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Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel + androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-κB transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44+ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44+ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evidence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer. Mol Cancer Ther; 13(5); 1270–84. ©2014 AACR.

PDF - 265K, Biochemical-relapse analysis according to gene expression levels of markers differentially expressed in treated vs non-treated patients.

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PDF - 72K, Genes expression values by qRT-PCR in treated patients vs non-treated patients; TLDA: TaqMan Low Density Arrays; FDR: False Discovery Rate.

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Docetaxel-based chemotherapy is the standard first-line therapy in metastatic castration-resistant prostate cancer (CRPC). However, most patients eventually develop resistance to this treatment. In this study, we aimed to identify key molecular genes and networks associated with docetaxel resistance in two models of docetaxel-resistant CRPC cell lines and to test for the most differentially expressed genes in tumor samples from patients with CRPC. DU-145 and PC-3 cells were converted to docetaxel-resistant cells, DU-145R and PC-3R, respectively. Whole-genome arrays were used to compare global gene expression between these four cell lines. Results showed differential expression of 243 genes (P < 0.05, Bonferroni-adjusted P values and log ratio > 1.2) that were common to DU-145R and PC-3R cells. These genes were involved in cell processes like growth, development, death, proliferation, movement, and gene expression. Genes and networks commonly deregulated in both DU-145R and PC-3R cells were studied by Ingenuity Pathways Analysis. Exposing parental cells to TGFB1 increased their survival in the presence of docetaxel, suggesting a role of the TGF-β superfamily in conferring drug resistance. Changes in expression of 18 selected genes were validated by real-time quantitative reverse transcriptase PCR in all four cell lines and tested in a set of 11 FFPE and five optimal cutting temperature tumor samples. Analysis in patients showed a noteworthy downexpression of CDH1 and IFIH1, among others, in docetaxel-resistant tumors. This exploratory analysis provides information about potential gene and network involvement in docetaxel resistance in CRPC. Further clinical validation of these results is needed to develop targeted therapies in patients with CRPC that can circumvent such resistance to treatment. Mol Cancer Ther; 11(2); 329–39. ©2011 AACR.

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PDF - 475K, Radiological-progression analysis according to gene expression levels of markers differentially expressed in treated vs non-treated patients.

PDF - 35K, Legends for Supplementary Figures 1 and 2.

Epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma are a frequent class of driver mutations. Single EGFR tyrosine kinase inhibitor (TKI) provides substantial clinical benefit, but almost nil radiographic complete responses. Patients invariably progress, although survival can reach several years with post-treatment therapies, including EGFR TKIs, chemotherapy or other procedures. Endeavours have been clinically oriented to manage the acquisition of EGFR TKI-resistant mutations; however, basic principles on cancer evolution have not been considered in clinical trials. For years, evidence has displayed rapidly adaptive mechanisms of resistance to selective monotherapy, posing several dilemmas for the practitioner. Strict adherence to non-small cell lung cancer (NSCLC) guidelines is not always practical for addressing the clinical progression that EGFR-mutant lung adenocarcinoma patients suffer. The purpose of this review is to highlight regulatory mechanisms and signalling pathways that cause therapy-induced resistance to EGFR TKIs. It suggests combinatorial therapies that target EGFR, as well as potential mechanisms underlying EGFR-mutant NSCLC, alerting the reader to clinical opportunities that may lead to a deeper and more durable response. Molecular reprogramming contributes to EGFR TKI resistance, and the compiled information is relevant in understanding the development of new combined targeted strategies in EGFR-mutant NSCLC.

Lung cancer (LC) is the leading cause of cancer deaths worldwide, being non-small lung cancer (NSCLC) sub-types the most prevalent. Since most LC cases are only detected during the last stage of the disease the high mortality rate is strongly associated with metastases. For this reason, the migratory and invasive capacity of these cancer cells as well as the mechanisms involved have long been studied to uncover novel strategies to prevent metastases and improve the patients' prognosis. This narrative review provides an overview of the main in vitro migration and invasion assays employed in NSCLC research. While several methods have been developed, experiments using conventional cell culture models prevailed, specifically the wound-healing and the transwell migration and invasion assays. Moreover, it is provided herewith a summary of the available information concerning chemical contaminants that may promote the migratory/invasive properties of NSCLC cells in vitro, shedding some light on possible LC risk factors. Most of the reported agents with pro-migration/invasion effects derive from cigarette smoking [e.g., Benzo(a)pyrene and cadmium] and air pollution. This review further presents several studies in which different dietary/plant-derived compounds demonstrated to impair migration/invasion processes in NSCLC cells in vitro. These chemicals that have been proposed as anti-migratory consisted mainly of natural bioactive substances, including polyphenols non-flavonoids, flavonoids, bibenzyls, terpenes, alkaloids, and steroids. Some of these compounds may eventually represent novel therapeutic strategies to be considered in the future to prevent metastasis formation in LC, which highlights the need for additional in vitro methodologies that more closely resemble the in vivo tumor microenvironment and cancer cell interactions. These studies along with adequate in vivo models should be further explored as proof of concept for the most promising compounds.

