EGFR-TK inhibitors (EGFR-TKIs) need to enter cancer cells and then competitively interact with the active site of tyrosine kinase receptors to suppress the downstream signaling pathway to inhibit tumor proliferation. In this study, in order to improve the tumor cell targeting ability of EGFR-TKI, EGFR-TKI erlotinib was conjugated with the cancer cell-targeting heptamethine cyanine dyes to form seventeen novel erlotinib-dye conjugates. The efficiency of tumor targeting properties of conjugates against cancer cell growth and EGFR-TK inhibition was evaluated in vitro. The result revealed that most erlotinib-dye conjugates exhibited stronger inhibitory effect on A549, H460, H1299 and MDA-MB-231 cell lines than the parent drug erlotinib. Meanwhile, representative compounds exhibited weak cytotoxicity on human normal mammary epithelial MCF-10A cells. Moreover, the conjugate CE17 also showed ~14-fold higher EGFR-TK inhibition activity (IC50 = 0.124 μM) than erlotinib (IC50 = 5.182 μM) in A549 cell line. Finally, molecular docking analysis verified that the erlotinib moiety of compound CE17 could form hydrogen bond with Met-769 and occupy active cavity of EGFR-TK. Therefore, we believed the integration strategy between heptamethine cyanine dyes and EGFR-TKI will contribute to enhancing the therapeutic effect of EGFR-TKI for NSCLC treatment.

Efficacy and safety of therapies for EGFR-mutant non-small cell lung cancer with brain metastasis: an evidence-based Bayesian network pooled study of multivariable survival analyses.

Preferable treatments for epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) with brain metastasis are elusive. The study intended to estimate the relative efficacy and safety of systemic therapies. Clinical trials about therapies for EGFR-mutant, brain-metastatic NSCLC were identified. Progression-free survival (PFS) and overall survival (OS) were analysed using random effects Bayesian network meta-analyses (NMAs) on the hazard ratio (HR)-scale. Nomogram and Kaplan-Meier plots based on clinical or individual factors are displayed using data obtained from the Surveillance Epidemiology and End Results (SEER) database. Third-generation EGFR- tyrosine kinase inhibitors (EGFR-TKI) (osimertinib), EGFR-TKIs + stereotactic radiosurgery (SRS)/whole brain radiotherapy (WBRT) (gefitinib/erlotinib + SRS/WBRT), and EGFR-TKIs (erlotinib) + anti-vascular endothelial growth factor receptor (anti-VEGFR) (bevacizumab) achieved superior PFS (HR: 0.30 (0.15-0.59); HR: 0.47 (0.31-0.72); HR: 0.50 (0.21-1.21) vs. deferring SRS/WBRT) and acceptability; EGFR-TKIs + SRS/WBRT was top ranking (vs. others) for OS followed by third-generation EGFR-TKI. In the dataset cohort of 1173 brain-metastatic NSCLC patients, the 6-month, 1-year, and 3-year survival rates were 59.8%, 41.3%, and 5.6%, respectively. Race and origin, and year of diagnosis were independent predictors of OS. Survival curves showed that the OS of patients varied significantly by histology and race. Third-generation EGFR-TKI and EGFR-TKIs + SRS/WBRT are more effective and potentially acceptable for EGFR-mutant NSCLC with brain metastases balancing OS and PFS. Surgeries without adjuvant therapies cannot significantly improve the OS of brain-metastatic NSCLC patients. The study highlights importance of osimertinib in these patients and provide a reference for clinical treatments.

Development of a Potent Brain-Penetrant EGFR Tyrosine Kinase Inhibitor against Malignant Brain Tumors.

The epidermal growth factor receptor (EGFR) is genetically altered in nearly 60% of glioblastoma tumors; however, tyrosine kinase inhibitors (TKIs) against EGFR have failed to show efficacy for patients with these lethal brain tumors. This failure is attributed to the inability of clinically tested EGFR TKIs to cross the blood-brain barrier (BBB) and achieve adequate pharmacological levels to inhibit various oncogenic forms of EGFR that drive glioblastoma. Through SAR analysis, we developed compound 5 (JCN037) from an anilinoquinazoline scaffold by ring fusion of the 6,7-dialkoxy groups to reduce the number of rotatable bonds and polar surface area and by introduction of an ortho-fluorine and meta-bromine on the aniline ring for improved potency and BBB penetration. Relative to the conventional EGFR TKIs erlotinib and lapatinib, JCN037 displayed potent activity against EGFR amplified/mutant patient-derived cell cultures, significant BBB penetration (2:1 brain-to-plasma ratio), and superior efficacy in an EGFR-driven orthotopic glioblastoma xenograft model.

