Epidermal Growth Factor Receptor Downstream Signaling Research Articles

EGFR bypass activation mediates acquired resistance to regorafenib in hepatocellular carcinoma

BackgroundRegorafenib, a tyrosine kinase inhibitor (TKI), is used in the treatment of unresectable hepatocellular carcinoma (HCC). However, the occurrence of acquired resistance limits its antitumor efficacy. While multiple studies have highlighted the crucial role of bypass activation in acquired TKI resistance, few have focused on bypass activation in regorafenib resistance in HCC.MethodsHigh-throughput proteomics was used to identify differential proteins associated with bypass activation between acquired regorafenib-resistant cells and parental cells. The ability of epidermal growth factor receptor (EGFR) bypass inhibition to reverse resistance was evaluated both in vitro and in vivo using direct microscopic observation, the CCK-8 assay, colony formation assay, Annexin V-FITC/propidium iodide double staining, cell cycle analysis, western blotting, and a xenograft model.ResultsThe expression of EGFR, a member of the receptor tyrosine kinase (RTK) family, was significantly increased in acquired regorafenib-resistant HCC cells compared with parental cells. Pharmacological inhibition of EGFR with gefitinib restored the sensitivity of regorafenib-resistant HCC cells to regorafenib. In a xenograft mouse model, gefitinib sensitized resistant tumors to regorafenib. Additionally, levels of RAS, RAF, and P-ERK1/2, components of the downstream EGFR signaling pathway, were positively associated with EGFR expression.ConclusionEGFR overexpression promotes acquired resistance to regorafenib through RAS/RAF/ERK bypass activation in HCC. Inhibition of EGFR restores sensitivity to regorafenib, and the combination of gefitinib and regorafenib demonstrates significant antitumor efficacy both in vivo and in vitro. These findings suggest that this combination could be a potential strategy for patients with advanced HCC.

Afatinib plus bevacizumab combination after osimertinib resistance in advanced EGFR-mutant non-small cell lung cancer: Phase II ABCD-study

IntroductionMany clinical studies showed a synergy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) and vascular endothelial growth factor inhibitors. We hypothesized afatinib plus bevacizumab exerts clinical potency after developing various osimertinib resistant mechanisms. MethodsEGFR-mutant non-small cell lung cancer patients were enrolled after osimertinib resistance. Afatinib at 30–40 mg/day and bevacizumab at 15 mg/kg tri-weekly were administered until progression. Plasma/histologic rebiopsied samples after osimertinib failure were analyzed to examine resistant mechanisms: gene alterations/copy-number gain using cancer personalized profiling by deep sequencing. ResultsBetween January 2018 and October 2020, 28 patients were enrolled. Response and disease control rates were 17.9 % and 78.6 %, respectively. Median duration of response was 9.0 (range, 4.2–22.3) months. Median progression-free and overall survivals were 2.7 and 9.3 months, respectively. Twenty-eight (100 %) plasma and/or 21 (75 %) histologic rebiopsies identified: 17 (61 %) TP53; 15 (54 %) T790M; 9 (32 %) uncommon EGFR; 9 (32 %) MET; 6 (21 %) C797S; 3 (11 %) BRAF; 2 (7 %) HER2; 2 (7 %) KRAS; and 2 (7 %) PI3K mutations. One (17 %) of 6 C797S patients showed complete response. Three (33 %) of 9 uncommon EGFR-mutated patients achieved radiographic response. Neither 15 T790M-positive nor 6 EGFR downstream signaling mutations: BRAF; KRAS; or PI3K-positive patients responded, but 5 (38 %) of 13 T790M-negative patients responded. Adverse events ≥ grade 3 and incidence ≥ 5 % were: hypertension (29 %); proteinuria (7 %); and diarrhea (7 %). There were neither treatment-related death nor interstitial lung disease. ConclusionsSelected population could obtain clinical benefit from afatinib plus bevacizumab, based on rebiopsy results after osimertinib resistance.

