EGFR Ligands Research Articles

A quantitative microscopy method to probe RTK activation.

Receptor tyrosine kinases (RTKs) are important for cell regulation, migration, differentiation, and proliferation. Activation of RTKs is characterized by phosphorylation at key tyrosine residues in the intracellular kinase domain. Phosphorylation is induced by ligand binding, which subsequently triggers multiple signaling cascades. We are seeking to develop a new method to quantify the activation of RTKs across the plasma membrane. While phosphorylation is typically characterized using western blotting, in this study we use a quantitative fluorescence technique to characterize the activation of EGFR and link ligand binding to the phosphorylation. EGFR plays an essential role in various biological responses such as differentiation and proliferation and is required in epithelization. One aim of this work is to investigate the activation of EGFR in direct response to ligand binding and the second aim is to investigate the effect of a mutation, known to cause a lung carcinoma, on EGFR activity.

Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels.

Transient receptor potential (TRP) channels are activated by various extracellular and intracellular stimuli and are involved in many physiological events. Because compounds that act on TRP channels are potential candidates for therapeutic agents, a simple method for evaluating TRP channel activation is needed. In this study, we demonstrated that a transforming growth factor alpha (TGFα) shedding assay, previously developed for detecting G-protein-coupled receptor (GPCR) activation, can also detect TRP channel activation. This assay is a low-cost, easily accessible method that requires only an absorbance microplate reader. Mechanistically, TRP-channel-triggered TGFα shedding is achieved by both of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and 17 (ADAM17), whereas the GPCR-induced TGFα shedding response depends solely on ADAM17. This difference may be the result of qualitative or quantitative differences in intracellular Ca2+ kinetics between TRP channels and GPCRs. Use of epidermal growth factor (EGF) and betacellulin (BTC), substrates of ADAM10, improved the specificity of the shedding assay by reducing background responses mediated by endogenously expressed GPCRs. This assay for TRP channel measurement will not only facilitate the high-throughput screening of TRP channel ligands but also contribute to understanding the roles played by TRP channels as regulators of membrane protein ectodomain shedding.

Molecular dynamic simulation reveals the molecular interactions of epidermal growth factor receptor with musk xylene are involved in the carcinogenicity.

Musk xylene (MX), a kind of personal care product, has become a new type of environmental contaminant in recent years. Long-term exposure to MX is associated with a variety of cancers, but the mechanism is still unclear. Meanwhile, our previous research showed that MX exposure could lead to malignant transformation of human liver cells L02 and up-regulation of multi genes which are involved in the MAPK signaling pathway, such as the epidermal growth factor receptor (EGFR). These findings indicated that the MAPK signaling pathway might be involved in the malignant transformation caused by MX, but the mechanism is also unclear. In this study, the underlying interaction mechanisms between EGFR and MX were investigated using molecular dynamics (MD) simulation. Results revealed that MX bound to the ECD of EGFR in four binding sites, which was mainly driven by van der Waals and nonpolar interactions, and the affinity of MX toward ECD was sIII > sI > sII > sIV. Further analysis through MD simulation found that s III, the site with the strongest binding, was coincidentally located at the binding area of EGF, which is the natural ligand of EGFR. Therefore, we speculated that MX may activate the MAPK signaling pathway by binding to EGFR in a similar way to EGF, and finally lead to tumorigenesis. In addition, the MM/PBSA method could also be utilized to calculate the hot residues in each binding site. The prediction of hot residues would provide some theoretical guidance for further study of the carcinogenesis mechanisms of MX both in MD simulation and experimental research.

Regulation of the alveolar regenerative niche by amphiregulin-producing regulatory T cells.

Following respiratory viral infection, regeneration of the epithelial barrier is required to preserve lung function and prevent secondary infections. Lung regulatory T (Treg) cells are critical for maintaining blood oxygenation following influenza virus infection through production of the EGFR ligand amphiregulin (Areg); however, how Treg cells engage with progenitors within the alveolar niche is unknown. Here, we describe local interactions between Treg cells and an Areg-responsive population of Col14a1+EGFR+ lung mesenchymal cells that mediate type II alveolar epithelial (AT2) cell-mediated regeneration following influenza virus infection. We propose a mechanism whereby Treg cells are deployed to sites of damage and provide pro-survival cues that support mesenchymal programming of the alveolar niche. In the absence of fibroblast EGFR signaling, we observe impaired AT2 proliferation and disrupted lung remodeling following viral clearance, uncovering a crucial immune/mesenchymal/epithelial network that guides alveolar regeneration.

Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis.

Immune cells are fundamental regulators of extracellular matrix (ECM) production by fibroblasts and have important roles in determining extent of fibrosis in response to inflammation. Although much is known about fibroblast signaling in fibrosis, the molecular signals between immune cells and fibroblasts that drive its persistence are poorly understood. We therefore analyzed skin and lung samples of patients with diffuse cutaneous systemic sclerosis, an autoimmune disease that causes debilitating fibrosis of the skin and internal organs. Here, we define a critical role of epiregulin-EGFR signaling between dendritic cells and fibroblasts to maintain elevated ECM production and accumulation in fibrotic tissue. We found that epiregulin expression marks an inducible state of DC3 dendritic cells triggered by type I interferon and that DC3-derived epiregulin activates EGFR on fibroblasts, driving a positive feedback loop through NOTCH signaling. In mouse models of skin and lung fibrosis, epiregulin was essential for persistence of fibrosis in both tissues, which could be abrogated by epiregulin genetic deficiency or a neutralizing antibody. Therapeutic administration of epiregulin antibody reversed fibrosis in patient skin and lung explants, identifying it as a previously unexplored biologic drug target. Our findings reveal epiregulin as a crucial immune signal that maintains skin and lung fibrosis in multiple diseases and represents a promising antifibrotic target.

Investigating isoform switching in RHBDF2 and its role in neoplastic growth in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths globally, and its prevalence rates are increasing daily. In the past, studies predicting therapeutic drug targets for cancer therapy focused on the assumption that one gene is responsible for producing one protein. Therefore, there is always an immense need to find promising and novel anti-cancer drug targets. Furthermore, proteases have an integral role in cell proliferation and growth because the proteolysis mechanism is an irreversible process that aids in regulating cellular growth during tumorigenesis. Therefore, an inactive rhomboid protease known as iRhom2 encoded by the gene RHBDF2 can be considered an important target for cancer treatment. Speculatively, previous studies on gene expression analysis of RHBDF2 showed heterogenous behaviour during tumorigenesis. Consistent with this, several studies have reported the antagonistic role of iRhom2 in tumorigenesis, i.e., either they are involved in negative regulation of EGFR ligands via the ERAD pathway or positively regulate EGFR ligands via the EGFR signalling pathway. Additionally, different opinions suggest iRhom2 mediated cleavage of EGFR ligands takes place TACE dependently or TACE independently. However, reconciling these seemingly opposing roles is still unclear and might be attributed to more than one transcript isoform of iRhom2. To observe the differences at isoform resolution, the current strategy identified isoform switching in RHBDF2 via differential transcript usage using RNA-seq data during breast cancer initiation and progression. Furthermore, interacting partners were found via correlation and enriched to explain their antagonistic role. Isoform switching was observed at DCIS, grade 2 and grade 3, from canonical to the cub isoform. Neither EGFR nor ERAD was found enriched. However, pathways leading to TACE-dependent EGFR signalling pathways were more observant, specifically MAPK signalling pathways, GPCR signalling pathways, and toll-like receptor pathways. Nevertheless, it was noteworthy that during CTCs, the cub isoform switches back to the canonical isoform, and the proteasomal degradation pathway and cytoplasmic ribosomal protein pathways were significantly enriched. Therefore, it could be inferred that cub isoform functions during cancer initiation in EGFR signalling. In contrast, during metastasis, where invasion is the primary task, the isoform switches back to the canonical isoform.

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.

The HIF-prolyl hydroxylases have distinct and nonredundant roles in colitis-associated cancer.

Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD). HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) control cellular adaptation to hypoxia and are considered promising therapeutic targets in IBD. However, their relevance in the pathogenesis of CAC remains elusive. We induced CAC in Phd1-/-, Phd2+/-, Phd3-/-, and WT mice with azoxymethane (AOM) and dextran sodium sulfate (DSS). Phd1-/- mice were protected against chronic colitis and displayed diminished CAC growth compared with WT mice. In Phd3-/- mice, colitis activity and CAC growth remained unaltered. In Phd2+/- mice, colitis activity was unaffected, but CAC growth was aggravated. Mechanistically, Phd2 deficiency (i) increased the number of tumor-associated macrophages in AOM/DSS-induced tumors, (ii) promoted the expression of EGFR ligand epiregulin in macrophages, and (iii) augmented the signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 signaling, which at least in part contributed to aggravated tumor cell proliferation in colitis-associated tumors. Consistently, Phd2 deficiency in hematopoietic (Vav:Cre-Phd2fl/fl) but not in intestinal epithelial cells (Villin:Cre-Phd2fl/fl) increased CAC growth. In conclusion, the 3 different PHD isoenzymes have distinct and nonredundant effects, promoting (PHD1), diminishing (PHD2), or neutral (PHD3), on CAC growth.

Abstract B078: PP2A activation drives alternative EGFR activation in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) stands to become the 2nd most deadly cancer by 2030. Over 90% of PDAC patients have oncogenic KRAS mutations with the most prevalent being KRASG12D which have proven difficult to therapeutically target. Several studies have implicated EGFR signaling as critical for PDAC tumorigenesis in KRAS mutant tumors. Consistent with these findings, the first FDA-approved targeted therapeutic for PDAC was the EGFR inhibitor, Erlotinib. However, single agent use of Erlotinib has shown minimal efficacy in the clinic suggesting that this signaling is complex and needs further interrogation. Studies have shown the serine/threonine phosphatase, Protein Phosphatase 2A (PP2A), negatively regulates several downstream targets of EGFR and KRAS. Our previous studies using small molecule activators of PP2A demonstrate a heterogeneous response to PP2A activation, with some PDAC cell lines downregulating oncogenic signals and others maintaining the oncogenic signaling. Using pharmacological activation of PP2A, as well as overexpression and knockdown studies, we have identified a novel signaling cascade in which PP2A activation leads to increased expression and secretion of EGFR ligands and EGFR activation in a subset of PDAC cell lines. Given this unique feedback mechanism, we combined EGFR inhibitors with PP2A activation and found that the combination killed more cells than either agent alone. Together, our studies identify a novel role for PP2A in regulating EGFR signaling and support the combined use of EGFR inhibitors and PP2A activators to increase efficacy of both agents. Citation Format: Claire M. Pfeffer, Sydney J. Clifford, Elizabeth G. Hoffman, Gagan K. Mall, Garima Baral, Brittany L. Allen-Petersen. PP2A activation drives alternative EGFR activation in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B078.

CSIG-03. EGFR LIGAND SHIFTS THE ROLE OF EGFR FROM ONCOGENE TO TUMOR SUPPRESSOR IN EGFR AMPLIFIED GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a devastating cancer largely because of its highly invasive nature. Although pathways that promote GBM invasion have been identified, no pathway that actively suppresses GBM invasion is known and there is no treatment that specifically blocks GBM invasion. EGFR is a prime oncogene and is frequently amplified and mutated in GBM. However multiple clinical trials of EGFR inhibition in GBM have failed. Here, we demonstrate that EGFR ligands activate a tumor suppressor for the EGFR in EGFR-amplified GBMs. Analysis of TCGA data reveals that GBM patients with EGFR amplification and a low level of EGFR ligands have a worse prognosis compared to those with EGFR amplification and a high level of EGFR ligands. We identify the cytoskeletal BAR domain protein BIN3 as a critical suppressor of invasion upregulated by EGFR ligand in EGFR amplified GBMs. BIN3 inhibits a DOCK-7 Rho GTPase pathway, resulting in small hyper-proliferating noninvasive tumors and a better prognosis, while constitutive EGFR wild type or EGFRvIII signaling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumors and worse survival in orthotopic models. Consistent with its role as a negative regulator of invasion, BIN3 is deleted in 10% of GBMs and increased expression of BIN3 confers a better prognosis. Thus, increased EGFR ligand shifts the role of EGFR from oncogene to tumor suppressor in EGFR amplified GBMs by suppressing invasion. We identify tofacitinib, a BBB penetrant drug as an upregulator of BIN3 and show that tofacitinib suppresses invasion and prolongs survival in orthotopic models. Our study identifies a new tumor suppressive function of EGFR in GBM and we propose the use of tofacitinib to directly target invasion in EGFR-amplified and EGFR ligand-poor GBMs.

