Molecular mechanism of action and potential biomarkers of growth inhibition of synergistic combination of afatinib and dasatinib against gefitinib-resistant non-small cell lung cancer cells.
ABSTRACT: Epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI) is the first choice of treatment for advanced non-small cell lung cancer (NSCLC) patients harbouring activating EGFR mutations. However, single agent usually has limited efficacy due to heterogeneous resistant mechanisms of cancer cells. Thus drug combination therapy would offer more benefits by synergistic interactions and avoidance of resistance emergence. In this study, we selected 8 NSCLC cell lines with different genetic characteristics as research models to investigate the efficacy of 4 agents (gefitinib, cetuximab, afatinib and dasatinib) and their combinations. As a single agent, both afatinib and dasatinib showed more inhibition against cell proliferation than gefitinib and cetuximab. Afatinib combined with dasatinib demonstrated significantly high efficacy against 7 gefitinib-resistant NSCLC cell lines. Moreover, it reversed the resistance to the 4 studied single agents in PTEN mutated NSCLC cells. By studying the activity of EGFR, Src and their downstream signalling pathways including PI3K/PTEN/Akt, Ras/Raf/MEK/ERK, Src/FAK and JAK/Stat, we demonstrated the synergistic interaction between afatinib and dasatinib was not only due to their blockage of different signalling pathways but also the complemental inhibition of the related signalling molecules such as Stat3. We also found that the level of Src, Stat3, and MAPK may be useful biomarkers predicating synergism between afatinib and dasatinib for the treatment of gefitinib-resistant NSCLC cells.
Project description:Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M.We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined.We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib-treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib.Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M.
Project description:Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non-Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs.
Project description:<h4>Background</h4>Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC) patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs.<h4>Results</h4>Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits cell adhesion-related response and greatly enhances the cell-killing effects of EGFR TKI (gefitinib for the PC9 cells; afatinib for the H1975 cells) in NSCLC cells, which would otherwise escape the TKI-induced apoptosis.<h4>Conclusion</h4>Results from this study indicate that NSCLC cells can employ the adhesion response as a survival pathway to survive under EGFR-targeted therapy. Simultaneous targeting of EGFR signaling and adhesion pathways would further boost the efficacy of EGFR-targeted therapy in NSCLC.
Project description:Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.
Project description:Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase-3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.
Project description:BACKGROUND:The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab. METHODS:NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index. RESULTS:EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA. CONCLUSIONS:EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.
Project description:Gefitinib, erlotinib or afatinib are the current treatment for non-small-cell lung cancer (NSCLC) harboring an activating mutation of the epidermal growth factor receptor (EGFR), but less than 5% of patients achieve a complete response and the median progression-free survival is no longer than 12 months. Early adaptive resistance can occur as soon as two hours after starting treatment by activating signal transducer and activation of transcription 3 (STAT3) signaling. We investigated the activation of STAT3 in a panel of gefitinib-sensitive EGFR mutant cell lines, and gefitinib-resistant PC9 cell lines developed in our laboratory. Afatinib has great activity in gefitinib-sensitive as well as in gefitinib-resistant EGFR mutant NSCLC cell lines. However, afatinib therapy causes phosphorylation of STAT3 tyrosine 705 (pSTAT3Tyr705) and elevation of STAT3 and RANTES mRNA levels. The combination of afatinib with TPCA-1 (a STAT3 inhibitor) ablated pSTAT3Tyr705 and down-regulated STAT3 and RANTES mRNA levels with significant growth inhibitory effect in both gefitinib-sensitive and gefitinib-resistant EGFR mutant NSCLC cell lines. Aldehyde dehydrogenase positive (ALDH+) cells were still observed with the combination at the time that Hairy and Enhancer of Split 1 (HES1) mRNA expression was elevated following therapy. Although the combination of afatinib with STAT3 inhibition cannot eliminate the potential problem of a remnant cancer stem cell population, it represents a substantial advantage and opportunity to further prolong progression free survival and probably could increase the response rate in comparison to the current standard of single therapy.
