EGF Receptor Research Articles

A review: drug-drug interactions of epithelial growth factor receptor-tyrosine kinase inhibitors

Mutations in the epithelial growth factor receptor (EGFR) is a driving factor that causes non-small cell lung carcinoma (NSCLC). The epithelial growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is a crucial discovery in the treatment of lung cancer, particularly the efficacy of EGFR-TKIs is superior to that of the standard chemotherapy for patients with EGFR mutation-positive advanced NSCLC. Patients with NSCLC use EGFR-TKIs and other medications simultaneously is commonly seen, especially among those with comorbidities, which increases the risk of drug-drug interactions (DDIs) of EGFR-TKIs. The most common mechanisms underlying the DDIs of EGFR-TKIs are modulations of cytochrome P450 (CYP) and drug transporters [including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)], as well as gastrointestinal acid-inhibitory drugs [proton pump inhibitors (PPIs) and H(2) receptor antagonists (H(2)RA)]. Inhibitors or inducers of CYP enzymes and drug transporters can inhibit or accelerate the metabolism of EGFR-TKIs, which increase or reduce the exposure of EGFR-TKIs, thereby affect the efficacy and safety of EGFR-TKIs. In addition, PPIs or H(2)RA can decrease the solubility, bioavailability and efficacy of EGFR-TKIs. This review summarizes the mechanisms of DDIs of gefitinib, erlotinib, icotinib, afatinib, dacomitinib and osimertinib; the management recommendations for DDIs of those EGFR-TKIs from the Chinese and global guideline, as well as from the recent pre-clinical and clinical studies, which provide the reference and evidence for managing the combination therapies of EGFR-TKIs and other medications in clinics.

435 Identification of the EGF receptor extracellular domain as a cell-surface sensor for ubiquitous organic pollutants

Chloracne, a persistent acne-like skin eruption, is a result of acute intoxication with dioxins and related halogenated aromatic hydrocarbons. The underlying pathomechanisms are not well understood, but may involve alterations in proliferation and differentiation of sebocytes and epidermal keratinocytes. The fact that in contrast to dioxin-like compounds (DLCs) some chloracnegenic agents do not bind to the aryl hydrocarbon receptor (AHR) implies the involvement of other cellular effector molecules.

The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

Studies on the mode of action of synthetic diindolylmethane derivatives against triple negative breast cancer cells.

Diindolylmethane (DIM) is a metabolic product of indole-3-carbinol (I3C), the major phytochemicals present in cruciferous vegetables, which can modulate multiple signalling pathways in cancer. The present study deals with the mechanism of action of two synthetic biaryl conjugates of DIM in triple negative breast cancer cells. Out of 12 DIM derivatives tested, two compounds, DIM-1 and DIM-4, exhibit cytotoxicity with GI50 values of 9.83 ± 0.2195 μM and 8.726 ± 0.5234 μM, respectively, in 2D culture. In 3D culture, DIM-1 and DIM-4 show GI50 values of 24.000 ± 0.7240 μM and 19.230 ± 0.3754 μM, respectively. The non-toxic nature of the compounds was also established by the toxicity studies using the zebrafish model system. The two compounds induced apoptosis and anoikis in the cancer cells, which was confirmed by morphological analysis, nuclear fragmentation, membrane integrity assay, caspase activity measurements and modulation of pro/anti-apoptotic proteins. The compounds inhibited cell migration and MMP-2 and MMP-9 activities indicating their anti-metastatic property. They also reduced the expression of active Ras, phosphorylated forms of PI3K, Akt and mTOR. Immunofluorescence studies revealed the reduced expression of EGFR and pEGFR in treated cells. To conclude, DIM-1 and DIM-4 induced anti-breast cancer effects by blocking EGF receptor and subsequently inhibiting Ras-mediated PI3K-Akt-mTOR signalling pathway.