Signaling dynamics in coexisting monoclonal cell subpopulations unveil mechanisms of resistance to anti-cancer compounds

BackgroundTumor heterogeneity is a main contributor of resistance to anti-cancer targeted agents though it has proven difficult to study. Unfortunately, model systems to functionally characterize and mechanistically study dynamic responses to treatment across coexisting subpopulations of cancer cells remain a missing need in oncology.MethodsUsing single cell cloning and expansion techniques, we established monoclonal cell subpopulations (MCPs) from a commercially available epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer cell line. We then used this model sensitivity to the EGFR inhibitor osimertinib across coexisting cell populations within the same tumor. Pathway-centered signaling dynamics associated with response to treatment and morphological characteristics of the MCPs were assessed using Reverse Phase Protein Microarray. Signaling nodes differentially activated in MCPs less sensitive to treatment were then pharmacologically inhibited to identify target signaling proteins putatively implicated in promoting drug resistance.ResultsMCPs demonstrated highly heterogeneous sensitivities to osimertinib. Cell viability after treatment increased > 20% compared to the parental line in selected MCPs, whereas viability decreased by 75% in other MCPs. Reduced treatment response was detected in MCPs with higher proliferation rates, EGFR L858R expression, activation of EGFR binding partners and downstream signaling molecules, and expression of epithelial-to-mesenchymal transition markers. Levels of activation of EGFR binding partners and MCPs’ proliferation rates were also associated with response to c-MET and IGFR inhibitors.ConclusionsMCPs represent a suitable model system to characterize heterogeneous biomolecular behaviors in preclinical studies and identify and functionally test biological mechanisms associated with resistance to targeted therapeutics.

Epidermal Growth Factor Receptor Targeting in Colorectal Carcinoma: Antibodies and Patient-Derived Organoids as a Smart Model to Study Therapy Resistance.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse-human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody-drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host's immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.

Potential role and prognostic value of Globo-H in lung cancer.

e20037 Background: Globo-H (GH), a globo-series carbohydrate antigen highly expressed on multiple epithelial cancers, is a promising target for cancer immunotherapy. Clinical trials have been conducted to evaluate the efficacy of anti-GH vaccine Adagloxad Simolenin/OBI-821 (in Ph 3) and OBI-833/OBI-821 (in Ph 2) in cancer patients. Epidermal growth factor receptor (EGFR) mutations are well known as a frequent class of driver mutations in lung cancer (LC), which are responsible for tyrosine kinase inhibitors. Besides high expression in cancer cells, GH has been implicated in regulation of EGFR signaling pathway. Knockdown of key enzyme involved in GH biosynthesis β1,3-galactosyltransferase V (β3GalT5) induces apoptosis through disruption of EGFR-related signaling protein complex. In addition, GH is reported to associate the EGFR mutant in LC patients. This study aims to further investigate the potential role of GH in LC. Methods: Clinical study: The level of GH expression was evaluated by immunohistochemistry (IHC) using H-score in clinical samples from 141 patients with lung adenocarcinoma. In vitro study: FACS and Western blotting methods were used to measure the levels of total EGFR, phospho-EGFRs or GH in LC cell lines. Microwestern protein array (MWA) analysis was performed in LC NCI-H1975 cells. Results: Clinical study: Positive GH expression (H score≥1; 18.4%) was significantly associated with poor differentiation grade (p = 0.044) in all samples and disease specific survival in patients with metastasis and stage 4 LC (p = 0.033). In vitro study: Inhibition of EGFR by an EGFR-specific shRNA resulted in a decrease of GH expression in LC cells while overexpression of EGFR resulted in an increase of GH levels. β3GalT5 knockout cells and anti-GH-antibody-treated cells showed a reduction of cell proliferation as well as the total level of EGFR and phosphor-EGFRs expression, which is consistent with the results from previous studies. MWA analysis on LC cells with β3GalT5 knockout, GH ceramide-treated β3GalT5 knockout cells and anti-GH Ab-treated LC cells indicated a positive correlation between the level of GH and EGFR-related signaling proteins including phosphor-JNK, phosphor-c-Jun, phosphor-PP2A, phosphor-PP2A, phosphor-AKT, and Cyclin D1 proteins. In addition, JNK signaling inhibitor partially reduced GH expression in NCI-H1975 cells. These results revealed the mechanistic link between GH regulation and tumor promoting signals in LC. Conclusions: GH expression was associated with the worse survival of LC patients in clinical setting. In addition, the level of GH is positively correlated with levels of EGFR and EGFR-related signaling proteins in vitro. Expression of GH can induce the expression of EGFR and vice versa in LC cells. Furthermore, EGFR downstream JNK signaling pathway might involve in GH regulation by EGFR. Taken together, GH-targeting therapy has a potential application for the treatment of LC patients.