Project description:OBJECTIVE:To compare the efficacy and safety of first line treatments for patients with advanced epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC). DESIGN:Systematic review and network meta-analysis. DATA SOURCES:PubMed, Embase, Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, and several international conference databases, from inception to 20 May 2019. ELIGIBILITY CRITERIA FOR SELECTING STUDIES:Published and unpublished randomised controlled trials comparing two or more treatments in the first line setting for patients with advanced EGFR mutated NSCLC were included in a bayesian network meta-analysis. Eligible studies reported at least one of the following clinical outcome measures: progression free survival, overall survival, objective response rate, and adverse events of grade 3 or higher. RESULTS:18 eligible trials involved 4628 patients and 12 treatments: EGFR tyrosine kinase inhibitors (TKIs; osimertinib, dacomitinib, afatinib, erlotinib, gefitinib, and icotinib), pemetrexed based chemotherapy, pemetrexed free chemotherapy, and combination treatments (afatinib plus cetuximab, erlotinib plus bevacizumab, gefitinib plus pemetrexed based chemotherapy, and gefitinib plus pemetrexed). Consistent with gefitinib plus pemetrexed based chemotherapy (hazard ratio 0.95, 95% credible interval 0.72 to 1.24), osimertinib showed the most favourable progression free survival, with significant differences versus dacomitinib (0.74, 0.55 to 1.00), afatinib (0.52, 0.40 to 0.68), erlotinib (0.48, 0.40 to 0.57), gefitinib (0.44, 0.37 to 0.52), icotinib (0.39, 0.24 to 0.62), pemetrexed based chemotherapy (0.24, 0.17 to 0.33), pemetrexed free chemotherapy (0.16, 0.13 to 0.20), afatinib plus cetuximab (0.44, 0.28 to 0.71), and gefitinib plus pemetrexed (0.65, 0.46 to 0.92). Osimertinib and gefitinib plus pemetrexed based chemotherapy were also consistent (0.94, 0.66 to 1.35) in providing the best overall survival benefit. Combination treatments caused more toxicity in general, especially erlotinib plus bevacizumab, which caused the most adverse events of grade 3 or higher. Different toxicity spectrums were revealed for individual EGFR-TKIs. Subgroup analyses by the two most common EGFR mutation types indicated that osimertinib was associated with the best progression free survival in patients with the exon 19 deletion, and gefitinib plus pemetrexed based chemotherapy was associated with the best progression free survival in patients with the Leu858Arg mutation. CONCLUSIONS:These results indicate that osimertinib and gefitinib plus pemetrexed based chemotherapy were associated with the best progression free survival and overall survival benefits for patients with advanced EGFR mutated NSCLC, compared with other first line treatments. The treatments resulting in the best progression free survival for patients with the exon 19 deletion and Leu858Arg mutations were osimertinib and gefitinib plus pemetrexed based chemotherapy, respectively. SYSTEMATIC REVIEW REGISTRATION:PROSPERO CRD42018111954.
Project description:First-generation, reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, represented an important addition to the treatment armamentarium for non-small-cell lung cancer (NSCLC) patients with activating EGFR mutations. However, all patients inevitably develop acquired resistance to these agents, primarily due to secondary EGFR mutations, molecular aberrations affecting other signaling pathways, or transformation to small-cell histology. It was hypothesized that development of second-generation TKIs with broader inhibitory profiles could confer longer-lasting clinical activity and overcome acquired resistance to first-generation inhibitors. Here, we review the development of afatinib, an irreversible ErbB family blocker that potently inhibits signaling of all homodimers and heterodimers formed by the EGFR, human epidermal growth factor receptor (HER)-2, HER3, and HER4 receptors. In two phase III trials in patients with EGFR mutation-positive NSCLC, first-line afatinib significantly improved progression-free survival (PFS) and health-related quality of life versus standard-of-care chemotherapy. Moreover, in preplanned sub-analyses, afatinib significantly improved overall survival in patients harboring EGFR Del19 mutations. Afatinib has also demonstrated clinical activity in NSCLC patients who had progressed on erlotinib/gefitinib, particularly when combined with cetuximab, and offers 'treatment beyond progression' benefit when combined with paclitaxel versus chemotherapy alone. Furthermore, a recent phase III study demonstrated that PFS was significantly improved with afatinib versus erlotinib for the second-line treatment of patients with squamous cell carcinoma of the lung. The activity of afatinib in both first-line and relapsed/refractory settings may reflect its ability to irreversibly inhibit all ErbB family members. Afatinib has a well-defined safety profile with characteristic gastrointestinal (diarrhea, stomatitis) and cutaneous (rash/acne) adverse events.
Project description:UNLABELLED:EGFR-mutant lung cancers responsive to reversible EGFR inhibitors (gefitinib/erlotinib) develop acquired resistance, mediated by second-site EGFR T790M mutation in >50% of cases. Preclinically, afatinib (irreversible ErbB family blocker) plus cetuximab (anti-EGFR monoclonal antibody) overcomes T790M-mediated resistance. This phase Ib study combining afatinib and cetuximab enrolled heavily pretreated patients with advanced EGFR-mutant lung cancer and acquired resistance to erlotinib/gefitinib. Patients provided post-acquired-resistance tumor samples for profiling EGFR mutations. Among 126 patients, objective response rate (overall 29%) was comparable in T790M-positive and T790M-negative tumors (32% vs. 25%; P = 0.341). Median progression-free survival was 4.7 months (95% confidence interval, 4.3-6.4), and the median duration of confirmed objective response was 5.7 months (range, 1.8-24.4). Therapy-related grade 3/4 adverse events occurred in 44%/2% of patients. Afatinib-cetuximab demonstrated robust clinical activity and a manageable safety profile in EGFR-mutant lung cancers with acquired resistance to gefitinib or erlotinib, both with and without T790M mutations, warranting further investigation. SIGNIFICANCE:This article reports the results of a trial combining afatinib and cetuximab in patients with acquired resistance and details the first clinical proof-of-concept for the preclinical hypothesis that a significant proportion of tumors in patients with acquired resistance to gefitinib/erlotinib remain dependent on EGFR signaling for survival.