N-glycosylation of GDF15 abolishes its inhibitory effect on EGFR in AR inhibitor-resistant prostate cancer cells

Castration-resistance of prostate cancer is one of the most challenging clinical problems. In the present study, we have performed proteomics and glycomics using LNCaP model. Growth differentiation factor-15 (GDF15) level is increased in androgen receptor (AR) inhibitor-resistant cells and the inhibitory effect of GDF15 on epithelial growth factor receptor (EGFR) pathway is relieved by GDF15 N70 glycosylation. Interference of GDF15 (siRNA or N70Q dominant negative) or EGFR pathway (inhibitor or siRNA for EGFR, SRC or ERK) decreases the resistant-cell survival in culture and tumor growth in mice. Our study reveals a novel regulatory mechanism of prostate cancer AR inhibitor resistance, raises the possibility of AR/SRC dual-targeting of castration-resistance of prostate cancer, and lays foundation for the future development of selective inhibitors of GDF15 glycosylation.

A scalable, open-source implementation of a large-scale mechanistic model for single cell proliferation and death signaling

Mechanistic models of how single cells respond to different perturbations can help integrate disparate big data sets or predict response to varied drug combinations. However, the construction and simulation of such models have proved challenging. Here, we developed a python-based model creation and simulation pipeline that converts a few structured text files into an SBML standard and is high-performance- and cloud-computing ready. We applied this pipeline to our large-scale, mechanistic pan-cancer signaling model (named SPARCED) and demonstrate it by adding an IFNγ pathway submodel. We then investigated whether a putative crosstalk mechanism could be consistent with experimental observations from the LINCS MCF10A Data Cube that IFNγ acts as an anti-proliferative factor. The analyses suggested this observation can be explained by IFNγ-induced SOCS1 sequestering activated EGF receptors. This work forms a foundational recipe for increased mechanistic model-based data integration on a single-cell level, an important building block for clinically-predictive mechanistic models.

The role of YAP1 target gene CTGF in the anoikis resistance of rheumatoid arthritis synovial fibroblasts.

To analyse pro-survival mechanisms elicited in RA synovial fibroblasts (RASFs) upon detachment from their extracellular matrix dependent on the disintegrin metalloproteinase ADAM15 and Yes-associated protein kinase 1 (YAP1). Detachment-induced apoptosis was determined by caspase 3/7 assays. Immunofluorescent stainings, cell surface biotinylation and immunoblotting were applied to analyse phosphorylated kinases and subcellular localization of YAP1 and connective tissue growth factor (CTGF). Caspase and transwell transmigration assays served to study CTGF function. Silencing of ADAM15 or YAP1 in RASFs leads to significantly increased levels of detachment-induced caspase activity. In non-silenced RASFs detachment causes simultaneous ADAM15-enhanced phosphorylation of YAP1 at S127, known for promoting its cytoplasmic localization, and Src-dependent phosphorylation at tyrosine Y357. The majority of nuclear YAP1 leaves the nucleus shortly after cell detachment, but prolonged detachment causes a marked nuclear re-entry of YAP1, resulting in significantly increased synthesis of CTGF. The newly synthesized CTGF, however, is not detectable in the supernatant, but is bound to the outside of the plasma membrane. In vitro studies demonstrated autocrine binding of CTGF to the EGF receptor and β1 integrin, with concomitant triggering of survival kinases, AKT1, ERK1/2, Src and focal adhesion kinase. Functional studies revealed anti-apoptotic effects of CTGF on detached RASFs and an enhancement of their potential for endothelial transmigration using HUVEC-coated transwells. The elucidation of a new molecular mechanism that protects RASFs in the highly pro-apoptotic environment of inflamed RA joints by promoting anoikis-resistance and transendothelial migration via ADAM15/YAP1-mediated CTGF upregulation uncovers potentially new targets for future therapeutic intervention.