Heparin binding epidermal growth factor-like growth factor is a prognostic marker correlated with levels of macrophages infiltrated in lung adenocarcinoma.

The interactions between tumor cells and the host immune system play a crucial role in lung cancer progression and resistance to treatment. The alterations of EGFR signaling have the potential to produce an ineffective tumor-associated immune microenvironment by upregulating a series of immune suppressors, including inhibitory immune checkpoints, immunosuppressive cells, and cytokines. Elevated Heparin-binding EGF-like growth factor (HB-EGF) expression, one EGFR ligand correlated with higher histology grading, worse patient prognosis, and lower overall survival rate, acts as a chemotactic factor. However, the role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the accumulation of immune cells in the tumor microenvironment remains unclear. The clinical association of HB-EGF expression in lung cancer was examined using the Gene Expression Omnibus (GEO) repository. HB-EGF expression in different cell types was determined using single-cell RNA sequencing (scRNA-seq) dataset. The correlation between HB-EGF expression and cancer-immune infiltrated cells was investigated by performing TIMER and ClueGo pathways analysis from TCGA database. The chemotaxis of HB-EGF and macrophage infiltration was investigated using migration and immunohistochemical staining. The high HB-EGF expression was significantly correlated with poor overall survival in patients with lung adenocarcinoma (LUAD) but not lung squamous cell carcinoma (LUSC). Moreover, HB-EGF expression was correlated with the infiltration of monocytes, macrophages, neutrophils, and dendritic cells in LUAD but not in LUSC. Analysis of scRNA-seq data revealed high HB-EGF expression in lung cancer cells and myeloid cells. Results from the pathway analysis and cell-based experiment indicated that elevated HB-EGF expression was associated with the presence of macrophage and lung cancer cell migration. HB-EGF was highly expressed in tumors and correlated with M2 macrophage infiltration in LUAD. HB-EGF is a potential prognostic marker and therapeutic target for lung cancer progression, particularly in LUAD.

A Retrospective Analysis of NRG/RTOG 0522: Low Myofibroblast Content in the Tumor-Associated Stroma may Predict Response to Epidermal Growth Factor Receptor (EGFR) Inhibitors

<h3>Purpose/Objective(s)</h3> Most head and neck cancers (HNC) overexpress EGFR, a feature associated with poor clinical outcome. Preclinical and clinical data suggest that peritumoral myofibroblasts (MF) express EGFR ligands and the presence of a high MF density in the tumor stroma is associated with statistically worse survival in patients with locally advanced HNC. We evaluated whether peritumoral MF density was prognostic of outcome in patients treated on RTOG 0522, a negative phase III trial evaluating the addition of C225 to cisplatin-based chemoradiation (CRT). We also evaluated whether MF density was predictive of benefit to C225. <h3>Materials/Methods</h3> Specimens from HPV-negative patients treated on RTOG 0522 were obtained from the NRG Oncology Biospecimen Repository and evaluated by a blinded pathologist (JW). MF density was determined as a percentage and the skewed data were normalized using Box-Cox transformation, from which the mean was calculated and used to stratify patients into low- and high-density groups. Overall survival (OS), progression-free survival (PFS), and distant metastasis (DM) rates were estimated using the Kaplan-Meier method and a multivariable analysis (MVA) by Cox proportional hazards model was used to test for the prognostic effect of this biomarker for patients with low vs. high MF density. To test for predictive effect of MF density on clinical outcomes in patients treated with CRT vs. CRT+C225, a Cox model with a treatment-by-biomarker interaction term was used. <h3>Results</h3> A total of 186 patients were evaluated, 94 were treated with CRT and 92 were treated with CRT+C225. On MVA, including primary site, age, PS, smoking history, T-stage, N-stage, and EGFR level, there was no significant difference in OS (Hazard ratio [HR], 0.65; 95% CI, 0.41-1.02; P=0.06), PFS (HR, 0.75; 95% CI, 0.50-1.12; P=0.16), or DM (HR, 0.76; 95% CI, 0.38-1.54; P=0.44) based on MF density. However, MVA Cox model including biomarker, treatment arm and biomarker-treatment interaction demonstrated that this interaction was significant (P=0.01). Within the low MF density subgroup, patients treated with CRT+C225 had significantly higher PFS (HR, 0.42; 95% CI, 0.21 to 0.82; P=0.01) and lower incidence of DM (HR, 0.18; 95% CI, 0.04 to 0.82; P=0.01), as well as numerically higher OS (HR, 0.48; 95% CI, 0.22 to 1.05; P=0.06) compared to patients treated with CRT. Patients with high MF density demonstrated no difference in OS, PFS, or DM based on treatment arm. <h3>Conclusion</h3> Peritumoral MF density was not prognostic of OS, PFS, or DM among patients treated on RTOG 0522. However, patients with low peritumoral MF density who received CRT+C225 had significantly higher PFS and lower incidence of DM compared to patients who received CRT alone. These results suggest that MF density is not a prognostic biomarker but may be predictive of benefit to C225. Further investigations are needed to validate these exploratory findings.

