EGFR-targeted Therapy Research Articles

Putative mechanisms of primary resistance to EGFR-targeted therapies: A retrospective study

BackgroundsAdvanced lung adenocarcinoma (LUAD) patient with EGFR mutations often experience resistance to first-line epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) therapy. Nonetheless, the mechanism and biomarkers of primary resistance remain unclear. Further exploration of independent prognostic factors will help clinicians identify patients who may not respond to EGFR-TKIs and select appropriate treatment strategies. MethodsA retrospective study involving 124 patients with stage IV LUAD harboring a common sensitizing EGFR mutation (exon 19 deletion or L858R mutation) who received EGFR-TKIs as first-line therapy was performed. All participants were tested by DNA-targeted sequencing in baseline samples, and there were 19 patients with progression-free survival (PFS) ≤ 3 months (cohort 1, C1, primary resistance), 22 patients with 3 < PFS < 8 months (cohort 2, C2, poor response) without known mutations associated with resistance, and 83 patients with PFS ≥ 8 months (cohort 3, C3, normal). ResultsThe most commonly mutated genes at baseline in patients prior to treatment within the entire study population. were TP53 (65 %), MYC (19 %), CDKN2A (12 %), MUC16 (12 %) and RBM10 (12 %). The baseline characteristics, except for the proportions of patients with EGFR L858R mutation and exon 19 deletion in C1 plus C2 compared to C3 (p = 0.036), were not significantly different among the cohorts. The frequencies of PIK3C2G, STK11, EPAS1, RARA and BTG2 variation were significantly higher in C1, the primary resistance group. Multivariate Cox analysis revealed that PIK3C2G (HR 15.70 95 % CI 3.24–76.05, p < 0.001), STK11 (HR 17.04, 95 % CI 3.68–78.92, p < 0.001), EPAS1 (HR 11.99, 95 % CI 2.57–56.03, p = 0.002), and BTG2 amplification (HR 9.53, 95 % CI 1.67–54.28, p = 0.011) were significantly associated with shorter PFS. ConclusionsThe genomic landscape varies significantly among patients with LUAD, which should be considered when making personalized treatment decisions. This information could provide insights into molecular changes and their effects on clinical treatment in diverse patients with LUAD harboring sensitizing EGFR mutations.

Reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant infection by blocking the epidermal growth factor receptor (EGFR) pathway.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants presents challenges in global efforts to transition from the pandemic to an endemic stage. The spike protein of the SARS-CoV-2 virus, which is pivotal for cell entry, exhibits significant mutations in its variants, potentially affecting infectivity and therapeutic efficacy. Recent findings indicate upregulation of the epidermal growth factor receptor (EGFR) pathway, a key target in cancer therapy, by the spike protein of SARS-CoV-2. This study aimed to investigate the activity of the EGFR pathway against SARS-CoV-2 variants and to assess the inhibitory effects of EGFR inhibitors using SARS-CoV variant pseudoviral particles to guide future therapeutic strategies. Omicron variant SARS-CoV pseudoviral particles exhibited heightened infectivity in human angiotensin-converting enzyme 2 (hACE2)-expressing HEK293 and A549 lung cancer cells accompanied by increased EGFR pathway activation in infected cells. Using the EGFR tyrosine kinase inhibitor, osimertinib, we observed a reduction in viral infection rates in hACE2-HEK293 and A549 cells infected with the SARS-CoV-2 variant pseudoviral particles. We conducted further experiments to confirm that the reduction in infection efficacy with osimertinib treatment was not associated to a decrease in cell viability. Furthermore, this inhibitory effect of osimertinib in cell lines was corroborated in a spheroid cell culture model derived from hACE2-A549 cells. These findings suggest the potential application of EGFR-targeted antiviral therapy against highly infectious SARS-CoV-2 variants.IMPORTANCEThe emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is concerning as vaccines designed for one variant need not essentially protect against other novel variants. Therefore, there is an urgent need to identify therapies that can act against multiple novel variants that have heightened virulence compared with the wild type. It has been reported that the spike protein of the SARS-CoV-2 virus elicits an increased expression of the epidermal growth factor receptor (EGFR) pathway. We used this information and examined whether treatment with an EGFR inhibitor, osimertinib, which is already approved for clinical use in cancer therapy, can reduce the infection caused by SARS-CoV-2, wild type, and Omicron and Delta variants, in two cell lines and one spheroid model. The results showed that osimertinib treatment successfully reduced infection efficacy, particularly in variants, and that this effect was not related to a reduction in cell viability, making this a promising strategy for treating SARS-CoV-2 infections.

Toxicity Profile of eBAT, a Bispecific Ligand-Targeted Toxin Directed to EGFR and uPAR, in Mice and a Clinical Dog Model.