Comparison of the Efficacy of EGFR Tyrosine Kinase Inhibitors Erlotinib and Low-dose Osimertinib on a PC-9-GFP EGFR Mutant Non-small-cell Lung Cancer Growing in the Brain of Nude Mice.

Brain metastases are found in approximately 30% of patients with epidermal-growth-factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). We compared the efficacy of two EGFR-tyrosine kinase inhibitors (TKIs), erlotinib and osimertinib on a PC-9-GFP EGFR mutant NSCLC growing in the brain of nude mice. The brain metastasis models were randomized into five groups and treated for 15 days: Control; 5 mg/kg erlotinib; 50 mg/kg erlotinib; 0.5 mg/kg osimertinib; 5 mg/kg osimertinib. Tumor volume was evaluated by non-invasive fluorescence imaging. Only 5 mg/kg osimertinib, a low-dose compared to the clinically-equivalent dose, showed significant tumor regression compared to the control. This study strongly supports the high activity of osimertinib for intracranial lesions of EGFR-mutant NSCLC.

Tetracyclines increase the survival of NSCLC patients treated with EGFR TKIs: a retrospective nationwide registry study

BackgroundWith the first and second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), clinical benefit and rash correlate together. EGFR TKI-induced rash can be alleviated with tetracyclines, but it is unknown whether the use of tetracyclines can increase the survival of non-small-cell lung cancer (NSCLC) patients treated with EGFR TKIs.MethodsWe collected all the patients (n=1271) who had reimbursement for EGFR TKIs (gefitinib, erlotinib and afatinib) in Finland 2011–2016, had purchased TKIs, and had data available at nationwide cancer registry. The survival was analysed from the first EGFR TKI purchase to death or end-of follow-up, and patients were stratified according to TKIs, purchases of antibiotics, their ATC class and timing.Results802 (63.1%) patients had antibiotic purchases −14 to +200 days from the first EGFR TKI purchase, 447 of these tetracyclines. 322 (25.3%) had had purchased antibiotics −14 to +14 days (prophylaxis) from the first EGFR TKI purchase, 188 of these tetracyclines. Purchase of antibiotics was associated with improved survival (HR 0.80, 95% CI 0.71 to 0.91), which limited to tetracycline purchases only (HR 0.72, 95% CI 0.64 to 0.82). The largest survival benefit was seen with the prophylactic use of tetracyclines (HR 0.74, 95% CI 0.62 to 0.88). The benefit from tetracyclines was limited to erlotinib only (HR 0.68, 95% CI 0.58 to 0.78) which was retained in multivariate analysis. Prophylactic use of tetracyclines was associated with a longer erlotinib treatment duration (HR 0.81, 95% CI 0.61 to 0.96) but not with dose reductions or treatment breaks.ConclusionsTetracyclines improve the survival of NSCLC patients treated with the first and second-generation EGFR TKIs and they should be considered as a prophylaxis when initiating EGFR TKIs with high incidence of rash.

Rare mutations of epidermal growth factor receptor in epidermal growth factor receptor-tyrosine kinase inhibitor-naive non-small cell lung carcinoma and the response to erlotinib therapy.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are considered to be effective treatments for advanced NSCLC patients with sensitizing EGFR mutations. There are many complex and rare mutations in the EGFR gene. The efficacy of the first-generation EGFR-TKI (erlotinib) is unknown for tumors harboring rare EGFR mutations. The purpose of this study was to investigate the clinical significance of rare EGFR mutations in EGFR-TKI-naive patients and the efficacy of erlotinib. Istanbul University, Istanbul Medical Faculty, Department of Medical Oncology, Istanbul/Turkey, and retrospective observational study. We retrospectively analyzed 117 non-small cell lung cancer (NSCLC) patients with EGFR mutations who had not previously used EGFR-TKIs. Exons 18-21 of EGFR were analyzed by polymerase chain reaction and subjected to direct sequencing methods. Survival estimates were calculated by the Kaplan-Meier method using SPSS 25 software (IBM SPSS, Chicago, USA). Of 117 patients who had EGFR mutations, 23 patients had rare and complex EGFR mutations. Only 9 of them were treated with erlotinib. Three patients (3.5%) with exon 20 mutations received erlotinib. Two with EGFR-p. Q787Q (SNP ID, rs10251977; c.2361G>A) synonymous mutation in exon 20 were responsive to erlotinib therapy in the second-line setting after first-line chemotherapy. To the best of our knowledge, the present two cases are the first to be reported with lung adenocarcinoma with EGFR-p. Q787Q synonymous mutation responding to erlotinib. NSCLC patients harboring rare EGFR mutations generally did not show consistent or favorable responses to EGFR-TKI. We suggest that this rare synonymous mutation (EGFR-p. Q787Q) is a sensitive EGFR mutation in NSCLC.