Plasma ctDNA biomarker study in patients with non-small cell lung cancer with EGFR exon 20 insertion mutation treated with sunvozertinib.

8563 Background: There are limited reports on biomarker studies of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutation (exon20ins). Sunvozertinib (DZD9008), an oral, potent, irreversible and selective EGFR tyrosine kinase inhibitor (TKI), has been granted conditional approval in China for the treatment of NSCLC with EGFR exon20ins in the ≥ second-line setting. Here we report biomarker analysis of EGFR exon20ins in large sample size, by pooling data from WU-KONG series of studies. Methods: Serial plasma ctDNA samples were collected from baseline until progressive disease (PD). Next generation sequencing was used to assess EGFR exon20ins in baseline plasma ctDNA, and to analyze genetic alterations in baseline and PD plasma ctDNA to identify potential resistance mechanisms to sunvozertinib. Droplet digital PCR was used to dynamically monitor the quantity of EGFR exon20ins during treatment. In addition, association between baseline EGFR exon20ins and disease characteristics as well as antitumor activity of sunvozertinib was analyzed. Furthermore, in vitro and in vivo experiments were performed to explore potential approach which may overcome the resistance to sunvozertinib. Results: A total of 121 patients were included in this biomarker analysis, among them 81 out of 121 (66.9%) had detectable EGFR exon20ins in baseline plasma ctDNA. There was a positive correlation between detectable EGFR exon20ins and higher number of metastasis sites. In addition, higher EGFR exon20ins abundance was positively correlated with more metastasis sites and brain metastasis. Patients with EGFR exon20ins negative in plasma ctDNA achieved a higher objective response rate (ORR) (68.0% vs. 45.8%) and longer median progression free survival (PFS) (7.4 months vs. 5.5 months) with sunvozertinib treatment. In patients with baseline detectable EGFR exon20ins, the mutant allele decreased over time with treatment in majority of patients (12/14, 85.7%), and the earliest clearance of EGFR exon20ins occurred after one week of treatment. In patients who developed disease progression, 12 of 18 (66.7%) maintained EGFR exon20ins at PD, and among them, 3 concurrently acquired EGFR C797S, all in cis with exon20ins. In addition, EGFR G724S and other genetic aberrations in EGFR downstream signaling pathway, were also detected. Exploratory work on overcoming resistance to sunvozertinib suggests that combination of golidocitinib, a selective JAK1 inhibitor, with chemotherapy could be a potential approach. Conclusions: This study showed that positive EGFR exon20ins in plasma ctDNA is associated with advanced disease characteristics. Sunvozertinib can effectively clear EGFR exon20ins in plasma ctDNA, confirming its direct effect on EGFR pathway. The resistance to sunvozertinib can be through EGFR-dependent and EGFR-independent mechanisms.

Abstract 7195: Repurposing non-oncology drugs with MET inhibitory effect to overcome osimertinib resistance in lung cancer