EGFR-targeted therapies are efficacious, but toxicity is common and can be severe. Urokinase type plasminogen activator receptor (uPAR)-targeted drugs are only emerging, so neither their efficacy nor toxicity is fully established. Recombinant eBAT was created by combining cytokines EGF and uPA on the same single-chain molecule with truncated Pseudomonas toxin. Its purpose was to simultaneously target tumors and their vasculature in the tumor microenvironment. In prior studies on mice and dogs, the drug proved efficacious. Here, we report the safety of eBAT in normal wildtype, uPAR knockout, and immunoreplete and immunodeficient tumor-bearing mice, as well as in dogs with spontaneous sarcoma that more closely mirror human cancer onset. In immunocompetent mice, tumor-bearing mice, uPAR knockout mice, and mice receiving species-optimized eBAT, toxicities were mild and self-limiting. Likewise, in dogs with life-threatening sarcoma given dosages found to be biologically active, eBAT was well tolerated. In mice receiving higher doses, eBAT was associated with dose-dependent evidence of liver injury, including portal biliary hyperplasia, oval cell proliferation, lymphoplasmacytic inflammation, periportal hepatocellular microvesicular change, hemorrhage, necrosis, and apoptosis. The results support continuing the clinical development of eBAT as a therapeutic agent for individuals with sarcoma and other cancers.

Functional loss of ERBB receptor feedback inhibitor 1 (MIG6) promotes glioblastoma tumorigenesis by aberrantactivation of epidermal growth factor receptor (EGFR).

Dysregulation of epidermal growth factor receptor (EGFR) is one of the most common mechanisms associated with the pathogenesis of various cancers. Mitogen-inducible gene 6 [MIG6; also known as ERBB receptor feedback inhibitor 1 (ERRFI1)], identified as a feedback inhibitor of EGFR, negatively regulates EGFR by directly inhibiting its kinase activity and facilitating its internalization, subsequently leading to degradation. Despite its proposed role as an EGFR-dependent tumor suppressor, the functional consequences and clinical relevance in cancer etiology remain incompletely understood. Here, we identify that the stoichiometric balance between MIG6 and EGFR is crucial in promoting EGFR-dependent oncogenic growth in various experimental model systems. In addition, a subset of ERRFI1 (the official gene symbol of MIG6) mutations exhibit impaired ability to suppress the enzymatic activation of EGFR at multiple levels. In summary, our data suggest that decreased or loss of MIG6 activity can lead to abnormal activation of EGFR, potentially contributing to cellular transformation. We propose that the mutation status of ERRFI1 and the expression levels of MIG6 can serve as additional biomarkers for guiding EGFR-targeted cancer therapies, including glioblastoma.

Differential expression of epidermal growth factor receptor in various pathological types of salivary gland cancers

ObjectiveWhile several studies reported epidermal growth factor receptor (EGFR) expression in salivary gland cancer (SGC), results varied due to a lack of unified definition of EGFR positivity. In this study, we assessed the EGFR expression level using both EGFR positive score and cumulative EGFR score in the patients with SGC. MethodsBetween January 2010 and April 2021, 102 patients with SGC who underwent surgical resection were reviewed retrospectively by immunohistochemistry. The membrane staining intensity was scored as follows: no staining (0), weak staining (1+), intermediate staining (2+), and strong staining (3+). The cumulative EGFR score was determined on a continuous scale of 0–300 using the formula:1 × (1+: percentage of weakly stained cells) + 2 × (2+: percentage of moderately stained cells) + 3 × (3+: percentage of strongly stained cells). ResultsEGFR expression in SGC varied widely even among the same as well as different histopathological types. The average EGFR positive scores were 46.0 %, 55.7 %, 51.6 %, 1.0 %, 26.8 %, 50 %, and 76.8 % for mucoepidermoid carcinoma (MEC), salivary duct carcinoma (SDC), adenoid cystic carcinoma (AdCC), acinic cell carcinoma (AcCC), adenocarcinoma NOS (ACNOS), carcinoma ex pleomorphic adenoma (CAexPA), and squamous cell carcinoma (SqCC), respectively. The average cumulative EGFR scores were 82, 91, 80, 1, 52, 93, and 185 for MEC, SDC, AdCC, AcCC, ACNOS, CAexPA, and SqCC, respectively. ConclusionsEGFR positive scores and cumulative EGFR scores in SGCs varied among the various histological types, and even in the same histological type. These scores may predict the clinical outcome of SGC treated with EGFR-targeting therapies, such as head and neck photoimmunotherapy, and need to be evaluated in future studies.

Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect

BackgroundThe dynamic interplay between tyrosine kinase inhibitors (TKIs) and the tumor immune microenvironment (TME) plays a crucial role in the therapeutic trajectory of non-small cell lung cancer (NSCLC). Understanding the...

Whole transcriptome analysis to reveal epithelial-mesenchymal transition after neoadjuvant therapy with or without additional anti-EGFR treatment: Subgroup analysis of esophageal squamous cell carcinomas of the SAKK 75/08 and the EORTC AIO POWER study.