Non-enzymatic role of SOD1 in intestinal stem cell growth

Superoxide dismutase 1 (SOD1) modulates intestinal barrier integrity and intestinal homeostasis as an antioxidant enzyme. Intestinal homeostasis is maintained by the intestinal stem cells (ISCs). However, whether and how SOD1 regulates ISCs is unknown. In this study, we established intestinal organoids from tamoxifen–inducible intestinal epithelial cell–specific Sod1 knockout (Sod1f/f; Vil-creERT2) mice. We found that loss of Sod1 in organoids suppressed the proliferation and survival of cells and Lgr5 gene expression. SOD1 is known for nearly half a century for its canonical role as an antioxidant enzyme. We identified its enzyme-independent function in ISC: inhibition of SOD1 enzymatic activity had no impact on organoid growth, and enzymatically inactive Sod1 mutants could completely rescue the growth defects of Sod1 deficient organoids, suggesting that SOD1-mediated ISC growth is independent of its enzymatic activity. Moreover, Sod1 deficiency did not affect the ROS levels of the organoid, but induced the elevated WNT signaling and excessive Paneth cell differentiation, which mediates the occurrence of growth defects in Sod1 deficient organoids. In vivo, epithelial Sod1 loss induced a higher incidence of apoptosis in the stem cell regions and increased Paneth cell numbers, accompanied by enhanced expression of EGFR ligand Epiregulin (EREG) in the stromal tissue, which may compensate for Sod1 loss and maintain intestinal structure in vivo. Totally, our results show a novel enzyme-independent function of SOD1 in ISC growth under homeostasis.

ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion.

Macrophages in the tumor microenvironment have a substantial impact on tumor progression. Depending on the signaling environment in the tumor, macrophages can either support or constrain tumor progression. It is therefore of therapeutic interest to identify the tumor-derived factors that control macrophage education. With this aim, we correlated the expression of A Disintegrin and Metalloproteinase (ADAM) proteases, which are key mediators of cell-cell signaling, to the expression of protumorigenic macrophage markers in human cancer cohorts. We identified ADAM17, a sheddase upregulated in many cancer types, as a protein of interest. Depletion of ADAM17 in cancer cell lines reduced the expression of several protumorigenic markers in neighboring macrophages in vitro as well as in mouse models. Moreover, ADAM17-/- educated macrophages demonstrated a reduced ability to induce cancer cell invasion. Using mass spectrometry-based proteomics and ELISA, we identified heparin-binding EGF (HB-EGF) and amphiregulin, shed by ADAM17 in the cancer cells, as the implicated molecular mediators of macrophage education. Additionally, RNA-Seq and ELISA experiments revealed that ADAM17-dependent HB-EGF ligand release induced the expression and secretion of CXCL chemokines in macrophages, which in turn stimulated cancer cell invasion. In conclusion, we provide evidence that ADAM17 mediates a paracrine EGFR-ligand-chemokine feedback loop, whereby cancer cells hijack macrophages to promote tumor progression.