Central nervous system metastases of lung adenocarcinoma harboring EGFR-activating mutations: survival results from a multidisciplinary approach, including EGFR-TKIs

OBJECTIVES: Central nervous system (CNS) metastases are frequent in advanced lung adenocarcinomas harboring EGFR-activating mutations. However, the best treatment approach must be defined. We aimed to evaluate the effectiveness of a multidisciplinary approach for CNS metastases in patients previously untreated with EGFR tyrosine kinase inhibitors (EGFR-TKIs). METHODS: We performed a retrospective analysis of EGFR-TKI naïve patients with CNS metastases from lung adenocarcinomas harboring EGFR-activating mutations treated in a Brazilian academic cancer center. Data were collected from electronic records. Overall survival (OS) and progression-free survival (PFS) curves were estimated using the Kaplan-Meier method and compared by logrank test. Cox model was used to evaluate prognostic factors. RESULTS: 35 consecutive patients were included. Treatment included an EGFR-TKI (erlotinib or gefitinib) for all patients, whole brain radiation therapy for 26 patients, stereotactic radiosurgery for 2 patients, and surgery for 8 patients. Median PFS and OS were 8.2 and 11.9 months, respectively. In a multivariable analysis, poor Eastern Cooperative Oncology Group performance status (3-4 vs 0-2) was associated with inferior OS (HR 2.86; 95% CI 1.12-6.74; p=0.016), while radiation therapy to treat brain lesions (yes vs no) showed a trend towards improved OS (HR 0.40; 95% CI 0.15-1.06; p=0.066). No difference was seen between upfront and salvage radiation therapy to the brain. CONCLUSIONS: A multidisciplinary treatment approach, including an EGFR-TKI, allowed promising outcomes for patients with CNS metastases of lung adenocarcinoma harboring EGFR-activating mutations, but the small number of patients here studied precludes definitive conclusions. Although radiation therapy to treat brain metastases has an important role, the best treatment sequence remains unclear. Currently, the approach must be individualized, considering patient characteristics, tumor biology and healthcare resources availability.

DDIS-17. DEVELOPMENT OF BRAIN-PENETRANT EGFR INHIBITORS FOR CNS MALIGNANCIES

Abstract The epidermal growth factor receptor (EGFR) is altered in nearly 60% of glioblastoma (GBM) tumors, however, EGFR tyrosine kinase inhibitors (TKIs) have failed to improve outcomes for patients with GBM. This can be attributed to the inability of clinically available EGFR TKIs (e.g., erlotinib, gefitinib, lapatinib, afatinib, cetuximab) to effectively cross the blood-brain-barrier (BBB) and reach adequate pharmacological levels for a tumor response. Herein, we performed a structure-activity relationship (SAR) to obtain EGFR TKIs with both high brain penetrance and potency against EGFR-activated GBM cells. From over 80 novel compounds synthesized, our lead EGFR TKI—JCN068—exhibited exceptional BBB penetration (350% brain to plasma) while also having optimal ADME properties (60% oral bioavailability, ~5 hr half-life, >100 µg/mL solubility, etc). Moreover, JCN068 demonstrated picomolar potency against purified EGFR kinase, 1000-fold selectivity for EGFR relative to other kinases, and nanomolar activity against EGFR-altered, GBM patient-derived cells in culture. Importantly, JCN068 demonstrated superior efficacy—with negligible toxicity—compared to clinically available small molecule EGFR TKIs (erlotinib and lapatinib) against multiple EGFR-altered patient-derived orthotopic GBM xenografts. Due to these excellent drug-like properties, JCN068 is currently progressing towards clinical development for EGFR-activated GBM patients.