4071 Background: In localized disease of esophageal squamous cell cancer (ESCC), a trend in higher pathological response rates and longer time to treatment failure was seen by addition of anti-EGFR treatment to radio-chemotherapy (RCTX) compared to RCTX alone (SAKK 75/08 trial and others). On the contrary, the effect of anti-EGFR treatment to chemotherapy (CTX) remains unclear as no survival benefit was detected within advanced patients (EORTC-AIO-POWER study). Since perfect discrimination and selection of subgroups with highest benefit for anti-EGFR treatment (AB) is missing, we investigated the predictive values of whole transcriptomic gene expression analysis (WTGE) applied on biopsy tissue. Methods: 50 pretherapeutic formalin fixed paraffin embedded specimens of ESCC patients from SAKK 75/08 study were analyzed for the effect of RCTX with and without anti-EGFR AB. As well as 26 historic controls of primary resected tumor stage equally distributed ESCC were analyzed. For the advanced setting, we analyzed 28 additional pretherapeutic biopsies and compared groups from CTX to CTX plus AB. The HTG EdgeSeq method was used for NGS-based WTGE. Response to treatment was assessed by histopathological tumor regression in localized disease and clinical courses. Response to treatment in the advanced tumor setting was assessed to the clinical course of stable disease and partial response versus non-responders. Results: Firstly, NGS-based WTGE failed to show significant differences in expression patterns between responding and non-responding ESCC patients, irrespective of treatment protocols and EGFR AB in the localized as well in advanced disease setting. Secondly however, comparison between pre-treatment biopsies and posttreatment residual tumor tissues detected 781 genes that were upregulated and 623 that were downregulated with gene sets associated with muscle processes and epithelial-mesenchymal transition (EMT) in the localized disease. EMT genes were also enriched in residual tumors after neoadjuvant RCTX compared to primary resection in an independent control cohort. Additionally performed immunohistochemical studies showed lower numbers of ZEB-1 positive ESCC in an independent collection of tumors treated by neoadjuvant RCTX compared to primary resection. Conclusions: Our Whole transcriptomic gene expression analyses suggest that EMT is involved in responses to neoadjuvant protocols including EGFR targeting therapies. Targeting EMT may be a molecular pathway to increase complete responses after neoadjuvant RCTX in ESCC.

SLC7A5 correlated with malignancies and immunotherapy response in bladder cancer

BackgroundMetabolic reprogramming contributes to bladder cancer development. This study aimed to understand the role of SLC7A5 in bladder cancer.MethodsWe systematically analyzed the correlation between SLC7A5 and bladder cancer through various approaches, including bioinformatics, western blotting, cell cycle analysis, cell proliferation assays, and invasion experiments. We also investigated the immunological features within the tumor microenvironment (TME), encompassing cancer immune cycles, immune modulators, immune checkpoints, tumor-infiltrating immune cells (TIIC), T cell inflammation scores, and treatment responses. Additionally, for a comprehensive assessment of the expression patterns and immunological roles of SLC7A5, pan-cancer analysis was performed using cancer genomics datasets.ResultsSLC7A5 was associated with adverse prognosis in bladder cancer patients, activating the Wnt pathway and promoting bladder cancer cell cycle progression, proliferation, migration, and invasion. Based on the evidence that SLC7A5 positively correlated with immunomodulators, TIIC, the cancer immune cycle, immune checkpoint and T cell inflammation scores, we also found that SLC7A5 was associated with the inflammatory tumor immune microenvironment. EGFR-targeted therapy, cancer immunotherapy, and radiation therapy were effective for patients with high SLC7A5 expression in bladder cancer. Low SLC7A5 patients were, however, sensitive to targeted therapies and anti-angiogenic therapy, such as blocking β-catenin network, PPAR-γ and FGFR3 signaling. Anti-SLC7A5 combined with cancer immunotherapy may have greater effectiveness than either therapy alone. Furthermore, we observed specific overexpression of SLC7A5 in TME of various cancers.ConclusionSLC7A5 can predict therapeutic response to immunotherapy, radiotherapy and chemotherapy in bladder cancer patients. Targeting SLC7A5 in combination with immunotherapy may be a potentially appropriate treatment option.

New Directions for Advanced Targeting Strategies of EGFR Signaling in Cancer.

Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling studies paved the way for a basic understanding of growth factor and oncogene signaling pathways and the development of tyrosine kinase inhibitors (TKIs). Due to resistance mutations and the activation of alternative pathways when cancer cells escape TKIs, highly diverse cell populations form in recurrent tumors through mechanisms that have not yet been fully elucidated. In this review, we summarize recent advances in EGFR basic research on signaling networks and intracellular trafficking that may clarify the novel mechanisms of inhibitor resistance, discuss recent clinical developments in EGFR-targeted cancer therapy, and offer novel strategies for cancer drug development.