Abstract 1626: The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells

Abstract Colorectal cancer is one of the most common cancer in the world. The mortality rate is the third among the top ten cancers. Among colorectal cancer patients, about 65-75% of patients have overexpression of epithelial growth factor receptors (EGFR). The antibody drug cetuximab that inhibits EGFR has also been developed. Moreover, overexpression of src protein is found in about 80% of patients with colorectal cancer. Higher levels of Src expression is related to the metastasis of colon cancer. Dasatinib is an oral taken tyrosine kinase inhibitor to inhibit the src phosphorylation. Therefore, in this study, the mechanism of cetuximab and dasatinib combined treatment in the orthotopic mouse model is investigated. The in vivo mechanism of EGFR and E-cadherin crosstalk in the mouse model is discussed. In this study, HT-29 colon cancer cells with BRAF V600E mutation were orthotopically injected into the cecum wall of BALB/c nude male mice. Two weeks later, cetuximab, dasatinib, and combined treatment of cetuximab and dasatinib were applied for three weeks. The tumor sizes were then recorded after sacrificed. Tumor Tissue were processed for tissue section with H&E staining. Immunohistochemistry (IHC) was applied to detect EGFR, p-Src, E-cadherin with specific antibodies. The protein expression levels were achieved by Western blot. In the results, the tumor size was smaller in cetuximab and dasatinib combined treated group. In IHC staining, the E-cadherin expression were not apparent in primary tumor site, however, were abundant in migrated cells. P-src expression was reduced in the dasatinib treated group and combined treatment group. In western blot result, the E-cadherin was increased after the treatment of dasatinib, and the p-src expression was inhibited after dasatinib treatment and combined treatment. Our results demonstrated cetuximab combined with dasatinib has the better result of suppression of BRAF mutated colon cancer cell growth and metastasis in the orthotopic mouse model. Citation Format: Yu-Cheng Weng, Yu-Lin Li, Xiang-Ling Hou, Hui Min Koo, Yi-Wen Lu, Viriya Adhiguna Winarso, Chia-Wen Lin, Wei-Ting Chao. The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1626.

Abstract 2875: Development of a new tumor-bearing humanized mouse model to evaluate the efficacy and toxicity of bispecific T cell engagers

Abstract Background: T cell-based immunotherapies, especially the bispecific T cell engager (BiTE), which recruit T cells to tumor cells, have been developed rapidly for treatment of malignancies. However, the wide application of BiTE therapies is limited by accompanying severe adverse effects such as cytokine release syndrome (CRS). Since human cancer cells are not incorporated in the CRS evaluation, currently used in vivo animal models and in vitro human immune cell activation assays do not reliably evaluate the induction of CRS by immune drugs that is seen in BiTE treated cancer patients. In addition, the differences between human and mouse immune systems hamper the development of new immune-oncology therapeutic drugs. Thus, establishing a predictive in vivo animal model that can screen both efficacy of cancer therapy and risk of developing CRS is critical for improving the safety of immune drug development for treatment of cancer patients. Method: Humanized mice were developed in NSG-MHC class I/II double knock out (DKO) mouse recipients following reconstitution with human peripheral blood mononuclear cells. The MDA-MB-231/Luciferase-2A-GFP stable breast cancer cell line (MDA), which expressed epidermal growth factor receptor (EGFR), were implanted in mice 5 days after PBMC engraftment. Following EGFRxCD3 BiTE treatment whole-body tumor burden was quantified by an in vivo imaging system. To evaluate the CRS levels in response to BiTE therapy, cytokine levels were analyzed by BD cytometric bead array. Clinical observations, CRS scores, body weight changes and human immune cell activation were also monitored for CRS assessment. Results: PBMC humanized models were successfully established with breast cancer cell engraftment. After treatment with EGFRxCD3 BiTE, the mice exhibited elevated serum levels of human IFNgamma, TNF-a, IL-6, IL-10, IL-2 and IL-4 compared to a vehicle control group. This cytokine production was dose-dependent and was associated with an increase of toxic mouse clinical evaluation score. In contrast, there was no significant inflammatory cytokine production in PBMC humanized mice without MDA cell implantation, suggesting T cell activation by BiTE followed binding to the cancer cells. BiTE treatment for 2-4 days significantly suppressed tumor growth in lung as measured by luciferin imaging. With serial doses of BiTE treatment, we observed a dose-dependent inhibition of tumor growth. Conclusion: We developed a novel in vivo PBMC humanized mouse model bearing human tumor cells that can be used to determine the optimal dose of BiTE for inhibition of tumor growth with low toxicity risk. This model provides a growth environment similar to that of patients and has important application values in simultaneously evaluating the efficacy and safety of any T cell-based immunotherapies including BiTE to define optimal approaches for clinical cancer treatment. Citation Format: Guoxiang Yang, Li-Chin Yao, Michael A. Brehm, Dale L. Greiner, Leonard D. Shultz, Danying Cai, MIngshan Cheng, James G. Keck. Development of a new tumor-bearing humanized mouse model to evaluate the efficacy and toxicity of bispecific T cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2875.