TAS6417/CLN-081 Is a Pan-Mutation-Selective EGFR Tyrosine Kinase Inhibitor with a Broad Spectrum of Preclinical Activity against Clinically Relevant EGFR Mutations.

Despite the worldwide approval of three generations of EGFR tyrosine kinase inhibitors (TKI) for advanced non-small cell lung cancers with EGFR mutations, no TKI with a broad spectrum of activity against all clinically relevant mutations is currently available. In this study, we sought to evaluate a covalent mutation-specific EGFR TKI, TAS6417 (also named CLN-081), with the broadest level of activity against EGFR mutations with a prevalence of ≥1%. Lung cancer and genetically engineered cell lines, as well as murine xenograft models were used to evaluate the efficacy of TAS6417 and other approved/in-development EGFR TKIs (erlotinib, afatinib, osimertinib, and poziotinib). We demonstrate that TAS6417 is a robust inhibitor against the most common EGFR mutations (exon 19 deletions and L858R) and the most potent against cells harboring EGFR-T790M (first/second-generation TKI resistance mutation). In addition, TAS6417 has activity in cells driven by less common EGFR-G719X, L861Q, and S768I mutations. For recalcitrant EGFR exon 20 insertion mutations, selectivity indexes (wild-type EGFR/mutant EGFR ratio of inhibition) favored TAS6417 in comparison with poziotinib and osimertinib, indicating a wider therapeutic window. Taken together, we demonstrate that TAS6417 is a potent EGFR TKI with a broad spectrum of activity and a wider therapeutic window than most approved/in-development generations of EGFR inhibitors. IMPLICATIONS: TAS6417/CLN-081 is a potent EGFR TKI with a wide therapeutic window and may be effective in lung cancer patients with clinically relevant EGFR mutations.

<p>Clinical evaluation of dacomitinib for the treatment of metastatic non-small cell lung cancer (NSCLC): current perspectives</p>

Systemic treatment of advanced non-small cell lung cancer (NSCLC) has undergone remarkable changes in the last decade, with the introduction of targeted therapies and immunotherapy. The identification of activating mutations in the epidermal growth factor receptor (EGFR) gene (deletions in exon 19 [Del19] and point mutation L858R in exon 21) has been the first important step toward molecularly guided precision therapy in lung cancer. Several randomized trials comparing EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, and afatinib) to standard chemotherapy in first-line treatment of advanced EGFR-mutant NSCLC showed significant improvement in progression-free survival (PFS) and in response rate, with lower rates of adverse events (AEs) and better symptom control. However, none of these trials showed significant improvement in overall survival (OS). Despite impressive responses with EGFR-TKI, disease invariably progresses after 9 to 13 months, due to acquired resistance. Dacomitinib is a potent, irreversible, highly selective, second-generation EGFR-TKI, which inhibits the signaling from both heterodimers and homodimers of all the members of the human epidermal growth factor receptor (HER) family. Here, we review the clinical development of dacomitinib from phase I to phase III, with particular attention to its toxicity and on its activity on T790M mutation. Then, we critically examine the results of ARCHER 1050, a study that was crucial for Food and Drug Administration (FDA) approval. ARCHER 1050 was the first randomized phase III study comparing dacomitinib with gefitinib, in first-line treatment of patients with advanced EGFR-mutated NSCLC. Dacomitinib was superior to gefitinib in terms of primary end-point (14.7 vs 9.2 months) and OS (34.1 vs 26.8 months). The incidence of diarrhea, skin rash, mucositis and, consequently, dose reductions was higher with dacomitinib, while hepatic toxicity was higher with gefitinib. Dacomitinib constitutes one of the standard first-line options in patients with advanced EGFR-mutated NSCLC.

Combined Treatment with Triptolide and Tyrosine Kinase Inhibitors Synergistically Enhances Apoptosis in Non-small Cell Lung Cancer H1975 Cells but Not H1299 Cells through EGFR/Akt Pathway.