Desmoglein 2 and desmocollin 2 depletions promote malignancy through distinct mechanisms in triple-negative and luminal breast cancer

BackgroundAberrant expressions of desmoglein 2 (Dsg2) and desmocollin 2(Dsc2), the two most widely distributed desmosomal cadherins, have been found to play various roles in cancer in a context-dependent manner. Their specific roles on breast cancer (BC) and the potential mechanisms remain unclear.MethodsThe expressions of Dsg2 and Dsc2 in human BC tissues and cell lines were assessed by using bioinformatics analysis, immunohistochemistry and western blotting assays. Wound-healing and Transwell assays were performed to evaluate the cells’ migration and invasion abilities. Plate colony-forming and MTT assays were used to examine the cells’ capacity of proliferation. Mechanically, Dsg2 and Dsc2 knockdown-induced malignant behaviors were elucidated using western blotting assay as well as three inhibitors including MK2206 for AKT, PD98059 for ERK, and XAV-939 for β-catenin.ResultsWe found reduced expressions of Dsg2 and Dsc2 in human BC tissues and cell lines compared to normal counterparts. Furthermore, shRNA-mediated downregulation of Dsg2 and Dsc2 could significantly enhance cell proliferation, migration and invasion in triple-negative MDA-MB-231 and luminal MCF-7 BC cells. Mechanistically, EGFR activity was decreased but downstream AKT and ERK pathways were both activated maybe through other activated protein tyrosine kinases in shDsg2 and shDsc2 MDA-MB-231 cells since protein tyrosine kinases are key drivers of triple-negative BC survival. Additionally, AKT inhibitor treatment displayed much stronger capacity to abolish shDsg2 and shDsc2 induced progression compared to ERK inhibition, which was due to feedback activation of AKT pathway induced by ERK inhibition. In contrast, all of EGFR, AKT and ERK activities were attenuated, whereas β-catenin was accumulated in shDsg2 and shDsc2 MCF-7 cells. These results indicate that EGFR-targeted therapy is not a good choice for BC patients with low Dsg2 or Dsc2 expression. Comparatively, AKT inhibitors may be more helpful to triple-negative BC patients with low Dsg2 or Dsc2 expression, while therapies targeting β-catenin can be considered for luminal BC patients with low Dsg2 or Dsc2 expression.ConclusionOur finding demonstrate that single knockdown of Dsg2 or Dsc2 could promote proliferation, motility and invasion in triple-negative MDA-MB-231 and luminal MCF-7 cells. Nevertheless, the underlying mechanisms were cellular context-specific and distinct.

Single cell lineage tracing reveals clonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer

BackgroundMost primary Triple Negative Breast Cancers (TNBCs) show amplification of the Epidermal Growth Factor Receptor (EGFR) gene, leading to increased protein expression. However, unlike other EGFR-driven cancers, targeting this receptor in TNBC yields inconsistent therapeutic responses.MethodsTo elucidate the underlying mechanisms of this variability, we employ cellular barcoding and single-cell transcriptomics to reconstruct the subclonal dynamics of EGFR-amplified TNBC cells in response to afatinib, a tyrosine kinase inhibitor (TKI) that irreversibly inhibits EGFR.ResultsIntegrated lineage tracing analysis revealed a rare pre-existing subpopulation of cells with distinct biological signature, including elevated expression levels of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2). We show that IGFBP2 overexpression is sufficient to render TNBC cells tolerant to afatinib treatment by activating the compensatory insulin-like growth factor I receptor (IGF1-R) signalling pathway. Finally, based on reconstructed mechanisms of resistance, we employ deep learning techniques to predict the afatinib sensitivity of TNBC cells. ConclusionsOur strategy proved effective in reconstructing the complex signalling network driving EGFR-targeted therapy resistance, offering new insights for the development of individualized treatment strategies in TNBC.

Clinical Outcomes of Patients with Non-Small Cell Lung Cancer Leptomeningeal Disease Following Receipt of EGFR-Targeted Therapy, Immune-Checkpoint Blockade, Intrathecal Chemotherapy, or Radiation Therapy Alone

BackgroundEGFR-targeted therapy (ETT) and immune-checkpoint blockade (ICB) have shown promising results in treating NSCLC brain metastases (BM). However, little is known of their effect in treating leptomeningeal disease (LMD). Patients and MethodsThis is a retrospective review of 80 patients diagnosed with NSCLC LMD from January 2014 to March 2021. Patients were grouped based on initial LMD treatment: radiotherapy (RT) alone, ETT, ICB, and intrathecal chemotherapy (ITC). ResultsEGFR mutation was present in 22 patients (28%). Twenty patients had positive cytology in cerebrospinal fluid, while 60 patients were diagnosed based on MRI with clinical correlation. The RT alone group consisted primarily of whole brain radiation (n = 20; 77%), stereotactic radiation (n = 3; 12%), and palliative spine radiation (n = 2; 7%). There were no significant differences amongst the treatment groups in age, performance status, or neurologic symptoms. Overall, the 6-month overall survival (OS) and craniospinal progression free survival (CS-PFS) were 35% and 24%, respectively. The 6-month OS for the ETT, ICB, ITC, and RT alone groups was 64%, 33%, 57%, and 29% respectively (log-rank P = .026). The 6-month CS-PFS for the ETT, ICB, ITC, and RT alone groups was 43%, 33%, 29%, and 19% respectively (log-rank P = .049). Upon univariate analysis, receipt of ETT compared to RT alone reached significance for OS (HR 0.35, P = .006) and CS-PFS (HR 0.39, P = .013). ConclusionsThe prognosis for patients with NSCLC LMD remains poor overall. However, the receipt of ETT for patients with EGFR-positive disease was associated with improved outcomes.