Abstract Background and Aim: Osimertinib is the only 3rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved for the 1st line therapy of advanced NSCLC patients with EGFR mutations. However, drug resistance is severely limiting its clinical efficacy. Aberrant MET activation is an important mechanism causing osimertinib resistance. There is no effective therapeutic option after osimertinib failure. This study investigated the repurposing of non-oncology MET inhibiting drug to overcome osimertinib resistance. Method: A few clinically-approved drugs with putative MET inhibitory effects were identified by a computational drug repurposing tool called “DRAR-CPI” via analysis of the chemical-protein interactome of known MET inhibitors (crizotinib, cabozantinib, foretinib, and tivantinib). Fexofenadine (FEX), originally indicated for allergic rhinitis and chronic urticaria, was chosen for detailed investigation in osimertinib-resistant NSCLC cell lines that exhibit aberrant MET activation with/without other EGFR-dependent osimertinib resistance mechanisms. The selectivity of kinase inhibition by FEX was evaluated by a KINOME profiling assay against a panel of human protein kinases. Western blot analysis was conducted to examine the activation status of Met and EGFR downstream signaling. RNA sequencing was performed to identify differentially expressed genes (DEGs) triggered by the most promising repurposed drug, presumably contributing to osimertinib resistance circumvention. Sulforhodamine B assay was used to evaluate cell proliferation. The circumvention of osimertinib resistance by FEX was further verified in a patient-derived tumor xenograft (PDX) from an advanced-stage osimertinib-refractory NSCLC patient. Result: FEX was found to inhibit recombinant MET kinase in cell-free assays in a dose- and time-dependent manner. It also inhibited MET phosphorylation and other downstream signaling molecules in osimertinib-resistant NSCLC cell lines harboring EGFR T790M+MET amplification or EGFR T790M loss+MET amplification. KINOME profiling revealed a similar kinase inhibition profile between FEX and cabozantinib using Spearman rank order correlation analysis. Among the tested osimertinib-resistant NSCLC cell lines, FEX showed the highest effectiveness in potentiating the anticancer effect of osimertinib in H820 cells (having MET amplification & EGFR T790M mutation). Transcriptome profiling analysis in H820 cells before and after FEX treatment revealed that the majority of DEGs were enriched in EMT-related biological processes and pathways. FEX was further shown to significantly potentiate the antitumor effect of osimertinib in a drug refractory PDX model, without inducing notable weight loss in the animals. Conclusion: These findings advocate the clinical evaluation of repurposing FEX to overcome osimertinib resistance. Citation Format: Kenneth K.W. To, Kwong Sak Leung, William C.S. Cho. Repurposing non-oncology drugs with MET inhibitory effect to overcome osimertinib resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7195.

Immunohistochemical Observation and Correlation of Epidermal Growth Factor Receptor Downstream Signaling Proteins with Plasma Medium-Chain Fatty Acids Levels in Breast Cancer

Abstract Aim: The aim of this study was to correlate the expression of epidermal growth factor receptor (EGFR) downstream signaling molecules with plasma medium-chain fatty acids in breast cancer (BC). Materials and Methods: One hundred and sixteen plasma samples were collected from BC and control subjects. mRNA was extracted and gene expression of mRNA was analyzed by reverse transcriptase-quantitative polymerase chain reaction. Plasma levels of phosphorylated EGFR (p-EGFR), p-Akt, and phosphorylated mammalian target of rapamycin (p-mTOR) were measured using an ELISA kit. Thirty BC tissue sections were used to analyze the expression of EGFR/p-EGFR, Akt/p-Akt, and mammalian target of rapamycin (mTOR)/p-mTOR by immunohistochemistry. Results: The relative mRNA expression levels of EGFR, Akt, and mTOR were high in BC compared to the control subjects, whereas plasma levels of phosphorylated EGFR downstream signaling molecules were upregulated in BC patients, which was statistically significant. The expression of p-EGFR was high in both the cytoplasm and the nuclei, and also, the higher the expression of the downstream molecules in BC tissue sections. Conclusion: In the present investigation, there was a noteworthy inverse relationship between plasma levels of caprylic and lauric acid and the expression of EGFR downstream signaling molecules in BC. These findings suggest that EGFR signaling in BC may be efficiently repressed by increasing the amount of caprylic and lauric acid supplements taken through diet.

Involvement of the AKT Pathway in Resistance to Erlotinib and Cabozantinib in Triple-Negative Breast Cancer Cell Lines.

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

Leveraging innate immunity with AFM24, a novel CD16A and epidermal growth factor receptor (EGFR) bispecific innate cell engager: Interim results for the non-small cell lung cancer (NSCLC) cohort.