EGFR Signaling Is Overactive in Pachyonychia Congenita: Effective Treatment with Oral Erlotinib

Pachyonychia congenita (PC) is a rare keratinizing disorder characterized by painful palmoplantar keratoderma for which there is no standard current treatment. PC is caused by dominant mutations in keratin (K) K6A, K6B, K6C, K16, or K17 genes involved in stress, wound healing, and epidermal barrier formation. Mechanisms leading to pain and painful palmoplantar keratoderma in PC remain elusive. In this study, we show overexpression of EGFR ligands epiregulin and TGF-α as well as HER1‒EGFR and HER2 in the upper spinous layers of PC lesions. EGFR activation was confirmed by upregulated MAPK/ERK and mTOR signaling. Abnormal late terminal keratinization was associated with elevated TGM1 activity. In addition, the calcium ion permeable channel TRPV3 was significantly increased in PC-lesional skin, suggesting a predominant role of the TRPV3/EGFR signaling complex in PC. We hypothesized that this complex contributes to promoting TGM1 activity and induces the expression and shedding of EGFR ligands. To counteract this biological cascade, we treated three patients with PC with oral erlotinib for 6‒8 months. The treatment was well-tolerated and led to an early, drastic, and sustained reduction of neuropathic pain with a major improvement of QOL. Our study provides evidence that targeted pharmacological inhibition of EGFR is an effective strategy in PC.

Abstract 331: Pre-clinical evaluation in endocrine resistant breast cancer of a novel antibody-drug conjugate targeting cell surface cleaved Amphiregulin

Abstract The EGFR ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We also show that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. Among ER-positive (n = 88) and ER-negative (n = 50) tumors evaluated, the proportions of tumors exhibiting medium/high intensity staining with 1A3 were essentially equivalent (69.3% v 70%, respectively). These data provide proof-of-principle that cell surface proteolytic cleavage products can represent good targets for antibody drug conjugate development and highlight the potential of using this antibody drug conjugate against tumors shedding high levels of Amphiregulin. Citation Format: Kristopher A. Lofgren, Sreeja Sreekumar, E. Charles Jenkins, Kyle J. Ernzen, Paraic A. Kenny. Pre-clinical evaluation in endocrine resistant breast cancer of a novel antibody-drug conjugate targeting cell surface cleaved Amphiregulin [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 331.

Abstract 5560: KIR-based CAR design showed durable antitumor efficacy by altered internalization kinetics following persistent antigenic stimulation

Abstract Poor persistence of functional chimeric antigen receptor (CAR) T cells in the tumor microenvironment can limit the effectiveness of the antitumor immune response. We previously showed that T cells expressing a multichain CAR design that uses an activating killer immunoglobulin-like receptor (KIR) transmembrane (TM) and cytoplasmic domain co-expressed with the ITAM-containing adapter, DAP12 (KIR-CAR) are able to control large, pre-established tumors that cannot be controlled by 2nd generation CD3ζ-based CAR T cells. This improved function is associated with maintenance of KIR-CAR expression on the tumor infiltrating lymphocytes compared with CD3ζ-based CARs. In the present study, we explored the mechanism for the improved KIR-CAR expression, which we hypothesized was related to reduced receptor internalization following receptor activation compared with standard CD3ζ-based CARs. To monitor receptor dynamics at the T cell surface, we took advantage of a surface protein tagging method that employs the SpyCatcher-SpyTag system to construct a KIR-based or CD3ζ-based CAR with the ability to pulse label the CAR at the cell surface with a fluorescent molecule. In this approach, a CAR ectodomain comprised of a SpyCatcher domain is linked to either the KIR TM and cytoplasmic domain (SpyKIR-CAR) or CD28 TM and cytoplasmic domain with CD3ζ (SpyCAR). The antigenic specificity of the CAR is conferred by addition of a fluorescently-labeled binder comprised of an antibody-like Designed Ankyrin Repeat Protein (DARPin) fused to a SpyTag sequence (SpyDARPin) that specifically binds the spyCatcher domain covalently via its spyTag and recognizes the EGFR antigen. The SpyKIR-CAR or Spy-CAR expression is confirmed by flow cytometry using an antibody to a myc-tag within the DARPin sequence. Uniform fluorescence labeling at the plasma membrane is observed following addition of the SpyDARPin as assessed by image cytometry and confocal microscopy. The kinetics of SpyCAR or SpyKIR-CAR fluorescence loss were similar in the absence of EGFR ligand with a half-life of 2 days consistent with normal membrane turnover by pinocytosis. Upon ligand-induced CAR activation, the SpyCAR fluorescence rapidly reduced beginning at 30 min post-activation to background fluorescence levels of unlabeled SpyCAR cells within 8 hours of activation. In contrast, the mean SpyKIR-CAR fluorescence showed no change in fluorescence at 30 min, while exhibiting a reduction to 80% of the mean baseline fluorescence at 8 hours and 4-fold above the background fluorescence. Confocal imaging confirmed the retention of labeled KIR-CAR at the plasma membrane over the time frame in which the labeled SpyCAR is lost. Our data support differences in CAR internalization upon activation as an important mechanism underlying the enhanced function of KIR-CAR T cells in solid tumors compared with traditional CD3ζ-based CAR designs. Citation Format: Qian Zhang, Eric Nigel, Alvin Yu, Morgan Hresko, Nicholas Minutolo, Daniel Powell, Michael C. Milone. KIR-based CAR design showed durable antitumor efficacy by altered internalization kinetics following persistent antigenic stimulation [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 5560.