Abstract 4113: Response to amivantamab, a bispecific EGF and MET receptor directed antibody, in a patient with an atypical EGFR mutated (G719X) non-small cell lung cancer (NSCLC) with leptomeningeal disease who progressed on osimertinib

Abstract Amivantamab is a bispecific antibody against EGF and MET receptors, approved for patients with locally advanced or metastatic NSCLC harboring EGFR exon 20 insertion after disease has progressed on or after platinum-based chemotherapy. Existing data is insufficient in establishing its efficacy on other rare EGFR mutated subtypes, central nervous system (CNS), or its resistance to 3rd generation tyrosine kinase inhibitor (TKI), osimertinib. Here, we report a case showing a good response on single agent amivantamab in an atypical EGFR mutation (G719A, exon18 substitution) NSCLC that progressed to CNS involvement on osimertinib. A 67-year-old male presented with stage IVB squamous cell carcinoma of lung with osseous metastases. CT chest, abdomen, and pelvis revealed a speculated 7.7 cm right upper lung lesion with hilar and subcarinal lymphadenopathies. MRI brain and spine showed multiple bone metastases without CNS involvement. Tissue biopsy confirmed poorly differentiated squamous cell carcinoma with PD-L1 IHC 75%. Tissue next generation sequencing (NGS) showed EGFR G719A. He was started on osimertinib. Two months later, he was hospitalized for drug-induced pneumonitis which prompted off osimertnib. CT angiogram chest showed new diffuse pericardial thickening and nodularity, suggestive of progressive disease. Two cycles of chemotherapy were delivered followed by immunotherapy with ipilimumab and nivolumab. In 10 weeks, progression of disease was revealed in MRI brain demonstrating new parenchymal and leptomeningeal metastases. Amivantamab monotherapy was initiated with discontinuation of immunotherapy. The patient tolerated amivantamab without major complications. His performance status remained the same before and after amivantamab. He denied fatigue, anorexia, nausea or vomiting, however, endorsed rash, which was managed with hydrocortisone cream. Repeat scans in 6 weeks showed decreased leptomeningeal enhancement, and reduction in the size of parenchymal lesions, lung mass, and lymphadenopathies. The highest variant allele fraction from circulating tumor DNA NGS assay from Guardant360 was significantly improved on amivantamab from 25.6% (EGFR G719A), at time of diagnosis, to non-detectable. Amivantamab monotherapy has shown an encouraging outcome in a patient with an atypical EGFR mutated (G719X) NSCLC with leptomeningeal disease who progressed on osimertinib. Our case has shown significant response on CNS involvements, which is contrary to known poor blood-brain barrier penetration of amivantamab. This supports current trial evaluating the efficacy of amivantamab for NSCLC with rare EGFR mutations such as G719X, and ones progressed on 3rd Gen TKI treatment. Additional studies evaluating the efficacy of amivantamab on CNS metastasis are warranted. Citation Format: Jinah Kim, Horyun Choi, Yeun Ho Lee, Leeseul Kim, Young Kwang Chae. Response to amivantamab, a bispecific EGF and MET receptor directed antibody, in a patient with an atypical EGFR mutated (G719X) non-small cell lung cancer (NSCLC) with leptomeningeal disease who progressed on osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4113.

Abstract 1525: Inhibition of HGF maturation overcomes cetuximab resistance in colorectal cancer

Abstract We have previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. These findings originated from our in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR. CC are sensitive to cetuximab, while SC and CC-CR are resistant. Both de novo and acquired modes of cetuximab resistance in SC and CC-CR, respectively, could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. Conversely, exogenous administration of MET ligand, HGF could transiently induce cetuximab resistance which could be further overcome by crizotinib addition. HGF/HGFL are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. To inhibit HGF/HGFL cleavage in cetuximab-resistant cells, we employed inhibitors of HGF/HGFL proteases (ZFH7116 and VD2173) and were able to overcome both de novo and acquired cetuximab resistance. A survey of TCGA datasets indicated that HGF/HGFL were overexpressed in several CRC CMS subtypes. We next expressed human HGF in cetuximab-sensitive CC cells and observed that HGF overexpression imparts cetuximab resistance. Moreover, cetuximab resistance induced by HGF overexpression could be overcome by the downstream MET inhibition (with crizotinib) and we are now testing if the upstream inhibition of HGF proteases (with ZFH7116/VD2173) also overcomes cetuximab resistance. Combined these results indicate that inhibition of HGF cleavage and maturation may be a novel way to overcome resistance to EGFR-targeted therapies in CRC. Citation Format: Ramona Graves-Deal, Galina Bogatcheva, Vivian T. Truong, Vishnu Damalanka, Lidija Klampfer, Robert J. Coffey, James W. Janetka, Bhuminder Singh. Inhibition of HGF maturation overcomes cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1525.