Background: The role of MET alterations in non-small cell lung cancer (NSCLC) is increasing and several targeted agents are under evaluation. MET exon 14 skipping mutations and MET amplifications are associated with potential sensitivity to MET inhibition, though resistance mechanisms are emerging. In MET addicted cells, MET inhibition leads to activation of proviral integration site for Moloney murine leukemia virus-1 (PIM1). PIM1 and proto-oncogene tyrosine-protein kinase Src (SRC) can regulate the expression of receptor tyrosine kinases (RTKs), potentially inducing resistance to MET inhibition through cross-activation. Methods: We evaluated the activity of class I-II MET inhibitors, the SRC inhibitor dasatinib, and panPIM inhibitors in four MET addicted cell lines. We assessed the effect of the dual MET/PIM and MET/ SRC inhibition on cell viability and at the protein level. We evaluated RNA expression profiles of the cell lines. Advanced NSCLCs were also screened for MET alterations. Results: All cell lines were sensitive to class I-II MET inhibitors. All cell lines were resistant to single PIM and SRC inhibition. Dual MET/PIM inhibition was synergistic or additive in MET amplified cell lines and dual MET/SRC inhibition was highly synergistic in all MET addicted cell lines. The addition of an SRC inhibitor partially prevents the RTKs cross-activation. MET alterations were found in 9 out of 97 evaluable samples (9.3%); median overall survival in MET altered patients was 5 months (95% CI, 3 m-NA). Conclusions: We identified a potential role of PIM inhibition in MET amplified tumors and of SRC inhibition in MET addicted tumors. Potential applications of this new treatment strategy warrant further evaluation.

Introduction p21-activated kinase 1 (PAK1) stimulates growth and metastasis in non-small cell lung cancer (NSCLC). Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models. Methods The effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models. Results The combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment. Conclusions Combination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.

Introduction Epidermal growth factor receptor (EGFR) pathway deregulation promotes the acquisition of stemlike properties in non–small-cell lung cancer. EGFR inhibition through NOTCH enriches lung cancer stem cells (CSCs). Src through Yes-associated protein 1 (YAP1) activates NOTCH. Signal transduction and activator of transcription 3 (STAT3) activation occurs upon EGFR blockade and regulates the generation of CSCs. Patients and Methods Using the Aldefluor assay kit, we investigated the enrichment of aldehyde dehydrogenase (ALDH)-positive cells in EGFR-mutation–positive cells treated with gefitinib, afatinib, and osimertinib. Western blot analysis was performed to evaluate changes in CSC marker expression upon EGFR blockade. We performed gene expression analysis in a cohort of EGFR-mutation–positive non–small-cell lung cancer patients. We evaluated the association of gene expression with treatment outcomes. Results The cell subpopulation surviving EGFR inhibition had high ALDH activity and elevated CSC marker expression. Concurrent inhibition of EGFR, STAT3, and Src diminished the CSC subpopulation in an EGFR-mutation–positive cellular model. In a cohort of 64 EGFR-mutation–positive patients, 2 ALDH1 isoforms and the NOTCH target hairy and enhancer of split 1 (HES1), when highly expressed, were predictive of worse outcome to EGFR blockade. The gene expression of B-cell–specific Moloney murine leukemia virus integration site 1 (Bmi-1) that maintains the self-renewal of stem cells was also related to treatment outcome. Conclusion Single EGFR inhibitors increase the population of CSCs. Combinatory therapy targeting STAT3 and Src may be of potential benefit. ALDH1, HES1, and Bmi-1 are essential biomarkers in the initial assessment of EGFR-mutation–positive patients.