Lung cancer is one of the most common malignant cancers in the world. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a second- or third-line therapy for mutated non-small cell lung cancer (NSCLC). It usually becomes drug resistance after a period of treatment. Triptolide (TPL) is an epoxy diterpenoid lactone compound extracted from Tripterygium wilfordii HOOK. F. and many studies demonstrated that TPL has a synergistic effect when combined with chemotherapy drugs. In this research, we plan to evaluate the combined effect of TPL and EGFR-TKIs (Gefitinib, Erlotinib, and Icotinib) and investigate the possible mechanisms. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to detect the cell viabilities, combined effect was evaluated by Combination Index. Molecular docking study was used to predict the binding ability of TPL. The expression of proteins was detected by Western blot. MTT results showed TPL had synergistic effect with three EGFR-TKIs at different concentrations on H1975 cells but not on H1299 cells. Molecular docking study demonstrated that TPL with T790M/L858R EGFR can form a more stable compound than that with wild type EGFR. Western blot results showed TPL inhibited the EGFR/Akt pathway and increased the expression of Bax and the ratio of Bax and Bcl-2 in H1975 cells. In conclusion, TPL had synergistic effect with three EGFR-TKIs on H1975 cells but not on H1299 cells, which may be due to the binding ability of TPL and different-type EGFR. The synergistic effect of TPL on H1975 cells may be partly related to the inhibition of the EGFR/Akt pathway.

Abstract 4930: PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC

Abstract Lung cancer management has been transformed by the discovery and effective targeting of driver mutations such as epidermal growth factor receptor (EGFR). Tyrosine Kinase Inhibitors (TKIs) that block EGFR kinase activity such as erlotinib and osimertinib cause dramatic tumor shrinkage and prolonged responses in patients whose tumors have activating EGFR mutations. However, resistance universally develops. Drug persister cells with properties of cancer stem cell-like cells play a major role in drug resistance. In tumors without pre-existing resistant clones, some tumor clonogens survive, despite expressing the driver mutation, to acquire target reactivation or bypass resistance mechanisms. Identification of biomarkers that are mechanistically linked to EGFR TKI resistant cell populations would have clinical utility in early determination of disease recurrence in EGFR mutant NSCLC patients. Previously, we have reported that EGFR-TKI treatment activates Notch3, and here we have identified that Notch3 activates β-catenin transcriptional target, PAI1, which could play a role in drug persistence and could be used for early identification of disease recurrence. Our in vitro ELISA studies have identified an increase in secreted PAI1 levels after treatment with the EGFR TKIs erlotinib and osimertinib demonstrate that measuring PAI1 levels could serve as an indicator of EGFR TKI persistence. To further validate PAI1 as a surrogate biomarker for EGFR TKI treatment, we performed a retrospective study in NSCLC patients (n = 42) with EGFR mutant tumors. Using ELISA to detect PAI1 protein in pre- and post-treatment serum samples as a mechanism-based indicator for β-catenin activation, we observed a broad range in pre-treatment PAI1 levels, spanning from 0.8 ng/ml to 134 ng/ml, suggesting a variable baseline β-catenin activation. Doing survival analysis after dichotomizing patients into low and high baseline PAI1 level groups, we found patients with low basal PAI1 levels had a significantly shorter PFS with EGFR TKI treatment compared to patients with high basal levels. Comparing patients with a ≥ 2-fold induction to those with less than 2-fold, there was a similar trend for worsened survival in those who showed significant induction of PAI1. Overall, we demonstrate that PAI1 expression is a candidate marker for β-catenin activation and the development of drug persisters; thus, it is a potential surrogate marker for poor outcome in patients receiving EGFR TKI therapy and a potential biomarker for selecting patients for β-catenin-targeted therapy. Citation Format: Rajeswara Rao Arasada, Walter Wang, Shankar Suman, David Paul Carbone. PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC [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 4930.