Abstract CT216: Anti-EGFR in combination with paclitaxel as second-line therapy for gastric cancer with EGFR overexpression

Abstract Background: We and other investigators reported that EGFR gene is amplified in 2-4% of gastric cancer with protein overexpression (Gastroenterology 2017 Aug;153(2):536-549; Cancer Cell 2019 Jan 14; 35:1:111:E10), but EGFR-targeted therapy has not entered into clinic. Patients and Methods: As a subprotocol of the investigator-initated of umbrella trial, patients with metastatic gastric cancer received weekly anti-EGFR monoclonal antibody (GC-1118 (GC Biopharma), 2.5mg/kg) in combination with paclitaxel 80mg/m2 (D1, D8, D15) every 4 weeks as a second-line therapy, if the gastric cancer tissue sample revealed EGFR protein overexpression (NCT04077255). Projected accrual was 19 based on minimax two-stage design (P1=45%; P0=15%; alpha and beta errors, 0.05 and 0.1, respectively). Results: Among 19 evaluable patients, 6 patients had objective clinical response (RR, 31.6%). Median progression-free survival was 4.7 months (95% CI, 1.8-5.1). Median overall survival was 7.6 months (95% CI, 4.2-11.1). Predictive biomarkers were explored by post hoc correlative analyses. Conclusion: EGFR inhibition, in combination with paclitaxel, demonstrated modest activity for gastric cancer refractory to first-line 5-FU/platinum-based chemotherapy. (This study was supported by a grant from the National R&amp;D Program for Cancer Control, Ministry of Health &amp; Welfare, Republic of Korea (HA22C0056.)) Citation Format: Dong-Hoe Koo, Jin-Young Kim, Jae-Joon Kim, Sung Y. Oh, Min-Hee Ryu, Dae Y. Zang, Hark K. Kim. Anti-EGFR in combination with paclitaxel as second-line therapy for gastric cancer with EGFR overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT216.

Oncogenic Roles of Laminin Subunit Gamma-2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation.

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal biliary epithelial cancer in the liver. Here, Laminin subunit gamma-2 (LAMC2) with important oncogenic roles in iCCA is discovered. In a total of 231 cholangiocarcinoma patients (82% of iCCA patients) across four independent cohorts, LAMC2 is significantly more abundant in iCCA tumor tissue compared to normal bile duct and non-tumor liver. Among 26.3% of iCCA patients, LAMC2 gene is amplified, contributing to its over-expression. Functionally, silencing LAMC2 significantly blocks tumor formation in orthotopic iCCA mouse models. Mechanistically, it promotes EGFR protein translation via interacting with nascent unglycosylated EGFR in the endoplasmic reticulum (ER), resulting in activated EGFR signaling. LAMC2-mediated EGFR translation also depends on its interaction with the ER chaperone BiP via their C-terminus. Together LAMC2 and BiP generate a binding "pocket" of nascent EGFR and facilitate EGFR translation. Consistently, LAMC2-high iCCA patients have poor prognosis in two iCCA cohorts. LAMC2-high iCCA cells are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs) treatment both in vitro and in vivo. Together, these data demonstrate LAMC2 as an oncogenic player in iCCA by promoting EGFR translation and an indicator to identify iCCA patients who may benefit from available EGFR-targeted TKIs therapies.

Abstract 684: DT-9045, a novel PAR2 inhibitor with best-in-class properties that reduces resistance to both EGFR-targeting therapies and immunotherapy in oncology models