Non-small cell lung cancer (NSCLC) tumors harboring mutations in EGFR ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs). Here, we show that resistant cells without the p.T790M or other acquired mutations are sensitive to the Aurora B (AURKB) inhibitors barasertib and S49076. Phospho-histone H3 (pH3), a major product of AURKB, is increased in most resistant cells and treatment with AURKB inhibitors reduces the levels of pH3, triggering G1/S arrest and polyploidy. Senescence is subsequently induced in cells with acquired mutations while, in their absence, polyploidy is followed by cell death. Finally, in NSCLC patients, pH3 levels are increased after progression on EGFR TKIs and high pH3 baseline correlates with shorter survival. Our results reveal that AURKB activation is associated with acquired resistance to EGFR TKIs, and that AURKB constitutes a potential target in NSCLC progressing to anti-EGFR therapy and not carrying resistance mutations., Non-small cell lung cancer with EGFR mutations are known to develop resistance to EGFR tyrosine kinase inhibitors. Here, the authors show AURKB activation to be associated with resistance in EGFR mutant lung cancer cells, and that AURKB is a therapeutic target in resistant tumours that lack the p.T790M or other acquired mutations.

Lung cancer (LC) is the leading cause of cancer deaths worldwide, being non-small lung cancer (NSCLC) sub-types the most prevalent. Since most LC cases are only detected during the last stage of the disease the high mortality rate is strongly associated with metastases. For this reason, the migratory and invasive capacity of these cancer cells as well as the mechanisms involved have long been studied to uncover novel strategies to prevent metastases and improve the patients’ prognosis. This narrative review provides an overview of the main in vitro migration and invasion assays employed in NSCLC research. While several methods have been developed, experiments using conventional cell culture models prevailed, specifically the wound-healing and the transwell migration and invasion assays. Moreover, it is provided herewith a summary of the available information concerning chemical contaminants that may promote the migratory/invasive properties of NSCLC cells in vitro, shedding some light on possible LC risk factors. Most of the reported agents with pro-migration/invasion effects derive from cigarette smoking [e.g., Benzo(a)pyrene and cadmium] and air pollution. This review further presents several studies in which different dietary/plant-derived compounds demonstrated to impair migration/invasion processes in NSCLC cells in vitro. These chemicals that have been proposed as anti-migratory consisted mainly of natural bioactive substances, including polyphenols non-flavonoids, flavonoids, bibenzyls, terpenes, alkaloids, and steroids. Some of these compounds may eventually represent novel therapeutic strategies to be considered in the future to prevent metastasis formation in LC, which highlights the need for additional in vitro methodologies that more closely resemble the in vivo tumor microenvironment and cancer cell interactions. These studies along with adequate in vivo models should be further explored as proof of concept for the most promising compounds.

Advanced NSCLC patients harboring EML4-ALK and CCDC6-RET rearrangements derive benefit from treatment with ALK and RET TKIs but not immune checkpoint inhibitors. New immunotherapeutic approaches, such as immunization against growth factors, can be of particular interest for combination treatment in these patients. Here, we investigated the effects of anti-EGF antibodies generated by vaccination (anti-EGF VacAbs), TKIs and combinations in EML4-ALK and CCDC6-RET NSCLC cell lines. We found that EGF and tumor growth factor alpha (TGFα) significantly decreased the antiproliferative activity of the RET inhibitor BLU-667 in CCDC6-RET cells and brigatinib, alectinib and crizotinib in EML4-ALK translocated cells. The addition of anti-EGF VacAbs reversed the effects of EGF and TGFα, potentiated the antitumor effects of the kinase inhibitors and delayed the appearance in vitro of resistant clones. Western blotting demonstrated that the combination of anti-EGF VacAbs with ALK or RET TKIs effectively suppressed EGFR downstream pathways in EML4-ALK translocated and CCDC6-RET cells, respectively. In conclusion, anti-EGF VacAbs significantly increased the antitumor activity of TKIs in ALK and RET-positive cell lines. Clinical trials of an EGF vaccine in combination with ALK and RET TKIs are warranted.