Abstract 5334: Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers

Abstract Background: Human epithelial growth factor receptor 2 (HER2) is a well-established therapeutic target in HER2-positive breast and gastric cancer. Recently, novel HER2-targeting agents are being developed after trastuzumab, and HER2-targeted therapies have been attempted in across solid tumor types, including biliary tract cancer. In these aspects, it is essential to understand and elucidate the resistant mechanism of the HER2-targeting strategies. Yes-associated protein (YAP), a transcription factor of the Hippo pathway, function as proto-oncoproteins by inducing target genes involved in cancer cell survival and proliferation (Zhao B, et al. Genes Develop 2008). Also, YAP is emerging as a resistance mechanism of cytotoxic and targeted drugs. In this study, we explore the role of YAP in overcoming resistance to trastuzumab in HER2-positive cancer cells. Methods: Four trastuzumab-resistant (HR) cell lines were established using HER2-postive gastric and biliary tract cancer cell lines (N87, SNU216, SNU2670, and SNU2773) (Jin MH, et al. Mol Cancer Ther. 2017). YAP siRNA and Verteporfin (YAP-TEAD inhibitor) were used to downregulate YAP. MTT assay, cell cycle analysis, western blot and migration assay were performed to analyze the antitumor effects through YAP downregulation. In order to elucidate tumor cell immune-modulation by YAP, human PBMC co-culture was used and immune markers, such as programmed death-ligand 1 (PD-L1), were analyzed in HR cell lines. Results: Increase of receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) expression was observed in the HR cells. Relatively high expression of pYAP, YAP, and transcriptional co-activator with PDZ-binding motif (TAZ) were observed in the HR cells (SNU216HR, SNU2670HR and SNU2773HR) compared to parental cells. Reducing overexpressed YAP in HR cells resulted in tumor cell growth inhibition. In cell cycle analysis, the increase of subG1 phase through YAP downregulation was observed, and cyclinD, B, A and BCL-XL were effectively decreased. Migration was also inhibited by the decrease in YAP expression. By verteporfin treatment, changes in immune markers including increase of CD80 and decrease of PD-L1 was found. In addition, p-STAT3 and IL6, Which regulate PD-L1, were also decreased through the reduction of YAP. In HR cells treated with siYAP, there was a tendency to increase CD8+ T cells upon PBMC co-culture. Conclusion: YAP is upregulated in trastuzumab-resistant (HR) cells and upregulated YAP induce PD-L1 expression. Inhibition of YAP shows anti-tumor effects and modulates immune status. Collectively, our results suggest that inhibition of YAP is one of promising strategies to overcome trastuzumab resistance in HER2-positive cancers. Citation Format: Ah Rong Nam, Jeesun Yoon, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Tae-Yong Kim, Do-Youn Oh. Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5334.

WX-0593 combined with an epithelial growth factor receptor (EGFR) monoclonal antibody in the treatment of xenograft tumors carrying triple EGFR mutations.