2533 Background: Patients with EGFR mutant NSCLC who progress on or after small molecule EGFR inhibitors have only limited treatment options. AFM24 is a tetravalent, bispecific innate cell engager that binds EGFR on solid tumor cells and CD16A on NK cells to redirect and enhance antitumor antibody-dependent cellular cytotoxicity, thus acting independently of the EGFR downstream signaling pathway and EGFR expression levels. The Phase 1 dose escalation phase of the presented study (NCT04259450) established the recommended Phase 2 dose of AFM24 monotherapy at 480 mg based on tolerability, receptor occupancy, and pharmacokinetics (PK). Correlative science data revealed an activation and enrichment of immune cells in the tumor microenvironment. Methods: The Phase 2a expansion phase is evaluating 480 mg AFM24 in patients with EGFR mutant NSCLC, relapsed or refractory to ≥1 prior lines of therapy; EGFR-positivity is defined as positive staining for EGFR in ≥1% of tumor cells per local immunohistochemistry assay. The study follows Simon’s two-stage design. The primary endpoint is overall response rate per local RECIST v1.1; secondary endpoints include safety, PK, and immunogenicity. Efficacy per iRECIST is an exploratory endpoint. AFM24 is administered intravenously once weekly in a 28-day cycle. Tumor assessments are performed at initial screening, cycles 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of December 2022, 14 patients (median [range] age 55 [37–82]; 12 male; 11 Asian, 3 White) were enrolled; the median number of prior therapies was 2.5 (1–12). Median treatment duration with AFM24 was 6.7 weeks (1.0–26.1). Best objective response in 10 evaluable patients was confirmed partial response in 1 patient (change from baseline –45%); 4 patients exhibited stable disease, 1 was confirmed. Tumor shrinkage occurred in 2 patients with stable disease (–6%, –18%). The tumor control rate was 50%. Treatment-related adverse events (TRAEs) were reported in 13 patients; infusion related reactions (IRRs, 12), dermatitis acneiform, neutrophil count decreased, decreased appetite, and myalgia (2 each) were most common. Grade ≥3 TRAEs occurred in 4 patients and included decreased neutrophil count (2), lymphopenia, and IRR (1 each). Grade 5 pneumonitis was observed in 1 patient with disease progression and multiple comorbidities; relation to AFM24 could not be ruled out. As of January 2023, enrollment is ongoing; results of the interim analysis will be presented. Conclusions: AFM24 demonstrated clinical activity and acceptable safety in heavily pretreated patients with EGFR mutant NSCLC. The novel mechanism of action of AFM24 could add to the therapeutic options for patients with EGFR expressing tumors and is under clinical evaluation as a single agent and in combination with atezolizumab or autologous NK cells. Clinical trial information: NCT04259450 .

Diarylheptanoid 35d overcomes EGFR TKI resistance by inducing hsp70-mediated lysosomal degradation of EGFR in EGFR-mutant lung adenocarcinoma

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) patients often respond to EGFR tyrosine kinase inhibitors (TKIs) initially but eventually develop resistance to TKIs. The switch of EGFR downstream signaling from TKI-sensitive to TKI-insensitive is a critical mechanism-driving resistance to TKIs. Identification of potential therapies to target EGFR effectively is a potential strategy to treat TKI-resistant LUADs. In this study, we developed a small molecule diarylheptanoid 35d, a curcumin derivative, that effectively suppressed EGFR protein expression, killed multiple TKI-resistant LUAD cells invitro, and suppressed tumor growth of EGFR-mutant LUAD xenografts with variant TKI-resistant mechanisms including EGFR C797S mutations invivo. Mechanically, 35d triggers heat shock protein 70-mediated lysosomal pathway through transcriptional activation of several components in the pathway, such as HSPA1B, to induce EGFR protein degradation. Interestingly, higher HSPA1B expression in LUAD tumors associated with longer survival of EGFR-mutant, TKI-treated patients, suggesting the role of HSPA1B on retarding TKI resistance and providing a rationale for combining 35d with EGFR TKIs. Our data showed that combination of 35d significantly inhibits tumor reprogression on osimertinib and prolongs mice survival. Overall, our results suggest 35d as a promising lead compound to suppress EGFR expression and provide important insights into the development of combination therapies for TKI-resistant LUADs, which could have translational potential for the treatment of this deadly disease.