Funding: supported by a La Caixa Foundation grant and the Spanish Association Against Cancer (PROYE18012ROSE) EGFR mutation-positive lung adenocarcinoma (LUAD) displays impaired phosphorylation of ERK and Src-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. We hypothesize that SHP2 expression could be predictive in patients positive with resected EGFR mutation versus patients with EGFR wild-type LUAD. We examined resected LUAD cases from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1, and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction. The activity of SHP2 inhibitors plus erlotinib were tested in EGFR -mutant cell lines and analyzed by cell viability assay, Western blot, and immunofluorescence. A total of 50 of 100 EGFR mutation-positive LUADs relapsed, among them, patients with higher SHP2 mRNA expression revealed shorter progression-free survival, in comparison with those having low SHP2 mRNA (hazard ratio: 1.83; 95% confidence interval: 1.05-3.23; p = 0.0329). However, SHP2 was not associated with prognosis in the remaining 167 patients with wild-type EGFR. In EGFR -mutant cell lines, the combination of SHP099 or RMC-4550 (SHP2 inhibitors) with erlotinib revealed synergism via abrogation of phosphorylated AKT (S473) and ERK1/2 (T202/Y204). Although erlotinib translocates phosphorylated SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocates SHP2 into the cytoplasm membrane, limiting AKT and ERK1/2 activation. Elevated SHP2 mRNA levels are associated with recurrence in resected EGFR mutation-positive LUADs, but not in EGFR wild-type. EGFR tyrosine kinase inhibitors can enhance SHP2 activation, hindering adjuvant therapy. SHP2 inhibitors could improve the benefit of adjuvant therapy in EGFR mutation-positive LUADs.

Introduction: Stage IIIB-IV non-small cell lung cancer patients with mutations in the EGF receptor gene (EGFR) usually derive clinical benefit from to tyrosine kinase inhibitors (EGFR TKIs) but ultimately relapse. In preclinical studies, we have showed that anti-EGF antibodies generated by vaccination significantly increased the antitumor activity of TKIs in EGFR-mut cell lines, blocking EGFR, Erk1/2, Akt and STAT3 activation and delaying emergence of resistance. Based on these findings, the EPICAL trial was initiated (ClinicalTrials.gov number, NCT03623750). Methods: The EPICAL was a single arm, phase 1b, single arm study to evaluate the safety and efficacy of first line anti-EGF vaccination combined with afatinib. The trial enrolled advanced NSCLC patients with sensitizing EGFR mutations confirmed in a central laboratory. Patients received 40 mg/day of afatinib and five intramuscular anti-EGF vaccinations every 14 days and then every three months until progression. Four medical centers in Spain participated, with a target enrollment of 30 patients. However, the COVID-19 outbreak forced an early termination of the study in March 2020 with only 23 patients included. Serial blood samples were collected and used to evaluate the levels of selected growth factors by ELISA and biological activity by addition of sera to in vitro cultures of EGFR-mut cells followed by Western blotting. Results: Of the 23 patients enrolled in the trial, nine (39%) had exon 19 in-frame deletions, twelve (52%) exon 21 substitutions and two (9%) exon 18 missense mutations. Combination treatment was well tolerated and no SAES related to anti-EGF vaccination were reported. Objective response and disease control rates were 78.3% (95%CI=53.6-92.5) and 95.7% (95%CI=78.1-99.9), respectively. At data cut-off, with a median follow-up of 11.4 months (95%CI=8.1-15.2), the median progression-free survival was 17.4 months (95% CI=13.22-NA) and median survival not reached (95% CI=15.21-NA). Median PFS for patients with exon 19 deletions and exon 21 point mutations were 13.9 months (95%CI=8.7-NR) and 17.4 months (95%CI=13.2-NR), respectively. Three months after initiation of treatment, high titers of anti-EGF antibodies were detected in all patients and serum EGF and TGFα levels were found to be significantly lower compared to baseline levels. Finally, treatment with post-vaccination patient’s sera inhibited EGFR, AKT and ERK1/2 phosphorylation in EGFR-mut cells growing in vitro. Conclusion: The combination of an anti–EGF vaccine with afatinib is well tolerated and induces a sustained immunogenic effect. Vaccination against EGF might enhance the clinical efficacy of EGFR TKIs. Keywords: anti-EGF vaccination, EGFR-mutant non-small cell lung cancer, EGFR inhibitors