BackgroundTo evaluate the safety and therapeutic efficacy of WX-0593, a newly developed potent anaplastic lymphoma kinase (ALK) inhibitor, in combination with an epithelial growth factor receptor (EGFR) monoclonal antibody (QL1203 or Vectibix) for the treatment of xenograft tumors carrying mutant EGFR and osimertinib-resistant mutations (EGFR/T790M/C797S).MethodsThe inhibition of tumor cell proliferation by WX-0593 and Vectibix alone or combined was evaluated in four EGFR triple-mutant cell lines: PC9 (EGFR Del19/T790M/C797S), NCI-H1975 (EGFR L858R/T790M/C797S), Ba/F3 (EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S). The in vivo antitumor efficacy of WX-0593 alone or combined with QL1203 or Vectibix was evaluated in xenograft tumor models of BALB/c nude mice developed from H1975 (EGFR-Del19/T790M/C797S) and Ba/F3 (EGFR-L858R/T790M/C797S) cell lines. Mice were randomized into groups and treated with or without WX-0593, QL1203, Vectibix, or their combination. The tumor volume, mouse body weight, and therapeutic side effects were monitored routinely. Blood samples were obtained from all mice at different time points after the last dosage of treatment to evaluate the pharmacokinetic parameters of the drugs.ResultsWX-0593 and Vectibix showed a strong synergistic inhibitory effect on the proliferation of two EGFR triple-mutant Ba/F3 cell lines (EGFR L858R/T790M/C797S and Del19/T790M/C797S), but little synergistic inhibitory effect on the proliferation of NCI-H1975 (EGFR L858R/T790M/C797S) and PC9 (EGFR Del19/T790M/C797S). In vivo, WX-0593 (25 mg/kg) showed a modest therapeutic effect when combined with QL1203 or Vectibix, but had no effect on tumor growth as a monotherapy at this dosage. WX-0593 (75 mg/kg) exhibited modest antitumor efficacy that was further enhanced in combination with QL1203 or Vectibix in both tumor models (H1975 and Ba/F3). No significant body weight alteration, any other side effect, or deaths were observed during treatment. Pharmacokinetic analysis showed that the serum level of QL1203 or Vectibix was significantly increased and lasted longer when combined with WX-0593.ConclusionsWX-0593 exhibited a synergetic effect with an EGFR monoclonal antibody on osimertinib-resistant EGFR-mutant non-small cell lung cancer (NSCLC) both in vitro and in vivo. Their combination showed potent antitumor efficacy and an acceptable safety profile, which may be a promising strategy for the treatment of patients with EGFR triple-mutant NSCLC resistant to osimertinib.

Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.

SUMMARYLigand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.

Effects of dabrafenib and erlotinib combination treatment on anaplastic thyroid carcinoma.

Dabrafenib is a BRAF kinase inhibitor approved for treatment of BRAF-mutated anaplastic thyroid carcinoma (ATC) in combination with trametinib. Erlotinib is a tyrosine kinase inhibitor of EGF receptor (EGFR). We evaluated effects of dabrafenib and erlotinib combination treatment on ATC cells in vitro and in vivo. Cell proliferation, colony formation, apoptosis, and migration of ATC cells harboring a BRAF mutation (BHT101, 8505C, and SW1736) were evaluated after treatment with dabrafenib in combination with erlotinib or trametinib. The changes in activation of mitogen extracellular kinase (MEK) and extracellular signal-related kinase (ERK) signaling were also evaluated by Western blot analysis. Effects of these combinations were also evaluated using an in vivo xenograft model. First, we detected EGFR activation in dabrafenib-resistant SW1736 cells using a phospho-receptor tyrosine kinase array. A dabrafenib and erlotinib combination synergistically inhibited cell proliferation, colony formation, and migration, with an induction of apoptotic cell death in all three ATC cells, compared with dabrafenib or erlotinib alone. This synergistic effect was comparable with a dabrafenib and trametinib combination. The dabrafenib and erlotinib combination effectively inhibited phosphorylated (p)-MEK, p-ERK, and p-EGFR expressions compared with dabrafenib or erlotinib alone, while the dabrafenib and trametinib combination only inhibited p-MEK and p-ERK expressions. The dabrafenib with erlotinib or trametinib combinations also significantly suppressed tumor growth and induced apoptosis in a BHT101 xenograft model. The dabrafenib and erlotinib combination could be a potential novel treatment regimen to overcome drug resistance to dabrafenib alone in patients with BRAF-mutated ATC.

Response to epithelial growth factor receptor inhibitor (EGFRi) treatment in patients with early-onset, treatment-naïve metastatic colorectal cancer (mCRC): An ARCAD database analysis.