Abstract P2-24-06: αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is more frequently seen in TNBC than other breast cancer subtypes, and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in both the MDA-MB-231-4175 and MDA-MB-468 TNBC xenograft models. To determine the mechanism of αEGFR-E-P125A action on inhibition of VM, we studied the effects of αEGFR-E-P125A on both the transcriptome and proteome. RNA-seq was conducted on MDA-MB-231-4175 TNBC cells plated on matrigel and undergoing VM, and on TNBC cells plated in matrigel and treated with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment downregulated genes on the KRAS, JAK-STAT, and angiogenesis signaling pathways, including EGF, VEGFA, STAT3, PTK2, ITGB1, ITGB3, ITGAV, and ITGA5. Phospho-array analysis was used to interrogate the proteome and demonstrated downregulation of phosphorylation on multiple sites downstream of the EGFR and the α5β1 integrin receptors, such as the EGFR Y1069 site, the FAK Y397 site, implicated in the regulation of endothelial and TNBC motility, and the STAT3 Y705 and S727 sites, known for their role in promoting the transcription of angiogenic genes. Since inhibition of EGFR signaling alone does not inhibit VM, we interrogated the mechanistic relationship between αEGFR-E-P125A and VM inhibition through the α5β1 integrin/FAK signaling pathway. Specific inhibition of FAK at the Y397 site using the small molecule inhibitor, PF-573228 inhibited TNBC VM in vitro. Transient siRNA knockdown of FAK in MDA-MB-231-4175 cells and shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM in vitro. To understand the effect of αEGFR-E-P125A on the EGFR and α5β1 integrin receptors, competition assays were conducted. αEGFR-E-P125A competed with EGF for the EGFR receptor and with the binding of fibronectin to α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A is bound to both EGFR and α5β1 integrin, simultaneously suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Rathin Das, Yu Zhang, Christian Elledge, Seung-Uon Shin, Joseph Rosenblatt. αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-06.

Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma.

Epidermal growth factor receptor (EGFR) amplification is found in nearly 40%-50% of glioblastoma cases. Several EGFR inhibitors have been tested in glioblastoma but have failed to demonstrate long-term therapeutic benefit, presumably because of acquired resistance. Targeting EGFR downstream signaling with mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitors would be a more effective approach to glioblastoma treatment. We tested the therapeutic potential of MEK1/2 inhibitors in glioblastoma using 3D cultures of glioma stem-like cells (GSCs) and mouse models of glioblastoma. Several MEK inhibitors were screened in an unbiased high-throughput platform using GSCs. Cell death was evaluated using flow cytometry and Western blotting (WB) analysis. RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and WB analysis were used to identify and validate neuronal differentiation. Unbiased screening of multiple MEK inhibitors in GSCs showed antiproliferative and apoptotic cell death in sensitive cell lines. An RNA-seq analysis of cells treated with trametinib, a potent MEK inhibitor, revealed upregulation of neurogenesis and neuronal differentiation genes, such as achaete-scute homolog 1 (ASCL1), delta-like 3 (DLL3), and neurogenic differentiation 4 (NeuroD4). We validated the neuronal differentiation phenotypes in vitro and in vivo using selected differentiation markers (β-III-tubulin, ASCL1, DLL3, and NeuroD4). Oral treatment with trametinib in an orthotopic GSC xenograft model significantly improved animal survival, with 25%-30% of mice being long-term survivors. Our findings demonstrated that MEK1/2 inhibition promotes neuronal differentiation in glioblastoma, a potential additional mechanism of action of MEK1/2 inhibitors. Thus, MEK inhibitors could be efficacious in glioblastoma patients with activated EGFR/MAPK signaling.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation invitro and invivo.

Structure-guided and phage-assisted evolution of a therapeutic anti-EGFR antibody to reverse acquired resistance

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFRS492R- or EGFRG465R-driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFRS492R, which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFRG465R). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFRS492R and EGFRG465R as well as EGFRWT. The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.