Depleting receptor tyrosine kinases EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer

BackgroundEpidermal growth factor receptor (EGFR) inhibitors, including cetuximab and panitumumab, are valuable therapeutics for colorectal cancer (CRC), but resistance to these inhibitors is common. The reason for such resistance is not well understood, which hampers development of better therapeutic strategies. Although activating mutations in KRAS, BRAF and PIK3CA are considered major drivers of CRC resistance to EGFR inhibitors, therapeutic targeting of these drug resistance drivers has not produced substantial clinical benefit.MethodsWe exploited cell lines and mouse tumor models (cell line xenografts and patient derived xenografts) for experiments of genetic and pharmacologic depletion of EGFR and/or its family member HER2, including EGFR mutants, inhibition of EGFR ligand shedding, and biochemical analysis of signaling proteins, to delineate the mechanism of CRC resistance to EGFR inhibitors and to assess the therapeutic activity of PEPDG278D, which is a recombinant human protein that induces the degradation of both EGFR and HER2.ResultsThe sensitivity of CRC cells to cetuximab and panitumumab correlates with the ability of these drugs to induce EGFR downregulation. PEPDG278D strongly inhibits oncogenic signaling and growth of CRC cells by causing profound depletion of EGFR and HER2, regardless of activating mutations of KRAS, BRAF and PIK3CA. siRNA knockdown of EGFR or HER2 also inhibits CRC cells resistant to EGFR inhibitors. Tumors harboring mutated KRAS, BRAF and/or PIK3CA also overexpress EGFR ligands, further suggesting that EGFR signaling remains important to the tumors. While excessive tumor-generated high-affinity EGFR ligands block target engagement by PEPDG278D, aderbasib, an inhibitor of ADAM10 and ADAM17, enables PEPDG278D to exert strong antitumor activity by inhibiting ligand shedding. Moreover, adding fluorouracil, which is commonly used in CRC treatment, to the combination of PEPDG278D and aderbasib further enhances tumor inhibition.ConclusionsOur study shows that CRC resistance to EGFR inhibitors results primarily from the inability of the inhibitors to downregulate their target and that a PEPDG278D-based combination treatment overcomes the resistance.

A biomarker enrichment trial of anti-EGFR agents in right primary tumor location (rPTL), RAS wild-type (RAS-wt) advanced colorectal cancer (aCRC): ARIEL (ISRCTN11061442).