3572 Background: Early onset colorectal cancer (eoCRC: disease diagnosed < 50) has been increasing over the past 2 decades. Currently, standard treatment recommendations for eoCRC patients (pts) with metastatic disease does not differ from late-onset CRC (loCRC) pts although outcomes data in eoCRC pts is limited. Methods: Individual patient data on 5,761 treatment-naive metastatic eoCRC pts was pooled from 8 phase II and III randomized EGFRi studies (2000 - 2012) from the ARCAD mCRC database. The distribution of demographics, clinicopathological features, and biomarkers were summarized by age groups. Progression-free survival (PFS) was compared between age groups by stratified Cox models, adjusting for potential confounders. Predictive value of age group was evaluated by testing interaction effect between treatment and age variables based on a subset of trials with concurrent randomizations between regimens with and without EGFRi Results: eoCRC (n=756) were more evenly distributed between gender, had improved performance status (PS), increased likelihood of metastatic resection, and distant lymph node metastasis, but were less likely to have lung metastasis or KRAS mutation compared to loCRC (n=5,005, table 1). eoCRC and loCRC patients had similar distribution of primary tumor sidedness, primary resection, liver and/or peritoneal involvement, number of metastatic sites involved, and BRAF mutations (MT). No difference in PFS for eoCRC versus loCRC pts was noted (7.8 vs. 7.9 months [M], adjusted hazard ratio [HRadj], 1.02, 95% confidence interval [CI], 0.93-1.11). Among pts with KRAS wild type (WT) and left sided primary tumors, univariable analysis of EGFRi demonstrated improved mPFS in loCRC (9.9 vs 8.5M, HR = 0.74, p<0.001), but this benefit was not seen in eoCRC (8.3 vs 8.9 months, HR 1.20, p=0.36). The same pattern was observed upon multivariable analysis (Table). Conclusions: In our pooled analysis, EGFRi + chemotherapy significantly improved PFS in treatment-naïve loCRC patients but not in left sided, KRAS WT, eoCRC patients. Further validation in an independent cohort is warranted. [Table: see text]

Identification of biomarker associated with Trop2 expression in breast cancer: Potential implication for targeted therapy.

e15120 Background: Trop2 has been confirmed to enhance tumor proliferation, metastasis and invasion in various epithelial tumors. Breast cancer (BC) patients with high Trop2 protein expression have better prognosis and objective response rates. Thus, this study aimed to explore potential biomarker associated with Trop2 protein expression in BC. Methods: BC patients with complete clinical parameters were enrolled, including age, estrogen receptor (ER) status, progesterone receptor (PR) status, human epithelial growth factor receptor 2 (HER2 status), molecular subtype, Ki67 status, T stage, N stage, TNM stage, PD-L1 and Trop2 protein expression level. Univariate and multivariate ordinal logistic regression analyses were performed to identify the independent factor for Trop2 protein expression level. Immunohistochemical analysis was conducted to detect the protein expression level of Trop2 in BC. Kaplan–Meier analysis and the log-rank test were executed to assess the survival difference between low- and high- gene expression level of Trop2. Results: A total of 65 BC patients from the Guangdong Provincial People’s Hospital were included. Based on the univariate and multivariate ordinal logistic regression analyses, Ki67 status was the only independent factor for Trop2 protein expression level (P < 0.05). This finding revealed that BC patients with higher Ki67 status may harbor higher Trop2 protein expression level. According to cBioPortal database, the mutation rate of Trop2 was 2.1% and mutation type was amplification. BC patients with low Trop2 gene expression level had significantly greater survival prognosis (P < 0.05). Conclusions: We performed the comprehensive analysis of the Trop2 in BC patients. It has the potential to guide individualized targeted treatment in BC patients.

Prophylactic Effect of Nitric Oxide Donors on Rat Models of EGFR Inhibitor‒Induced Cutaneous Toxicities

EGF receptor (EGFR) inhibitors have been established as first-line standard-of-care therapies for nonsmall cell lung cancer but are frequently accompanied by adverse dermatological effects, in particular, acneiform rash. There is no effective clinical intervention, partially because of its poorly understood etiology. In this study, we show that inhibition of EGFR initiated keratinocyte HaCaT cell cycle arrest and apoptosis, which fueled a robust secondary inflammatory response. Rats gavaged with EGFR inhibitor showed a phenotype similar to that of clinical patients, which was in line with the interrupted functions observed in HaCaT keratinocytes. We found that a nitric oxide donor, nitroglycerin, was a feasible treatment alternative for EGFR inhibitor‒induced rash. Restoration of epidermal extracellular signal‒regulated kinase and a reduction in signal transducer and activator of transcription 3 signaling through nitroglycerin treatment rescued the cellular functions that had been damaged invitro and further ameliorated the rash in rat models. In addition, the efficacy of nitroglycerin was superior to that of existing clinical interventions. These data highlighted the importance of epidermal EGFR signaling and led to the identification of a small-molecule nitric oxide donor as a mediator that can maintain EGFR pathway functions during anti-EGFR therapies, providing a therapeutic anchor point for adverse EGFRI-induced skin effects.