Polysaccharide Peptide Induced Colorectal Cancer Cells Apoptosis by Down-Regulating EGFR and PD-L1 Expression.

Colorectal cancer (CRC) is the most frequent death-causing disease in the world. The Trametes versicolor mushroom, a traditional Chinese medicine, has been used as anti-cancer medicine with long history. Its cultured mycelia extracts, namely polysaccharide peptide (PSP) as the major active component in Trametes versicolor, is widely used in eastern countries to stimulate the immune system and treat deadly cancers, including CRC. This study aimed to explore the mechanism through which PSP inhibits CRC cells proliferation. In vitro, cell proliferation and cytotoxicity of PSP were assessed using human CRC cell lines (HCT116 and HT29). The real-time polymerase chain reaction (PCR), western blot, and immunofluorescence methods were used to examine the expression of epidermal growth factor receptor (EGFR), programmed cell death-ligand 1 (PD-L1), activator of transcription 3 (STAT3), c-Jun, and NF-κB in the PSP treated CRC cells. Human peripheral blood mononuclear cells (PBMC), which were activated with CD3/CD28/CD2 T cell activator and interleukin 2 (IL-2), were co-cultured with HCT116, which was pre-treated with PSP to reduce PD-L1 expression. The synergic effect of T-cells killing was evaluated using the terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) method. Polysaccharide peptide significantly inhibited proliferation of HCT116 and HT29 cell line in vitro. Polysaccharide peptide strongly reduced the expression and phosphorylation level of EGFR. In addition, PSP pretreatment significantly decreased the expression of downstream molecules PD-L1 and EGFR signaling pathways (c-Jun and STAT3) in HCT116. Polysaccharide peptide pretreatment enhanced the T-cells killing effect induced by co-culture PBMC on HCT116 cells. Polysaccharide peptide may be used as a prophylactic and therapeutic agent against CRC via down-regulating PD-L1 and EGFR signaling pathway.

THE INSIGHT OF IN SILICO AND IN VITRO EVALUATION OF OYSTER MUSHROOM (PLEUROTUS OSTREATUS) AGAINST COLORECTAL CANCER TARGETING TIMP-1

Targeted therapy treatment in Colorectal Cancer (CRC) works specifically on a protein or target cell in cancer. It plays a role in the survival of cancer cells. Targeted therapy involves several monoclonal antibodies, one of which is cetuximab which targets the Epidermal Growth Factor Receptor (EGFR). However, there are cases of cetuximab resistance caused by activities of the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1). TIMP-1 can activate the downstream EGFR signaling pathway, thereby that TIMP-1 activities need to be inhibited. Polysaccharide compounds in Pleurotus ostreatus have potential as an immunomodulator and anticancer. This study aimed to determine the potential interaction, types of bonds, the binding energy between the polysaccharide compounds Pleurotus ostreatus and TIMP-1, and survival assay of human adenocarcinoma colorectal (HT-29) cells. This research method compounds selection, data mining, compounds and proteins preparation, docking and visualization of docking results, and cell viability assay. To the study results, TIMP-1 interacted with α-glucan, β-glucan, and galactomannan, which were mediated by hydrophobic and hydrogen bonds. We found new two-point interaction of TIMP-1 with α-glucan and β-glucan on Glu156 and Lys157 that plays a role in the interaction with CD63. Therefore, TIMP-1 can be a candidate for a new ligand on colon cancer targeted therapy. Cetuximab and Pleurotus ostreatus Extract (POE) combinations inhibited the survival of HT-29 cells. The α-glucan and β-glucan may partially inhibit TIMP-1 so that the possibility of resistance due to the interaction of TIMP-1 with CD63 can be inhibited and polysaccharide from Pleurotus ostreatus has anticancer activity on HT-29 cells.