Abstract Protease-activated receptor 2 (PAR2) is a promising therapeutic target in oncology and immuno-oncology. It is upregulated and associated with poor prognosis in several cancer types. A pan-cancer meta-analysis showed that PAR2 is one of the genes most significantly associated with resistance to immune checkpoint blockade (ICB) and T cells dysfunction in cancer patients. Mechanistically, being expressed by various cells of the tumor microenvironment, PAR2 promotes both survival and proliferation of cancer cells and dampens the anti-cancer immune response. Domain Therapeutics has developed a novel PAR2 inhibitor, DT-9045, with best-in-class properties. Indeed, compared to its most advanced competitors, DT-9045 is a small molecule, orally bioavailable, insurmountable, biased, and active in tumor-like conditions (i.e. acidic pH and high level of activating proteases). This compound is also highly potent and selective on PAR2. Pharmacokinetic properties are suitable with a once-a-day oral administration. In vitro, DT-9045 completely prevented PAR2-mediated resistance to EGFR-targeting drugs. In vivo, its combination with anti-PD1 therapy showed a significant increase in efficacy over monotherapies in preclinical syngeneic mouse cancer models. This result was similar to the one observed in PAR2 knockout mice, indicating that oral administration of DT-9045 induces a complete inhibition of the receptor. We have further demonstrated that treatment with a PAR2 inhibitor enhances dendritic cell-mediated T cell activation and intratumoral infiltration of CD4+ and CD8+ T cells as well as a decrease in M2 macrophage infiltration. In conclusion, Domain Therapeutic has identified a novel PAR2 inhibitor, DT-9045, with clear competitive advantages. This preclinical candidate has shown strong potency in alleviating the resistance to both EGFR-targeting therapies and immunotherapy in preclinical cancer models. IND-enabling studies are currently ongoing to bring this new hope for cancer patients to the clinic. Citation Format: Thibaut Brugat, Maleck Kadiri, Samya Aouad, Anne-Laure Blayo, Baptiste Rugeri, Antoine Mousson, Aurélie Janvier, Edith Steinberg, Luc Baron, Mandy Recolet, Xavier Wirth, Maria Jesus Garcia-Leon, Quentin Ruet, Meriem Semache, Laurent Sabbagh, Orphée Blanchard, Christel Franchet, Stanislas Mayer, John Stagg, Nathalie Lenne, Stephan Schann. DT-9045, a novel PAR2 inhibitor with best-in-class properties that reduces resistance to both EGFR-targeting therapies and immunotherapy in oncology models [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 684.

Abstract 7203: Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors

Abstract Mutant epidermal growth factor receptor (EGFR), a major oncogenic driver in non-small cell lung cancer (NSCLC), is found in the tumors of 15% (western countries) to 50% (Asian countries) of patients. EGFR-targeted therapy using tyrosine kinase inhibitors (TKIs) is a standard of care for those patients who developed metastatic or recurrent diseases. However, despite having an 80% initial response rate, most patients had partial tumor regression and their disease would progress when acquired resistance emerges. Thus, developing more effective treatment is an unmet need. The clinical response of these patients can be recapitulated in mouse models of patient-derived xenograft (PDX) tumor bearing sensitizing EGFR mutations. In this study, treating established PDX tumors with osimertinib, a third generation EGFR TKI, induced partial tumor regression (50% to 70% size reduction from baseline) followed by progression with a median progression-free survival (PFS) of 26 to 28 days. Interestingly, co-administering an antibody against hepatoma-derived growth factor (HDGF) with osimertinib resulted in enhanced tumor regression (98% to 100% size reduction from baseline) which translated into significantly extended animal survival with a median PFS of 103 or 135 days. Immunohistochemical and western blot examination of tumors collected at different treatment timepoints indicated that AKT/mTOR and MAPK pathways were reactivated after initial suppression in TKI monotherapy arm despite persistent EGFR inhibition. Since AKT/mTOR and MAPK pathways are key survival and proliferative signaling pathways, their reactivation can promote TKI tolerance of tumor cells and the emergence of TKI resistance. However, blocking HDGF attenuated the reactivation of these pathways which could partially explain the enhanced efficacy of osimertinib plus anti-HDGF antibody combination treatment seen in this study. Our results suggest tumor-derived HDGF could play a crucial role in the rise of acquired resistance to osimertinib. Identifying the exact cells and molecular pathways utilized by HDGF to promote tumor cell tolerance to TKI will help us understand how TKI sensitive tumor cells survive EGFR blockade and emerge as resistant cells. Citation Format: Cindy Q. Zhou, Ariel Li, Kaoru Ri, Ahmed Sultan, Hening Ren. Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors [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 7203.

Abstract 2619: A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation

Abstract EGFR and HER3 are receptor tyrosine kinases that are highly expressed in multiple epithelial tumors. EGFR point mutations and small insertions within the kinase domain are common in lung cancer. HER3 is overexpressed in patients who are resistant to EGFR-TKI therapy, and is one of the common resistance mechanisms of EGFR and HER2-targeted therapy. We hypothesize that simultaneous targeting of EGFR and HER3 with a bispecific ADC will have the potential to overcome the resistance caused by HER3 after EGFR-targeted therapy and obtain better efficacy than the combination of EGFR monotherapy and HER3 monotherapy. We first generated fully human bispecific antibodies (bsAbs) targeting EGFR and HER3 using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. The anti-EGFR × HER3 bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, pancreatic, colorectal, and breast cancer cell lines. The anti-EGFR × HER3 bsAb also showed high internalization activity in cell lines expressing EGFR and HER3. The anti-EGFR × HER3 bsAb was then conjugated with vc-MMAE (valine-citrulline-monomethyl auristatin E) for proof-of-concept studies, as well as BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and highly hydrophilic protease-cleavable linker, to generate EGFR- and HER3-targeting bispecific ADC candidates BCG019. In vivo efficacy of the bsADC candidates were evaluated in cell-derived NSCLC and gastric cancer xenografts and in patient-derived gastric and colorectal xenograft models. Both bsADCs showed superior anti-tumor activity when compared to benchmark ADCs. Collectively, these results suggest that our novel anti-EGFR × HER3 bsADC has therapeutic potential for EGFR and HER3 co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation [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 2619.