Background: The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGFR mutations. However, acquired resistance appears invariably due to several mechanisms. The strategy of using EGF-targeted nanobodies (Nbs) to block the initial step of the EGFR pathway constitutes a new research area. Nbs also offer several advantages compared to traditional mAbs, such as their reduced size, higher stability and tissue penetration, low production costs and lack of Fc effector function, which provide key advantages for targeting soluble tumoral growth factors. In this study we investigated the efficacy of anti-EGF Nbs to reduce osimertinib-resistance. Experimental procedures: Cell viability and colony formation assays were performed in PC9 and PC9-osimertinib-resistant NSCLC cell lines tested with anti-EGF Nbs alone or in combination with osimertinib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study protein expression and activation. Summary of data: Two anti-EGF Nbs, generated in our laboratory, inhibited cell viability and colony formation in PC9 and PC9-derived osimertinib-resistant cell lines. The combination of these Nbs with osimertinib improved the antitumoral efficacy of this EGFR-TKI in cell viability and colony formation experiments. The combination of anti-EGF Nbs with osimertinib clearly decreases the activation of proteins involved in the EGFR pathway, such as, EGFR, Akt and Erk 1/2. In addition, it increases cellular apoptosis, and decreases the expression of Hes1, a marker of cancer stem cells involved in metastasis and osimertinib resistance mechanisms. Conclusions: We conclude that the combination of anti-EGF nanobodies and osimertinib exhibits a good efficacy in inhibiting cell viability and colony formation, while, inhibiting expression and activation of proteins involved in osimertinib resistance. Citation Format: Salvador Guardiola, Macarena Sánchez-Navarro, Ernest Giralt, Rafael Rosell, Jordi Codony-Servat. Nanobodies against EGF enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1839.

Monotherapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) still leads to incomplete responses in most EGFR-mutation positive non-small cell lung cancer (NSCLC) patients, often due to acquired resistance through activation of parallel compensatory pathways. We have previously shown that co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3), and Src-yes-associated protein 1 (YAP1) was highly synergistic in vitro and in vivo. In the present study, we treated EGFR-mutation positive cell lines with the combination of osimertinib plus a natural compound, pterostilbene, which has been reported to abrogate Src and STAT3 activation. Methods: Cell viability assays and immunoblotting were performed to reveal the mechanisms of action of pterostilbene, osimertinib and pterostilbene plus osimertinib in five EGFR-mutation positive NSCLC and one triple negative breast cancer (TNBC) cell lines. Results: Osimertinib plus pterostilbene yielded synergistic effects in all EGFR-mutation positive NSCLC cell lines investigated. Surprisingly, pterostilbene alone did not inhibit, nor downregulate Src phosphorylation in the EGFR-mutation positive NSCLC cell lines or the TNBC cell line, MDA-MB-231. However, the double combination of osimertinib plus pterostilbene reversed the osimertinib-induced STAT3, YAP1, and CUB domain-containing protein-1 (CDCP1) phosphorylation and slightly suppressed Src phosphorylation in PC9 and H1975 cells. Conclusion: The results of this study indicate that pterostilbene may be used to abrogate the activated resistance pathways of single osimertinib treatment in EGFR-mutation positive NSCLC. Future studies should focus on in vivo translation and confirmation of these results.

Background: The activation of multiple signaling pathways jeopardizes the clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutation positive non-small cell lung cancer (NSCLC). Integrin-1 inked kinase (ILK) regulates the interactions between tumor cells and extracellular environment to activate signaling pathways and promote cell proliferation, migration, and epithelial-mesenchymal transition. Src homology 2 domain-containing phosphatase 2 (SHP2) is essential for receptor tyrosine kinase signaling and mitogenactivated protein kinase (MAPK) pathway activation. Methods: We analyzed tumor ILK,13-receptor subunit glycoprotein 130 (gp130), SHP2, and stromal hepatocyte growth factor (HGF) and interleukin-6 (IL-6) mRNA expression in baseline tumor specimens of advanced EGFR-mutation positive NSCLC patients treated with EGFR TKIs. Results: ILK, when highly expressed, was an independent poor prognostic factor for the progression-free survival of the patients, both in the univariate (hazard ratio [HR for disease progression, 2.49; 95% Cl, 1.37-452; P .0020]) and in the multivariate (HR 3.74; 95% Cl, 1.33-10.56; P.0126) Cox regression model. Patients with high SHP2 expression had an almost 13-month shorter progression-free survival (P.0094) and an 18 month shorter overall survival (P.0182) in comparison to those with low SHP2 mRNA expression. Interpretation: The levels of ILK and SHP2 could be predictive for upfront combinatory therapy of EGFR TKIs plus SHP2 or ILK inhibitors. Fund: A grant from La Caixa Foundation, an Institute de Salud Carlos III grant (RESPONSE, PIE16/00011), an Institute de Salud Carlos III grant (PI14/01678), a Marie Sklodowska-Curie Innovative Training Networks European Grant (ELBA No 765492) and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE). (C) 2018 The Authors. Published by Elsevier B.V.