Abstract 2413: αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling

Abstract Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used either alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). Our laboratory previously developed a novel targeted therapy, αEGFR-E-P125A, an antibody-endostatin fusion protein, by linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and decreases metastatic tumor growth (lung metastasis) in both the MDA-MB-231-4175 and MDA-MB 468 TNBC xenograft models. Preliminary studies of the mechanism of αEGFR-E-P125A action indicate that αEGFR-E-P125A downregulates VM through combined inhibition of EGFR and integrin signaling. Phospho-array analysis of αEGFR-E-P125A treated MDA-MB-231-4175 cells demonstrated downregulation of phosphorylation of EGFR at Y1069, a site of aberrant EGFR signaling which promotes TNBC motility. The phospho-array also demonstrated inhibition of focal adhesion kinase (FAK) phosphorylation at Y397. FAK signaling occurs downstream of β1 and β3 integrins, and the Y397 site is implicated in the regulation of endothelial and TNBC motility, VM, and metastatic behavior. We specifically inhibited FAK at the Y397 site using a small molecule inhibitor, PF-573228, and demonstrated that FAK inhibition at Y397 alone inhibited the TNBC VM in vitro. shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM formation in vitro. Immunoprecipitation (IP) of EGFR detected the presence of β1 integrin, and IP of β1 integrin detected EGFR in both MDA-MB-231-4175 and MDA-MB-468 TNBC cells treated with αEGFR-E-P125A. αEGFR-E-P125A treatment increased levels of bound β1 integrin following IP of EGFR relative to untreated or Cetuximab treated TNBC cells. These results indicate that αEGFR-E-P125A is binding to both EGFR and β1 integrin simultaneously, suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and β1 integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hyun Mi Cho, Hava Gil-Henn, Sundaram Ramakrishnan, Rathin Das, Christian Elledge, Yu Zhang, Seung-Uon Shin, Joseph D. Rosenblatt. αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling [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 2413.

A Mechanistic Role for RECK in the Regulation of Hepatocellular Inflammation

RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane‐anchored glycoprotein, modifies the extracellular matrix involved in various cancers, including hepatocellular carcinoma (HCC). Its role in regulating inflammatory and fibrogenic processes has also been postulated. Both inflammation and fibrosis contribute to progression of nonalcoholic steatohepatitis (NASH) to HCC. Here, we tested the hypothesis that inducing or sustaining RECK expression will inhibit proinflammatory amphiregulin and epidermal growth factor receptor (EGFR) signaling, progression of NASH and the development of HCC using an in vitro cell culture model employing isolated adult mouse primary hepatocytes. Ectopic RECK overexpression (gain‐of‐function) was achieved by the adenoviral (Adv) transduction of murine RECK cDNA. Adv.GFP served as a control. RECK expression was silenced (loss‐of‐function) by transducing RECK‐specific siRNA using lipofectamine. Nonspecific siRNA served as a control. Hepatocytes were exposed to TNFα in the presence or absence of inhibitors against ADAM 10 (A Disintegrin and Metalloproteinase Domain‐Containing Protein 10) and/or ADAM17 – two well‐established pro‐inflammatory sheddases. Results show that RECK overexpression inhibited TNFα‐induced ADAM10/17 activity, and amphiregulin (an EGFR ligand) and EGFR expression. In the presence of ADAM10/17 inhibitors, no further reduction was observed in amphiregulin and EGFR activity, suggesting a maximal inhibitory effect of RECK on EGFR activity. In contrast, silencing RECK significantly increased amphiregulin secretion, and this effect was reversed by inhibition of ADAM10/17, indicating RECK signals via regulation of these sheddases. Thus, RECK not only regulates sheddase activity of ADAM10 and 17, but also downstream amphiregulin and EGFR signaling under pro‐inflammatory stimuli. Because increased and sustained EGFR activity contributes to progression of NASH to HCC in preclinical models, our results indicate that inducing RECK has the potential to inhibit hepatocellular inflammation in the setting of NASH and HCC.

Clathrin coated pits as signaling platforms for Akt signaling.

Signaling by the activated epidermal growth factor receptor (EGFR) results in diverse cell fates. In this issue, Cabral-Dias et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.201808181) demonstrate how plasma membrane clathrin coated pits can act as a signaling platform for one branch of EGFR downstream signaling.