Abstract 4552: EGFR inhibitors increase LGR5 expression and enhance potency of LGR5 antibody-drug conjugates targeting colorectal cancer stem cells

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-associated deaths in the United States. One of the primary challenges in treating CRC is therapy resistance thought to be mediated by cancer stem cells (CSCs), a subpopulation of cancer cells with infinite replicative potential that can differentiate to drive tumorigenesis and disease relapse. Antibody-drug conjugates (ADCs) are amongst the fastest growing classes of anticancer drugs and utilize the specificity of monoclonal antibodies (mAbs) to hone cytotoxic payloads to cancer cells. We generated ADCs directed against leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-defined biomarker of CSCs that is highly overexpressed in CRCs. LGR5 ADCs demonstrated high specificity and efficacy in CRC cells and xenograft models, though tumor relapse after treatment withdrawal was a major obstacle. Interestingly, FDA-approved therapy targeting epidermal growth factor receptor (EGFR) has been shown to increase LGR5 mRNA expression levels in patient-derived models of CRC. This study aims to determine the mechanism of EGFR-mediated regulation of LGR5 expression and evaluate the therapeutic efficacy of combination treatments targeting EGFR and LGR5 for the improved treatment of CRC. To first analyze the effect of EGFR inhibition on LGR5 expression, a panel of CRC cell lines of different KRAS and PI3CKA mutational statuses were treated with FDA-approved EGFR-targeted mAbs or small molecule inhibitors. LGR5 protein expression was shown to be upregulated by EGFR-targeted therapies in a dose- and time-dependent manner, independent of mutation status. Notably, treatment with EGF, MEK1/2 inhibitor trametinib, and EGFR-directed siRNA all resulted in concomitant reduction in EGFR and LGR5 protein levels, suggesting EGFR and LGR5 are co-degraded. Co-immunoprecipitation and immunocytochemistry experiments identified a novel EGFR-LGR5 interaction and co-internalization mechanism in CRC cells, respectively. Furthermore, we showed EGFR-LGR5 interaction is enhanced by treatment with the EGFR-targeting mAb, cetuximab (CTX). To evaluate efficacy of combination therapies targeting EGFR and LGR5, CRC cells were treated with LGR5 ADCs with or without CTX. Importantly, we showed CTX significantly enhanced the potency of LGR5 ADCs incorporating different classes of cytotoxic payloads. These results suggest combining LGR5 ADCs with EGFR inhibitors may be a more effective approach for the treatment of CRC and eliminating CSCs to overcome resistance and relapse. Citation Format: Peyton C. High, Zhengdong Liang, Kendra S. Carmon. EGFR inhibitors increase LGR5 expression and enhance potency of LGR5 antibody-drug conjugates targeting colorectal cancer stem cells [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 4552.

Abstract 5799: Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response

Abstract Aberrant activation of epidermal growth factor receptor (EGFR) signaling pathways is associated with tumor growth and progression. EGFR overexpression occurs in various types of solid cancers and correlates with poor outcome. Unfortunately, the clinical benefit from EGFR-targeted therapies is usually short-lived, due to the development of resistance. TargImmune has developed first-in-class Targeted Apoptotic Immunomodulators (TAIMs), which harness the body’s anti-viral responses to treat solid tumors. Our novel nanoparticle platform technology, Ta:RNA , selectively delivers synthetic dsRNA, polyinosinic:polycytidylic acid (polyIC) to tumor cells that overexpress a target receptor. PolyIC and other pattern recognition receptor (PRR) agonists have been used as adjuvants or for intratumoral administration but have limited efficacy as single agents. TargImmune’s platform allows systemic delivery of dsRNA, tailoring the nanoparticles to various solid cancers by simply changing the targeting moiety. Here we describe EGFR-Ta:RNA, which is targeted to tumors that overexpress EGFR. EGFR-Ta:RNA does not inhibit EGFR per se, and therefore activation of alternative or downstream oncogenes is not expected to cause resistance. The mechanism of action of EGFR-targeted Ta:RNA nanoparticles was extensively studied in vitro and in vivo, using confocal imaging, large-scale RNA sequencing studies, Western blot analysis and ELISA assays. In vitro, EGFR-Ta:RNA potency and selectivity were established by comparing cells with high receptor and low receptor expression. Imaging studies showed that the nanoparticles are internalized selectively into EGFR-overexpressing cancer cells by receptor-mediated endocytosis. RNAseq studies revealed: (i) a common transcriptional signature in EGFR-Ta:RNA-treated tumours and cancer cells; (ii) enrichment in pathways related to anti-viral response, inflammation, type I interferon signaling, and NFΚB signaling; (iii) enrichment in genes coding for cytokines. Western blot analysis and ELISA assays confirmed that the mechanism of action of EGFR-Ta:RNA is mediated via the induction of PRR downstream signaling, which results in cytokine secretion, apoptosis and immune cell activation. The efficacy of EGFR-Ta:RNA was demonstrated in multiple animal models, including both xenograft and immunocompetent models. The pharmacodynamic effect, exemplified by target engagement and activation of PRR signaling in tumors, was verified in vivo. In conclusion, TargImmune’s EGFR-Ta:RNA nanoparticles deliver polyIC in a tumor-targeted manner to evoke PRR signaling, inducing targeted tumor cell apoptosis as well as immune cell recruitment and activation against the heterogenous tumor. EGFR-Ta:RNA represents a novel, multimodal treatment approach for EGFR overexpressing cancers. Citation Format: Derrick Broka, Caroline De Feyter, Shoshana Klein, Meera Saxena, Joerg Schreiber, Alexei Shir, Michal Skowicki, Yaakov Benenson, Alexander Levitzki, Cornelia G. Palivan, Esteban Pombo-Villar, Babette Schade, Maya Zigler. Novel nanoparticle technology targets the delivery of polyIC to EGFR overexpressing cancers and induces a multimodal anti-tumor response [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 5799.