Background: Osimertinib improve therapy for non-small cell lung cancer (NSCLC). However, invariable acquired resistance appears. Methods: MTT assay was used to analyze cell viability. Protein expression and activation was detected by Western blotting. In addition, the effects of heat shock protein 90 (Hsp90) inhibitors and osimertinib were studied in colony formation assays. Results: Our laboratory generated osimertinib resistant cell lines from PC9 cell line and overexpression or activation of several proteins was detected. Hsp90 inhibitors, ganetespib and luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines and combination of these inhibitors with osimertinib achieved to enhance this cell viability and colony formation inhibition. Luminespib downregulated the expression of the several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression of several of these proteins, such as Stat3, Yap, Akt, EGFR and Met. Osimertinib activated the phosphorylation of several membrane receptors and downstream molecules that was partially inhibited by luminespib. In addition, a lung cancer patient with an EGFR eon 20 mutation had a partial radiographic response to ganetespib. Conclusions: Hsp90 inhibitors and osimertinib exhibits a good efficiency to inhibit cell viability, colony formation and inhibits expression and activation of proteins involved in osimertinib-resistance and may represent an effective strategy for NSCLC with intrinsic resistance to osimertinib inhibition.

Background: Recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) has a dismal prognosis with limited progressionfree survival and overall survival, even when treated with different combinations of chemotherapy, targeted therapies and immunotherapy. We explored in vitro and in vivo the effect of the epidermal growth factor (EGFR) inhibitor, osimertinib, alone and in combination with dihydroartemisinin (DHA) in HNSCC. Methods: The combination of osimertinib with DHA was tested in the FaDu and CAL27 HNSCC cell lines. Tumor cell proliferation assays were conducted in cultured cells and mouse xenografts. Western blotting analysis of related signal pathways was performed to investigate the molecular mechanisms of the inhibitory effect of DHA and the combination. Other compounds, which inhibit signal transducer and activator of transcription 3 (STAT3), Src-family kinases (SFKs), sphingosine kinase 1 (SPHK1), or the receptor tyrosine kinase (RTK) AXL were also combined with osimertinib in vitro. Results: Osimertinib exerted synergistic cytotoxicity toward FaDu and CAL27 HNSCC cells when combined with DHA. DHA reversed the osimertinibinduced STAT3 and Src phosphorylation. The double combination inhibited AXL expression. The anticancer potential of osimertinib plus DHA combination was validated in vivo on FaDu and CAL27 xenografts in mice with significant tumor regression. Interpretation: The results illustrate that the combinatory therapy of osimertinib and DHA, as a repurposing anticancer drug, could be a novel therapeutic strategy for recurrent and/or metastatic HNSCC patients. The findings strongly indicate that a clinical trial is warranted to confirm the benefit of the combination. Funding Statement: Work in Dr. Rosell's laboratory is partially supported by a grant from La Caixa Foundation, an Instituto de Salud Carlos III grant (RESPONSE, PIE16/00011), an Instituto de Salud Carlos III grant (PI14/01678), a Marie Sklodowska-Curie Innovative Training Networks European Grant (ELBA No 765492) and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE). Work in Dr. Cao's laboratory is partially supported by the Major National Science and Technology Program of China for Innovative Drug (2017ZX09101002-002-006), the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine) grant and Key R&D Program of Jiangsu Province (BE2018755). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: All procedures were based on Guide for Care and Use of Laboratory Animals of National Institutes of Health and approved by Institutional Animal Care and Use Committee of Jiangsu Province Academy of Traditional Chinese Medicine (SYXK 2016-0018).