Abstract 331: Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas

Abstract Glioblastoma (GBM) is a devastating primary brain tumor with limited treatment options. Extensive molecular characterization has revealed two particularly frequent mutations: CDKN2A deletion (50-60%) and EGFR (40-50%). EGFRvIII (~35%) is a constitutively active truncation mutant with exons 2-7 deleted. EGFR is a particularly attractive therapeutic target due to frequent activating mutations, such as EGFRvIII, and ready availability of multiple targeted inhibitors. Several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used genetically-engineered mouse astrocytes harboring homozygous deletions of Cdkn2a, as well as EGFRvIII (CEv3). CEv3 astrocytes were made intrinsically resistant to the EGFR TKI gefitinib or erlotinib via long-term exposure, both in vitro and in vivo. We found that long-term gefitinib or erlotinib exposure conferred variable levels of cross resistance to a panel of second- and third-generation EGFR TKI (ΔIC50 1.12-36.1-fold), relative to non-resistant parent lines. We have previously shown that dynamic kinome reprogramming may be responsible for TKI resistance in glioblastoma. Therefore, we used a chemical proteomics method, multiplexed inhibitor beads and mass spectrometry (MIB-MS), to examine changes in the expressed and functional kinome, in both the presence or absence of one of several EGFR TKI known to penetrate the blood-brain barrier. Additionally, we performed RNA sequencing (RNA-seq) to inspect transcriptomic alterations in response to these drugs. RNA-seq showed that resistant CEv3 mouse astrocytes clustered separately from their non-resistant in vitro and in vivo counterparts. Acquired resistance also induced transcriptome alterations governing cellular metabolism, including upregulation of metabolic pathways and downregulation of RNA processing genes. Importantly, the kinase transcriptome was rewired, as 67 kinases were differentially expressed across parental and resistant cell lines (Q<0.001). Probing the dynamic kinome response to afatinib, an EGFR TKI, using RNA-seq identified two potential kinases involved in acute, adaptive resistance to afatinib, Bmx and Ntrk3. Integrated kinome profiling using RNA-seq and MIB-MS in murine models of GBM with defined mutational profiles provides a powerful framework to define novel therapeutic targets that could significantly alter current treatment paradigms. Citation Format: Abby Shelton, Erin Smithberger, Madison Butler, Alex Flores, Ryan Bash, Steve Angus, Noah Sciaky, Harshil Dhruv, Gary L. Johnson, Michael E. Berens, Frank Furnari, C. Ryan Miller. Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas [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 331.

<p>Rhein sensitizes human colorectal cancer cells to EGFR inhibitors by inhibiting STAT3 pathway</p>

BackgroundActivation of epidermal growth factor receptor (EGFR) has been reported in a variety of cancer types, including colorectal cancer (CRC), and represents a potential chemotherapeutic drug target. EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been increasingly applied in the clinical treatment of CRC, but development of drug resistance during the treatment has greatly limited their application. Signal transducer and activator of transcription 3 (STAT3) and its mediated signal transduction pathway play an important role in the occurrence, development and metastasis of CRC, and are related to the development of EGFR-TKI resistance in CRC.MethodsCell viability, colony formation and cellular morphology were examined to evaluate the potent antiproliferative effect of the STAT3 inhibitor napabucasin, LY5 and rhein on the human CRC cell lines HCT116, SW620, RKO and DLD-1. Flow cytometry-based analysis was employed to determine whether rhein can affect the cell cycle and apoptosis. The expression level of phosphorylated STAT3 (P-STAT3), and cell cycle- and apoptosis-related proteins BCL2, CDC2 BAX, Cyclin D1 and Cyclin B1 were detected by Western blot analysis.ResultsThis study revealed that rhein can significantly reduce cell viability and stimulate apoptosis in human CRC cells in a dose-dependent manner. In addition, rhein induced cell cycle arrest at the G2/M phase in CRC cells and dose-dependently inhibited the expression of cell cycle-related proteins. Additionally, it was found that napabucasin, LY5 and rhein considerably sensitized cells to the EGFR-TKI erlotinib, thus suppressing CRC cell proliferation. Rhein also inhibited the phosphorylation of its downstream target STAT3. Inhibition of STAT3 and EGFR phosphorylation was also observed after treatment with a combination of rhein and EGFR inhibitors.ConclusionThis study confirmed the synergistic effect of STAT3 inhibitor and EGFR inhibitor in CRC cell lines. Additionally, we found that rhein sensitizes human CRC cells to EGFR-TKIs by inhibiting STAT3 pathway. When combined with EGFR-TKIs, rhein may be a novel STAT3 inhibitor in CRC.

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A phase II randomized control trial of erlotinib in combination with celecoxib in patients with operable oral squamous cell carcinoma (OSCC): Erlo-Xib Study.