Abstract 1221: Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma

Abstract In lung adenocarcinoma (LUAD), EGFR-targeted therapies often result in incomplete tumor responses followed by disease progression caused by acquired resistance. As EGFR mutations are truncal (i.e., present in every cell of a tumor), it remains unclear why a fraction of tumor cells can survive in the presence of drug treatment. It is increasingly recognized that drug-tolerant persister cells (DTPCs) can escape selective drug pressure and are important drivers of therapy failure and tumor relapse. The mechanisms underlying tumor cell adaptation and survival of DTPCs during therapy are not fully understood.Using EGFR-mutant LUAD cell lines treated with osimertinib, we unexpectedly observed that DTPCs display uncoupled mRNA and protein variations of PBK (PDZ Binding Kinase), a spindle assembly serine/threonine protein kinase. Mechanistically we discovered that although global protein synthesis is decreased upon osimertinib treatment, PBK translation is selectively maintained in DTPCs.The establishment and survival of DTPCs following EGFR inhibition is critically dependent on PBK, as genetic depletion of PBK reduces persistence to osimertinib treatment in vitro. PBK knockout leads to suppression of ERK1/2 reactivation following EGFR inhibition, and results in decreased expression of ZEB1 and Vimentin, key effectors of epithelial-to-mesenchymal transition (EMT), a hallmark of the DTPC phenotype. Consistently, combined treatment with osimertinib and OTS514 (PBK inhibitor) in vitro, reduces the DTPC fraction and decreases the enrichment of EMT-related transcriptional signatures. Similar results were obtained in LUAD patient-derived organoids, used to recapitulate the phenotype of residual disease in patients. In PDX treated with osimertinib, PBK is activated at both residual disease and acquired resistance. Concordantly, combined EGFR/PBK pharmacological inhibition was also effective in reverting osimertinib acquired resistance in vitro. Finally, in matched diagnosis and relapse samples from 7 EGFR-mutant LUAD patients treated with EGFR inhibitors, we observed increased PBK activation at the time of acquired resistance in 5 cases, including 3 EGFRT790M-positive cases, suggesting that PBK activation may co-occur with genetic resistant alterations.Our findings unveil a novel mechanism by which EGFR-mutant lung cancer DTPCs selectively translate PBK mRNA as an adaptive survival mechanism to persist during osimertinib treatment; and provide a therapeutic target of non-genetic drug resistance otherwise unlooked-for in routine transcriptomic analyses. A deeper understanding on the mRNA translational reprogramming in DTPCs is currently performed to uncover therapeutic vulnerabilities to reduce residual disease and delay or revert the emergence of acquired resistance. Citation Format: Camille Leonce, Anne-Laure Désage, Nour El-Houda Mourksi, Geneviève De Souza, Béatrice Vanbervliet, Camille Charrondière, Constance Nicq, Jérémy Néri, Aurélie Swalduz, Laurie Tonon, Eric Cumunel, Nicolas Gadot, Gabriel Ichim, Jean-Jacques Diaz, Christine Lovly, Julien Mazières, Mayeul Tabutin, Maurice Pérol, Sylvie Lantuejoul, Olivier Calvayrac, Fabien Forest, Virginie Marcel, Pierre Saintigny, Sandra Ortiz-Cuaran. Selective translational activation of PBK (PDZ- binding kinase) reveals a molecular vulnerability of drug-tolerant persister cells to targeted therapies in EGFR-mutant lung adenocarcinoma [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 1221.