TPS3633 Background: Meta-analysis of 6 RCTs indicates that anti-EGFR agents are ineffective in rPTL RAS-wt aCRC (Arnold D, et al. Ann Oncol. 2017;28:1713-1729). However, data from the phase III PICCOLO and COIN trials suggest high tumor expression of the EGFR ligands, EREG and AREG, confers sensitivity to anti-EGFR agents in a subset of this population (Adams RA, et al. J Clin Oncol. 2012;30(30_suppl):32-32; Seligmann JF, et al. Ann Oncol. 2020;31:1021-1029). More data is needed before ligand assessment can be integrated into routine care: to date, EREG/AREG mRNA has only been assessed retrospectively, and feasibility of timely delivery of results must be demonstrated. The ARIEL trial aims to determine whether first-line chemotherapy plus cetuximab or panitumumab is more effective than chemotherapy alone in achieving early tumor shrinkage (ETS) after 8 weeks of treatment in patients (pts) with EREG/AREG-high rPTL RAS-wt aCRC. Methods: ARIEL is a multicentre, phase IV, open label, biomarker enrichment RCT. Pts with previously untreated rPTL RAS-wt (or RAS-unknown) aCRC are eligible for registration and EREG/AREG assessment using archival FFPE tumor tissue. Those confirmed as RAS-wt EREG/AREG-high (expression above 30th centile based on PICCOLO)3 are eligible for randomization to chemotherapy alone (fluoropyrimidine backbone plus irinotecan or oxaliplatin) vs chemotherapy (FOLFOX or FOLFIRI) plus anti-EGFR therapy (panitumumab or cetuximab) (options at physician’s discretion). Pts with EREG/AREG-low tumors are not eligible for randomization but may consent to translational research and follow-up. The primary endpoint is ETS at 8 weeks (≥30%, yes vs no). Secondary endpoints are depth of response at 16 weeks, overall survival, overall treatment utility, pt-reported quality of life, cost per QALY, pt acceptability of trial procedures, and safety. Pre-trial work-up included cross-validation of the EREG/AREG RT-qPCR assay at trial laboratories in Leeds and Birmingham, UK demonstrating reproducibility of biomarker results. Recruitment to an internal pilot phase is currently ongoing to demonstrate feasibility of timely delivery of biomarker results to sites (lower limit of 90% CI of mean result delivery time for first 20 pts must include 3 weeks). Mean monthly recruitment rate will be assessed at 18 months to determine likelihood of completion of the trial within the 3 year recruitment period. ARIEL is funded by the UK National Institute for Health Research (NIHR) and opened the first of 40 sites in February 2022. 440 pts will be registered for biomarker assessment in order to randomize 162 pts. All pts will be followed-up to 1 year post-randomisation, with a final assessment in all pts when the last pt has completed a year of follow-up (median 3.5 years). ARIEL is participating in the NIHR Associate PI scheme. Clinical trial information: 11061442.

Repair of the Murine Tympanic Membrane Displays Hallmarks of Regeneration

BackgroundThe tympanic membrane (TM) has a remarkable ability to repair itself, with perforations typically closing in days to weeks in all mammalian species studied. The cellular and molecular mechanisms underlying TM repair remain largely unknown, however. This study looked to thoroughly characterize the repair process of the injured TM and uncover mechanisms of repair to inform future treatments for TM disorders. We hypothesized that the TM does not undergo canonical “wound healing” but regenerates in response to injury.MethodsCells from TMs injured on adult mice 14, 7, 3, and 1 day prior to sacrifice were submitted for single‐cell RNA sequencing using the 10x Genomics scRNA‐seq platform. Using Seurat clustering and the CellFindR algorithm, unbiased cell clusters were generated with matrices of differentially expressed genes for each cluster. Protein detection via immunofluorescence (IF) and RNA detection via RNAscope were used to biologically validate and define the anatomic locations of distinct cellular populations. The K5Cre‐ERT2 promoter was utilized in mouse models to genetically manipulate and fluorescently label keratinocyte (KC) populations. The mTmG and Confetti reporters were utilized as fluorescent labels.ResultsRNA expression data from all timepoints of perforation were merged and analyzed, revealing 8 distinct major populations of cells and revealing time‐dependent transcriptional shifts in each layer of the TM. From both cross‐sectional and whole‐mount views, the TM shows a rapid, proliferative response to injury by 18 hours post‐injury, predominantly in the KCs. 3 days after perforation, there are large transcriptional shifts in the immune, mesenchymal, and mucosal populations. The multi‐layered tissue shows a large volumetric increase by day 7 but quickly remodels and restores the original volume of the TM by day 14. At slightly longer timepoints, the radial and circular collagen patterning of the TM is also restored, creating a scar‐free structure. We identified a regeneration‐induced “wounded epithelial” population, characterized by a combination of distinct marker genes. A K5Cre‐ERT2;Confetti mouse model shows that the population migrates from known stem cell regions of the organ to the site of injury. Based on expression values and immunostaining, EGFR signaling is upregulated during regeneration, corresponding with increased expression of EGFR ligands and processing co‐factors. When EGFR is deleted in vivo, using a K5‐CreERT2;Egfrfl/fl; R26mTmG/mTmG mouse model, TMs no longer display proliferation post‐injury and cannot repair perforations.ConclusionsTM perforation healing displays a complex and coordinated series of transcriptional, morphological, and patterning events, ultimately culminating in the restoration of normal tissue architecture to the TM. These characteristics are more typical of epimorphic regeneration, rather than more typical scar induced mammalian wound healing.