Multiomic Profiling Identified EGF Receptor Signaling as a Potential Inhibitor of Type I Interferon Response in Models of Oncolytic Therapy by Vesicular Stomatitis Virus.

Cancer cell lines responded differentially to type I interferon treatment in models of oncolytic therapy using vesicular stomatitis virus (VSV). Two opposite cases were considered in this study, glioblastoma DBTRG-05MG and osteosarcoma HOS cell lines exhibiting resistance and sensitivity to VSV after the treatment, respectively. Type I interferon responses were compared for these cell lines by integrative analysis of the transcriptome, proteome, and RNA editome to identify molecular factors determining differential effects observed. Adenosine-to-inosine RNA editing was equally induced in both cell lines. However, transcriptome analysis showed that the number of differentially expressed genes was much higher in DBTRG-05MG with a specific enrichment in inflammatory proteins. Further, it was found that two genes, EGFR and HER2, were overexpressed in HOS cells compared with DBTRG-05MG, supporting recent reports that EGF receptor signaling attenuates interferon responses via HER2 co-receptor activity. Accordingly, combined treatment of cells with EGF receptor inhibitors such as gefitinib and type I interferon increases the resistance of sensitive cell lines to VSV. Moreover, sensitive cell lines had increased levels of HER2 protein compared with non-sensitive DBTRG-05MG. Presumably, the level of this protein expression in tumor cells might be a predictive biomarker of their resistance to oncolytic viral therapy.

Myeloid Expressed Epidermal Growth Factor Receptor (EGFR) is Critical to Heart Homeostasis and Limits Adverse Outcomes Following Ischemic Attack

The healthy heart comprises immune cells, mainly macrophages of differing class and origin. While these myeloid cells remain an area of continuous investigation, we have come to appreciate that a subset of tissue residing macrophages (TRMs) seed the heart during development and are maintained through self‐renewal. Further, TRMs continue to be linked to the maintenance of cardiac homeostasis, for instance, through facilitating crosstalk with other cardiac cells, and participating in immunosurveillance. In the case of injury however, such as a myocardial infarction (MI), TRMs are largely replaced by circulating and recruited myeloid cells. Post‐infarct macrophages (mf) are greater in number and class, and functionally, debris clearance and initiation of cardiac repair mechanisms becomes their utmost priority to limit the severity of heart failure (HF). Given these significant roles, it has become critical to understand how cardiac myeloid cells are regulated, and what mechanisms contribute to their known functions both at steady‐state and following injury. Epidermal growth factor receptor (EGFR) is a cell surface tyrosine kinase receptor known to govern cell function through myriad processes. Although historically studied as an oncogene, EGFR has been shown to regulate cardiac physiology, and notably mf activation, survival and function. However, it is currently unknown whether EGFR influences myeloid homeostasis within the heart. Thus, we have generated myeloid‐specific EGFR knockout mice (EGFRmylKO) by crossing floxed EGFR (EGFRf/f) with LysM Cre mice and performed comparative analyses between the lines at baseline and following injury. In response to MI, EGFRmylKO mice exhibit altered inflammatory responses, resulting in aggravated cardiac dysfunction, exacerbated hypertrophic remodeling, and limited angiogenic repair. These functional and structural outcomes are preceded by a disruption in the post‐injury myeloid cell infiltration continuum that favors adequate repair, resulting in decreased transcripts of reparative factors including IL‐10. Additionally, EGFRmylKO mouse hearts show modest signs of stress at baseline, in the absence of injury, including cardiomyocyte hypertrophy with an associated increase in expression of signature fetal gene transcripts. Whole transcriptome analysis of cardiac resident myeloid (Cd11b+) cells by RNASeq revealed over 700 differentially expressed genes that were significantly altered between EGFRmylKO and EGFRf/f control mice. Among these are IGFBP family members 5 and 7, each known to regulate IGF availability, which itself has been recently shown to influence cardiac hypertrophy. In sum, our results reveal that myeloid cell‐expressed EGFR plays previously unknown roles in the regulation of the cardiac homeostasis baseline as well as the chronic remodeling response to ischemic injury.