6054 Background: When combined with COX-2 inhibitors, the EGFR tyrosine kinase inhibitor Erlotinib has shown a better antitumor response in preclinical studies. Since high volume hospitals in many countries usually have a longer waiting period for surgery, neoadjuvant targeted therapy may be helpful in reducing disease progression and downstaging oral squamous cell cancers. Methods: Sixty-four treatment-naïve operable oral cancer patients were randomized into a four-arm window of opportunity study consisting of treatment with erlotinib 150mg daily, celecoxib 200mg twice daily, the combination of both or observation alone (NCT02748707). Since the regular wait period for surgery at our hospital was four to five weeks, we planned a 21-day drug treatment versus observation followed by definitive surgery in the fourth week. MRI scans and biopsies were done before and after drug treatment. Post-operative adjuvant treatments were given as per the standard guidelines used for regular patients. Results: There were 10 females and 54 males with a mean and median age of 44 and 45 years respectively. Taking a 20% reduction in the maximum tumor dimension after drug treatment (assessed clinically and radiologically) as partial response, the combination arm had a 60% partial response and a 25% stable disease. Whereas, 60% in the control arm had disease progression. The ratio of the longest tumor dimension at day 21 versus day 0 (Clinical & MRI assessment) also showed a significant difference between the observation vs erlotinib arms (p < 0.001) using Mann-Whitney Test. Grade II/III rashes was the commonly observed toxicity predominantly in the combination arm. Though not powered for survival analysis, a significant difference (p = 0.048) was observed for two-year overall survival for celecoxib + control (60%) versus erlotinib + combination groups (86%) using Kaplan Meier estimator. Biomarker analysis (transcriptome sequencing and IHC) is being done on pre and post-treatment tumor specimens and the final results will be presented. Conclusion: Preoperative targeted therapy with erlotinib and celecoxib combination can arrest disease progression and downstage tumors with possible impact on survival. The identified biomarkers can further refine a future cohort for effective neoadjuvant targeted therapy in oral cancers. Clinical trial information: CTRI/2012/07/002828.

The repression and reciprocal interaction of DNA methyltransferase 1 and specificity protein 1 contributes to the inhibition of MET expression by the combination of Chinese herbal medicine FZKA decoction and erlotinib

Ethnopharmacological relevanceThe Chinese herbal medicine Fuzheng Kang-Ai (FZKA) decoction obtained from Guangdong Kangmei Pharmaceutical Company, which contains 12 components with different types of constituents, has been used as part of the adjuvant treatment of lung cancer for decades. We previously showed that FZKA decoction enhances the growth inhibition of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant non-small cell lung cancer (NSCLC) cells by suppressing glycoprotein mucin 1 (MUC1) expression. However, the molecular mechanism underlying the therapeutic potential, particularly in sensitizing or/and enhancing the anti-lung cancer effect of EGFR-TKIs, remains unclear. Materials and methodsCell viability was measured using 3-(4, 5-diMEThylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 5-ethynyl -2′-deoxyuridine (EdU) assays. Western blot analysis was performed to examine the protein expressions of DNA methyltransferase 1 (DNMT1), specificity protein 1 (SP1), and MET, an oncogene encoding for a trans-membrane tyrosine kinase receptor activated by the hepatocyte growth factor (HGF). The expression of MET mRNA was measured by quantitative real-time PCR (qRT-PCR). Exogenous expression of DNMT1 and SP1, and MET were carried out by transient transfection assays. The promoter activity of MET was tested using Dual-luciferase reporter assays. A nude mouse xenografted tumor model further evaluated the effect of the combination of FZKA decoction and erlotinib in vivo. ResultsThe combination of FZKA and erlotinib produced an even greater inhibition of NSCLC cell growth. FZKA decreased the expressions of DNMT1, SP1, and MET (c-MET) proteins, and the combination of FZKA and erlotinib demonstrated enhanced responses. Interestingly, there was a mutual regulation of DNMT1 and SP1. In addition, exogenously expressed DNMT1 and SP1 blocked the FZKA-inhibited c-MET expression. Moreover, excessive expressed MET neutralized FZKA-inhibited growth of NSCLC cells. FZKA decreased the mRNA and promoter activity of c-MET, which was not observed in cells with ectopic expressed DNMT1 gene. Similar findings were observed in vivo. ConclusionFZKA decreases MET gene expression through the repression and mutual regulation of DNMT1 and SP1 in vitro and in vivo. This leads to inhibit the growth of human lung cancer cells. The combination of FZKA and EGFR-TKI erlotinib exhibits synergy in this process. The regulatory loops among the DNMT1, SP1 and MET converge in the overall effects of FZKA and EGFR-TKI erlotinib. This in vitro and in vivo study clarifies an additional novel molecular mechanism underlying the anti-lung cancer effects in response to the combination of FZKA and erlotinib in gefitinib-resistant NSCLC cells.