Background: We have recently shown that antibodies generated by vaccination (anti-EGF VacAbs) potentiate the effects of tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor mutant (EGFR-mut) cell lines (1) and a Phase I/II clinical trial of an anti-EGF vaccine in combination with afatinib has been initiated. In this study we aimed to determine the efficacy of anti-EGF VacAbs to improve the antitumor activity of RET, BRAF, MEK and PI3K inhibitors in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines. Methods: Cell lines with RET translocations (LC-2/ad) and BRAF, KRAS and PIK3CA mutations (HT29, DLD1, LS174T and H508) were used. Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant human EGF. Cell lines were treated with anti-EGF VacAbs alone and in combination with RET, BRAF, MEK and PI3K inhibitors. Cell viability was determined by MTT, cell cycle was analyzed by flow cytometry, changes of total and phosphorylated proteins by Western blot and emergence of resistance by direct microscopic examination in low density cultures. Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and blocked EGFR transduction signaling pathway in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of BLU667 in LC-2/ad, trametinib and encorafenib in HT29, trametinib in DLD1 and LS174T and trametinib, taselisib, alpelisib and copanlisib in H508 cells. In these cell lines, anti-EGF VacAbs in combination with kinase inhibitors suppressed EGFR, Akt and Erk 1/2 phosphorylation. Cell cycle experiments revealed that anti-EGF VacAbs significantly increased the antiproliferative effects of the kinase inhibitors, measured as the percentage of cells in G2+M. Finally, the addition of the anti-EGF VacAbs to the culture medium significantly delayed the emergence resistant clones to trametinib in DLD1 cells. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of RET, MEK, BRAF and PI3K inhibitors in tumor cell lines. Our data provide a rationale for clinical trials testing the combination of anti-EGF VacAbs with kinase inhibitors in RET-translocated, KRAS, BRAF and PIK3CA mutant NSCLC and CRC patients.(1)“Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” Codony-Servat J, García-Roman S, Molina-Vila MA, et al. J Thorac Oncol. 2018. Citation Format: Silvia Garcia Roman, Jordi Codony Servat, Miguel Angel Molina Vila, Jordi Bertran Alamillo, Monica Garzon, Alejandro Martínez Bueno, Santiago Viteri, María González Cao, Andrés Aguilar, Juan José García, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies significantly improve the activity of RET, BRAF, MEK and PI3K kinase inhibitors in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6645.

9540 Background: Epidermal growth factor (EGFR)-mutant lung adenocarcinomas (LUADs) display impaired phosphorylation of extracellular signal-regulated kinase (ERK) and SRC-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. However, the function of SHP2 in early EGFR-mutant LUADs and EGFR wild-type LUADs has not been reported. We posit that SHP2 mRNA expression could be a predictive marker in resected EGFR-mutant LUADs versus EGFR wild-type patients (pts). Methods: We examined 267 resected LUADs from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1 and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction (PCR). EGFR mutant cell lines were investigated for their activity of SHP2. Results: Among the 267 enrolled pts, 100 (37.3%) were EGFR-mutant LUADs. Five-year recurrence-free survival (RFS) and overall survival (OS) were lower for EGFR-mutant LUADs with high SHP2 mRNA levels (hazard ratio = 1.83 and 2.28, respectively. p = 0.03 and p = 0.04). However, SHP2 was not associated with RFS nor OS in the 167 wild-type EGFR LUADs. In EGFR-mutant cells, RMC-4550 (SHP2 inhibitor) plus erlotinib showed synergism via inhibition of AKT (S473) and ERK1/2 (T202/Y204). While erlotinib translocates SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocalizes SHP2 into the cytoplasm membrane, limiting AKT and ERK activation. Conclusions: High SHP2 mRNA is related to shorter RFS and OS in EGFR-mutant LUADs, but not in EGFR wild-type LUADs. The findings indicate that the addition of SHP2 inhibitors could improve adjuvant therapy in EGFR-mutant LUADs.

Epidermal growth factor receptor (EGFR)-mutation-positive non-small cell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p = 0.0407) and overall survival (hazard ratio of 2.23, p = 0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing., Graphical Abstract Unlabelled Image, Highlights • AXL and CDCP1 are co-expressed in treatment-naïve EGFR-mutation-positive NSCLC patients. • AXL and CDCP1 are related to shorter progression-free survival with EGFR inhibitors and shorter overall survival. • Src family kinases and YAP1 are regulatory nodes for AXL and CDCP1 expression. • The combination of EGFR TKI with TPX-0005 is synergistic in EGFR-mutation-positive lung tumors in culture and in vivo. We explore the molecular changes that occur after the application of an EGFR inhibitor in EGFR-mutation positive tumors. The tumors do not acquire secondary drivers to overcome a primary driver but, counter-regulatory nodes observable before treatment, are immediately made apparent by pathway-specific intervention. The expression of the receptor tyrosine kinase AXL and the transmembrane protein CDCP1 in baseline samples of EGFR-mutation positive NSCLC patients can provide us with information on the treatment outcome. The upfront combination of an EGFR inhibitor with a multikinase inhibitor, that controls the regulatory nodes for RTKs activation, is a therapeutic approach that deserves to be further explored.