Relative efficacy of interventions in the treatment of second-line non-small cell lung cancer: a systematic review and network meta-analysis

BackgroundLocally advanced or metastatic non-small cell lung cancer (NSCLC) that has progressed after first-line treatment has a poor prognosis. Recent randomized clinical trials (RCTs) have demonstrated survival benefits of alternative treatments to docetaxel. However, information is lacking on which patients benefit the most and what drug or regimen is optimal. We report a systematic review and network meta-analysis (NMA) of second-line treatments in all subgroup combinations determined by histology, programmed death ligand 1 (PD-L1) expression, and epidermal growth factor receptor (EGFR) mutation.MethodsMEDLINE, PubMed, EMBASE, Biosciences Information Service (using the Dialog Platform), Cochrane Library, and abstracts from scientific meetings were searched for RCTs published up to September 2015. Key outcomes were overall survival (OS) and progression-free survival (PFS). Bayesian hierarchical exchangeable NMAs were conducted to calculate mean survival times and relative differences for eight subgroups, using docetaxel as the reference comparator. For OS, the NMA was based on hazard ratios applied to a first-order fractional polynomial model fitted to the reference treatment. For PFS, a second-order fractional polynomial model was fitted to reconstructed patient-level data for the entire network of evidence.ResultsThe search identified 30 studies containing 17 different treatment regimens. Docetaxel plus ramucirumab was associated with a significant improvement in OS and PFS, relative to docetaxel, regardless of patient type. Docetaxel plus nintedanib showed similar efficacy to docetaxel plus ramucirumab in the nonsquamous populations. EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib showed superior levels of efficacy in EGFR mutation-positive populations and the one PD-1 immunotherapy (nivolumab) studied showed superior efficacy in the populations exhibiting high PD-L1 expression.ConclusionsIn the absence of head-to-head comparisons, we performed a mixed-treatment analysis to synthesize evidence of the efficacy of each treatment. Benefits are optimized by targeting specific treatments to individual patients guided by histology, PD-L1 expression, and EGFR mutation status.Systematic review registrationThis review is registered in PROSPERO (registration number: CRD42014013780 available at www.crd.york.ac.uk/PROSPERO).

Development of treatment options for Chinese patients with advanced squamous cell lung cancer: focus on afatinib.

Lung cancer is the leading cause of cancer death in China, and approximately one third of these cancers are squamous cell carcinoma (SqCC) of the lung. Ethnic diversity and country-specific environmental factors can account for interindividual variations in response to and tolerability of anticancer therapies. Although several targeted therapies have recently been approved for patients with relapsed/refractory SqCC of the lung, only afatinib, an irreversible ErbB family blocker, has data of Chinese patients. In the Phase III LUX-Lung 8 trial, afatinib demonstrated a significant clinical benefit vs the reversible first-generation EGFR tyrosine kinase inhibitor erlotinib in both the overall population and the Chinese subset, with a manageable safety profile. Emerging biomarker data from LUX-Lung 8 suggest that patients with ErbB mutations, especially ErbB2, and those classified as “good” in the VeriStrat® proteomic test, may benefit from afatinib treatment in particular, regardless of ethnicity, and may get a long-term response. In conclusion, afatinib is a valid second-line option for Chinese patients with SqCC of the lung, and specific biomarkers may help guide in treatment decision-making. Ongoing studies will provide further guidance on afatinib’s place in the treatment algorithm, alongside the other novel targeted therapies.

Role of osimertinib in the treatment of EGFR-mutation positive non-small-cell lung cancer.

Mutations in the EGFR occur in approximately 10-35% of non-small-cell lung cancer (NSCLC) patients. Osimertinib is a third-generation oral small molecule inhibitor of EGFR, active against the common targetable activating EGFR mutations in L858R and exon 19 deletion; it also inhibits the T790M mutation. It was initially developed and approved for the treatment of acquired resistance to EGFR inhibition mediated by the T790M pathway activation. Recently, the FLAURA trial showed significantly improved progression-free survival with osimertinib compared with the first generation EGFR tyrosine kinase inhibitors gefitinib or erlotinib; this has led to its approval by US FDA and European Medicines Agency (EMA) as frontline therapy. Ongoing studies will define the resistance mechanisms to osimertinib, novel combination approaches and role in earlier stages of NSCLC.