Introduction: Mutations in EGFR correlate with impaired response to immune checkpoint inhibitors and the development of novel immunotherapeutic approaches for EGFR mutant NSCLC is of particular interest. Immunization against epidermal growth factor (EGF) has shown efficacy in a phase III trial including unselected NSCLC patients, but little was known about the mechanisms involved in the effects of the anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) or their activity in tumor cells with EGFR mutations. Methods: The EGFR-mutant, NSCLC cell lines H1975, and PC9, together with several gefitinib and osimertinib-resistant cells derived from PC9, were treated with anti-EGF VacAbs and/or EGFR tyrosine kinase inhibitors (TKIs). Cell viability was analyzed by proliferation assays, cell cycle by fluorescence-activated cell sorting analysis, and levels of RNA and proteins by quantitative retro-transcription polymerase chain reaction and Western blotting. Results: Anti-EGF VacAbs generated in rabbits suppressed EGF-induced cell proliferation and cycle progression and inhibited downstream EGFR signaling in EGFR-mutant cells. Sera from patients immunized with an EGF vaccine were also able to block activation of EGFR effectors. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of all TKIs tested, suppressed Erk1/2 phosphorylation, blocked the activation of signal transducer and activator of transcription 3 (STAT3) and downregulated the expression of AXL receptor tyrosine kinase (AXL). Finally, anti-EGF VacAbs significantly delayed the emergence in vitro of EGFR TKI resistant clones. Conclusions: EGFR-mutant patients can derive benefit from immunization against EGF, particularly if combined with EGFR TKIs. A phase I trial of an EGF vaccine in combination with afatinib has been initiated. (C) 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Background The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGR mutations. However, invariable acquired resistance appears due to several molecular mechanisms. Hsp90 protein clients are involved in these processes. Methods Cell viability and colony formation assays were performed in PC9, H1975 and PC9-derived osimertinib-resistant NSCLC cell lines tested with osimertinib plus hsp90 inhibitors, luminespib or ganetespib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study the protein expression and activation. Results In our laboratory five osimertinib-resistant cell lines were generated from PC9 NSCLC cell line and overexpression or activation of several proteins, such as, AXL, Yap, bcl2, Akt, Stat3 and IGF-1R were detected. Hsp90 inhibitors, Ganetespib and Luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines, and the combination of these inhibitors with osimertinib achieved enhanced cell viability reduction. Luminespib downregulated the expression of several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression in several of these proteins, such as, Stat3, Yap, Akt, EGFR and Met. In addition, osimertinib activated the phosphorylation of several membrane receptors and downstream molecules, such as, Met, Yap, Stat3, Akt and Src, and its activation was partially inhibited by Luminespib. Conclusions We conclude that hsp90 inhibitors and osimertinib exhibit good efficiency in inhibiting cell viability and colony formation, while at the same time, inhibiting expression and activation of proteins involved in osimertinib-resistance. Our preclinical study shows that the combination of hsp90 inhibitors and osimertinib may represent an effective strategy for NSCLC patients with resistance to osimertinib treatment. Legal entity responsible for the study Pangaea Oncology. Funding Pangaea Oncology. Disclosure All authors have declared no conflicts of interest.

Oxaliplatin-resistant LoVo colon cancer cells overexpressing c-MET and VEGFR-1 were selected to study several signaling pathways involved in chemoresistance, as well as the effect of increasing amounts of VEGF in the regulation of c-MET. In comparison with chemosensitive LoVo colon cancer cells, oxaliplatin-resistant cells (LoVoR) overexpress and phosphorylate c-MET, upregulate the expression of transmembrane and soluble VEGFR-1 and, unexpectedly, downregulate VEGF. In addition, LoVoR cells activate other transduction pathways involved in chemoresistance such as Akt, β-catenin-TCF4 and E-cadherin. While c-MET is phosphorylated in LoVoR cells expressing low levels of VEGF, c-MET phosphorylation decreases when recombinant VEGF is added into the culture medium. Inhibition of c-MET by VEGF is mediated by VEGFR-1, since phosphorylation of c-MET in the presence of VEGF is restored after silencing VEGFR-1. Dephosphorylation of c-MET by VEGF suggests that tumors coexpressing VEGFR-1 and c-MET may activate c-MET as a result of anti-VEGF therapy.

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 progressionfree 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 gefitinibresistant EGFR mutant NSCLC cell lines. However, afatinib therapy causes phosphorylation of STAT3 tyrosine 705 (pSTAT3(Tyr705)) and elevation of STAT3 and RANTES mRNA levels. The combination of afatinib with TPCA-1 (a STAT3 inhibitor) ablated pSTAT3(Tyr705) 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.

Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms., EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo.