EGFR-targeted Therapy Research Articles

Abstract 549: PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation

Abstract Background: Triple-negative breast cancer (TNBC) is lack of effective targeted therapies. Epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. However, TNBC is resistant to EGFR-targeted drugs in clinical trials, and the tolerance mechanism remains unclear. This study aimed to elucidate the possible resistance mechanism for TNBC to EGFR-targeted therapy and how sensitize TNBC cells to erlotinib. Methods: PDZ domain containing 1 (PDZK1) correlated with TNBC development was screened out by bioinformatics analysis and its correlation with resistance to erlotinib was identified by western blotting (WB). Its interaction with EGFR and structural basis were determined by GST-Pull down and coimmunoprecipitation. Its regulation on EGFR signaling activation and expression level were detected by WB in PDZK1 overexpression and knockdown TNBC cells, xenograft tissues and correlation analyses. Correlation was performed by gene set enrichment analysis (GSEA) and immunohistochemistry of tissue microarray followed by Pearson analysis. TNBC-suppressing effects of PDZK1 and its sensitizing effects on erlotinib were investigated using cell viability assay, colony-forming assay, wound healing assay, Matrigel invasion assay and xenograft model in combination with PDZK1 wild type/mutant transfection and rescue experiment. Results: Here we found that PDZK1 was correlated with TNBC development and its level was downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation was related to erlotinib resistance in TNBC. PDZK1 bound with EGFR. Through the interaction, PDZK1 promoted EGFR degradation by enhancing the binding of EGFR with c-Cbl and inhibited EGFR phosphorylation by hindering EGFR dimerization. PDZK1 was specifically downregulated in TNBC tissues and was correlated with poor prognosis of TNBC. Functional assays in vitro and in vivo showed that PDZK1 suppressed TNBC development. Restoring EGFR expression and kinase inhibitor treatment reversed the malignancy caused by PDZK1 overexpression and knockdown, respectively. PDZK1 wild type, but not mutant overexpression enhanced the inhibitory effect of erlotinib on EGFR signaling and TNBC malignancy in vitro and in vivo. Conclusion: PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for TNBC to reverse the tolerance of TNBC cells to erlotinib. Citation Format: Junfang Zheng, Yuanzhen Ma, Zhiyu Fang, Yijun Qi. PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation [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 549.

Abstract 4728: Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer

Abstract Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody (mAb), results in beneficial, yet limited, clinical improvement in metastatic colorectal cancer (mCRC) patients with wild-type (WT) KRAS gene. Numerous studies have shown that circRNAs are aberrantly expressed in tumors and play crucial roles in tumor growth and metastasis in various human malignancies, including colorectal cancer (CRC). One such circRNA, circ-EGFR, has been identified as having associations with tumor progression and chemotherapy response in diverse cancer types. However, exploring the functional role and underlying mechanisms of circ-EGFR in the context of the response to EGFR-targeted therapy may offer new therapeutic avenues for mCRC. Methods: The expression level of circ-EGFR (hsa_circ_0080229) was evaluated in cetuximab-sensitive and resistant RAS and BRAF WT CRC cell lines by using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The biological function of circ-EGFR was investigated by loss- and gain-of-function assays in cultured cells and an animal model. The potential targets of circ-EGFR were identified through in-silico analysis, and the circ-EGFR/miR-942-3p/GAS1 axis pathway was verified using the luciferase reporter assay and RT-qPCR. The association of circ-EGFR expression with clinical prognosis in mCRC patients with different cetuximab responses was analyzed by Kaplan-Meier survival analysis. Results: The expression of circ-EGFR was remarkedly higher in sensitive (DiFi and SW48) compared with resistant cell lines (CaCO2 and SNU-C1) after exposure of cells to cetuximab (sensitive vs. resistant: p < 0.001). Biologically, genetic depletion of circ-EGFR significantly enhanced cell proliferation, migration [DiFi: sh-circ-EGFR vs. shNC: Foldchange (FC) = 1.97, p < 0.001; SW48: FC = 1.96, p < 0.001], and invasion (DiFi: sh-circ-EGFR vs. shNC: FC = 2.23, p < 0.001; SW48: FC = 1.78, p < 0.001) after 24-48 hours of cetuximab treatment in DiFi and SW48 cell lines, and its overexpression suppressed tumor growth in vivo under cetuximab treatment. Mechanically, we identified circ-EGFR as a sponge for miR-942-3p to regulate the expression of Growth Arrest Specific 1 (GAS1), which promoted sensitivity to cetuximab treatment in CRC. Notably, our findings identified circ-EGFR as a promising therapeutic indicator in clinical applications. Conclusion: Our study illustrated that circ-EGFR plays a vital role in affecting cetuximab efficacy by regulating the miR-942-3p/GAS1 axis in CRC. This holds promise for improving patient selection and management in this malignancy. Citation Format: Silei Sui, Caiming Xu, Yuan Li, Wenyi Wei, Joan Maurel, Man Li, Ajay Goel. Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal 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 4728.

Abstract 5853: Regulatory network inference of lung adenocarcinoma drug-tolerant persister cells

Abstract Despite the effectiveness of the latest generation of cancer drugs, partial tumor response and subsequent disease relapse is commonly observed in patients. Recent evidence suggests that treatment resistance emerges from drug-tolerant persister cells (DTPC) that survive through non-genetic tumor cell adaptation. Although DTPC are mostly slow-cycling, a rare subset can re-enter the cell cycle under constitutive presence of treatment. Understanding how DTPC dynamically remodel their molecular portraits at the single-cell level may provide high-resolution insights into the molecular circuits associated with adaptive survival to therapy.Here, we use single-cell RNA sequencing (scRNA-seq) to identify potential mechanisms that promote survival of DTPC and decipher their underlying gene regulatory networks (GRNs), in a model of EGFR-mutant lung adenocarcinoma treated with osimertinib (EGFR inhibitor). Gene expression analysis revealed fewer cell states in DTP compared to treatment-naïve cells (TN). We confirmed the acquisition of DTPC hallmarks (e.g., high epithelial-mesenchymal transition, slow-cycling phenotype, deficient DNA repair). We also identified subpopulations of cells that display intermediate transcriptional states: a pre-existing DTP-like state in a bulk of TN cells, and DTPC with enriched cell cycle signatures, evoking a phenotype of cycling DTPC. To characterize the GRN that govern TN cells, DTPC and the intermediate cell states, we applied the network inference tool PYSCENIC, which associates transcription factors (TFs) to their candidate target genes (TGs) on the basis of the scRNA-seq data and enrichment of TF binding motifs in the genomic regions around TGs. The resulting (GRN) consisted of 5 TF modules clustered on the basis of correlated TF activity.This analysis revealed that, compared to TN cells, DTPC and the intermediate cell states display a marked decreased expression of TFs and TGs involved in mRNA splicing, and more specifically in the initiation of spliceosome assembly through E-complex and A-complex formation. Interestingly, the splicing-related TFs all appear to have affinity for binding to a GCCxC consensus motif or close variant in genes (i.e., SNRPB and SNRPE) that code for key effectors of these initiation complexes. This pilot study highlights that scRNA-seq analyses coupled with inference of regulatory networks might enable the identification of molecular mechanisms that potentially trigger cell state transitions during drug-tolerance. The role of altered mRNA splicing in persistence to EGFR-targeted therapies will be explored experimentally in patient-derived cell lines and 3D organoid cultures. Citation Format: Lisa Chabrier, Camille Leonce, Raphael Schneider, Cyril Degletagne, Pierre Saintigny, Anton Crombach, Sandra Ortiz-Cuaran. Regulatory network inference of lung adenocarcinoma drug-tolerant persister 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 5853.

Recent Advances in Therapeutic Strategies to Improve Colorectal Cancer Treatment.

Colorectal cancer (CRC) is the second-leading cause of cancer-related mortality worldwide. CRC mortality results almost exclusively from metastatic disease (mCRC) for which systemic chemotherapy is often a preferred therapeutic option. Biomarker-based stratification of mCRC enables the use of precision therapy based on individual tumor mutational profiles. Activating mutations in the RAS/RAF/MAPK pathway downstream of EGFR signaling have, until recently, limited the use of EGFR-targeted therapies for mCRC; however, the development of anti-RAS and anti-RAF therapies together with improved strategies to limit compensatory signaling pathways is resulting in improved survival rates in several highly lethal mCRC sub-types (e.g., BRAF-mutant). The use of fluoropyrimidine (FP)-based chemotherapy regimens to treat mCRC continues to evolve contributing to improved long-term survival. Future advances in chemotherapy for mCRC will need to position development relative to the advances made in precision oncology.

Genome-scale CRISPR-Cas9 screen identifies PAICS as a therapeutic target for EGFR wild-type non-small cell lung cancer.

Epidermal growth factor receptor-targeted (EGFR-targeted) therapies show promise for non-small cell lung cancer (NSCLC), but they are ineffective in a third of patients who lack EGFR mutations. This underlines the need for personalized treatments for patients with EGFR wild-type NSCLC. A genome-wide CRISPR/Cas9 screen has identified the enzyme phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), which is vital in de novo purine biosynthesis and tumor development, as a potential drug target for EGFR wild-type NSCLC. We have further confirmed that PAICS expression is significantly increased in NSCLC tissues and correlates with poor patient prognosis. Knockdown of PAICS resulted in a marked reduction in both in vitro and in vivo proliferation of EGFR wild-type NSCLC cells. Additionally, PAICS silencing led to cell-cycle arrest in these cells, with genes involved in the cell cycle pathway being differentially expressed. Consistently, an increase in cell proliferation ability and colony number was observed in cells with upregulated PAICS in EGFR wild-type NSCLC. PAICS silencing also caused DNA damage and cell-cycle arrest by interacting with DNA repair genes. Moreover, decreased IMPDH2 activity and activated PI3K-AKT signaling were observed in NSCLC cells with EGFR mutations, which may compromise the effectiveness of PAICS knockdown. Therefore, PAICS plays an oncogenic role in EGFR wild-type NSCLC and represents a potential therapeutic target for this disease.

Design, Synthesis, and Biological Evaluation of Dual Inhibitors of EGFRL858R/T790M/ACK1 to Overcome Osimertinib Resistance in Nonsmall Cell Lung Cancers.

Activation of the alternative pathways and abnormal signaling transduction are frequently observed in third-generation EGFR-TKIs (epidermal growth factor receptor tyrosine kinase inhibitors)-resistant patients. Wherein, hyperphosphorylation of ACK1 contributes to EGFR-TKIs acquired resistance. Dual inhibition of EGFRL858R/T790M and ACK1 might improve therapeutic efficacy and overcome resistance in lung cancers treatment. Here, we identified a EGFRL858R/T790M/ACK1 dual-targeting compound 21a with aminoquinazoline scaffold, which showed excellent inhibitory activities against EGFRL858R/T790M (IC50 = 23 nM) and ACK1 (IC50 = 263 nM). The cocrystal and docking analysis showed that 21a occupied the ATP binding pockets of EGFRL858R/T790M and ACK1. Moreover, 21a showed potent antiproliferative activities against the H1975 cells, MCF-7 cells and osimertinib-resistant cells AZDR. Further, 21a showed significant antitumor effects and good safety in ADZR xenograft-bearing mice. Taken together, 21a was a potent dual inhibitor of EGFRL858R/T790M/ACK1, which is deserved as a potential lead for overcoming acquired resistance to osimertinib during the EGFR-targeted therapy.

Abstract IA003: Composing each patient's equation: Deciphering brain tumor dynamics and evolution by integrating multi-regional image-localized biopsies

Abstract Glioblastoma is a uniquely challenging and aggressive cancer that is practically considered uniformly fatal. This aggressiveness is driven by heterogeneity between and within patients and the diffuse invasion of tumor cells deep into the normal appearing brain tissue surrounding the frank tumor abnormality. We use our unique collection of 1000+ image-localized biopsies across nearly 200 patients to detect sex-distinct patterns of mapping between regional imaging features and underlying regional tumor biology. Specifically, we build mathematical models that fuse locoregional changes on MRI to underlying changes in tumor biology. We apply the resultant spatio-temporal models to serial imaging of patients receiving a variety of standard of care and novel therapeutics to track tumor dynamics under said therapies. This allows us unprecedent insight into the dynamics of each patients tumor under therapy. This includes detecting clonal loss through targeted therapies as well as shifts in the tumor ecosystem under immunotherapies. In this presentation, I will demonstrate how these methods can be brought together to help us detect, decipher and predict tumor dynamics and evolution in patients. Specifically, I will demonstrate the value of these novel combined resources in predicting response to treatment for EGFR-targeted therapies in 3 clinical trials and differentiating response to novel dendritic cells vaccines and CAR T-cell therapies in 2 additional clinical trials. Taken together, these complimentary approaches provide a platform to incorporate inter- and intra- tumoral heterogeneity into accurate predictive models that advance evolutionary- and ecologically-adaptive precision oncology for glioblastoma patients. Citation Format: Kristin R. Swanson. Composing each patient's equation: Deciphering brain tumor dynamics and evolution by integrating multi-regional image-localized biopsies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA003.

A phase 1/2 study of REGN7075 (EGFRxCD28 costimulatory bispecific antibody) in combination with cemiplimab (anti–PD-1) in patients with advanced solid tumors, including colorectal cancer: Trial in progress update.

TPS239 Background: REGN7075 is a human costimulatory bispecific antibody designed to bridge epidermal growth factor receptor (EGFR)-expressing tumor cells with CD28 on the surface of T cells to drive co-stimulation and recognition of endogenous tumor antigens. EGFR-targeted therapies are approved in head and neck squamous cell carcinoma, non-small cell lung cancer (NSCLC), and colorectal cancer (CRC). Methods: This is an open-label, Phase 1/2, first-in-human, international study evaluating the safety, tolerability, PK, and anti-tumor activity of REGN7075 ± cemiplimab (anti–PD-1) in patients (pts) with solid tumors (NCT04626635). Here we focus on the cohorts of pts with microsatellite-stable-CRC (MSS-CRC). The study includes a dose-escalation phase (Part 1) and a dose-expansion phase (Part 2). In Part 1, pts will receive a 3-week REGN7075 monotherapy once-weekly (QW) lead-in prior to combination therapy with REGN7075 QW or every 3 weeks (Q3W) + cemiplimab Q3W. Concurrent Q3W dosing with cemiplimab may be conducted for dose levels for which the lead-in is tolerable; to date, this has been up to a dose of 900 mg. After a tolerable dose and schedule of interest is identified in Part 1, Part 2 may consist of eight tumor-specific expansion cohorts, including two cohorts of pts with metastatic MSS-CRC or locally advanced CRC (not candidates for curative surgery/radiation), who have received at least one prior line of therapy (anti–EGFR for RAS wild type disease, anti–VEGF, or documented reason why not appropriate). One cohort will include pts with MSS-CRC with active liver and/or peritoneal metastases, and the other will include pts with MSS-CRC with only lung and/or lymph node metastases. Overall, key inclusion criteria are: evidence of a protocol-defined advanced solid tumor, an ECOG performance status of 0 or 1, and no previous anti–PD-1/anti–PD-(L)1 therapy (Part 2 only). The analyses of Part 1 will be descriptive and exploratory in nature. Primary endpoints are safety and tolerability of REGN7075 ± cemiplimab (Part 1), and objective response rate per RECIST 1.1 (Part 2). Secondary endpoints include: REGN7075 concentrations in serum and overall survival (both parts); incidence and severity of treatment-emergent adverse events (AEs), AEs of special interest, serious AEs, and Grade ≥3 laboratory abnormalities; incidence and titer of ADA to REGN7075 + cemiplimab over time; pt-reported quality of life, symptoms, functioning, and general health status (Part 2 only). The study is expected to enroll ~769 pts: ~221 pts in Part 1 and ~548 pts in Part 2, including 74 pts in Cohort E and 73 pts in Cohort F. As of September 5, 2023, 93 pts were enrolled in Part 1. This study is ongoing and currently open to enrollment. Clinical trial information: NCT04626635 .

Real-World Data Presenting the Descriptive Analysis of the Use of Tyrosine Kinase Inhibitors (TKIs) Among Metastatic Non-Small-Cell Lung Cancer (mNSCLC) Patients in Qatar: A Nationwide Retrospective Cohort Study.

There has been significant improvement in treating metastatic non-small-cell lung cancer (mNSCLC) over the past 2 decades. The aim of this study is to describe the use of tyrosine kinase inhibitors (TKIs) in Qatar. This study focuses on the objective response rate (ORR) and reported adverse drug events (ADEs) of TKIs used for the management of patients with mNSCLC. This is a descriptive retrospective cohort study. All non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) mutations who received TKIs between 2015 and 2019 in Qatar were included. The TKIs used during this period include EGFR inhibitors such as afatinib, erlotinib, gefitinib, and osimertinib and ALK inhibitors such as alectinib and crizotinib. The response on each TKI was identified by reporting the ORR (as the sum of the complete response [CR] and the partial response [PR]), in addition stable disease (SD) and disease progression (DP) were reported. While ADEs were reported using the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE). A total of 63 patients were included, of which 36 cases (57.1%) expressed EGFR mutation, and 27 patients (42.9%) expressed ALK rearrangement. The ORR in EGFR inhibitors was as follows: osimertinib 40%, gefitinib 33%, afatinib 22%, and erlotinib 18%. However, the response to the ALK-targeted therapy was 43% with alectinib and 40% with crizotinib. A total of 112 ADEs were reported. They were distributed as 63.4% (71 of 112) with the anti-EGFR and 36.6% (41 of 112) ADEs with the ALK inhibitors. In the anti-EGFR group, the most common types of ADEs were dermatological toxicity 30%, whereas, in the anti-ALK group, gastrointestinal toxicity was the most common (29%). The EGFR-targeted and ALK-targeted therapies appear to have acceptable clinical response rate and safety profile in our population. Close and frequent monitoring of adverse events is advised to ensure a good quality of life and prevent serious complications.

TARGETING EPIDERMAL GROWTH FACTOR RECEPTOR FOR CANCER TREATMENT

The Epidermal Growth Factor Receptor (EGFR) is a pivotal player in cellular signalling pathways and its dysregulation is implicated in various cancers. Targeting EGFR has emerged as a promising strategy for cancer treatment. This abstract provides Comprehensive analysis of EGFR-targeted therapies, including small molecule tyrosine kinase inhibitors and monoclonal antibodies, reveals their efficacy in suppressing aberrant signalling cascades associated with tumor growth. Mechanisms of resistance, biomarkers for patient stratification, and ongoing clinical trials are discussed to highlight the evolving landscape of EGFR-targeted cancer therapies. The multifaceted nature of EGFR signaling necessitates a personalized approach, emphasizing the need for precision medicine to optimize therapeutic outcomes. This abstract encapsulates the significance and challenges of targeting EGFR for cancer treatment, providing a foundation for further research and clinical advancements in the field. KEYWORDS: Epidermal Growth Factor Receptor (EGFR), Cancer Treatment, Tyrosine Kinase Inhibitors, Monoclonal Antibodies, Biomarkers, Precision Medicine

Relevance and mechanism of STAT3/miR-221-3p/Fascin-1 axis in EGFR TKI resistance of triple-negative breast cancer.

The epidermal growth factor receptor 1 (EGFR) plays a crucial role in the progression of various malignant tumors and is considered a potential target for treating triple-negative breast cancer (TNBC). However, the effectiveness of representative tyrosine kinase inhibitors (TKIs) used in EGFR-targeted therapy is limited in TNBC patients. In our study, we observed that the TNBC cell lines MDA-MB-231 and MDA-MB-468 exhibited resistance to Gefitinib. Treatment with Gefitinib caused an upregulation of Fascin-1 (FSCN1) protein expression and a downregulation of miR-221-3p in these cell lines. However, sensitivity to Gefitinib was significantly improved in both cell lines with either inhibition of FSCN1 expression or overexpression of miR-221-3p. Our luciferase reporter assay confirmed that FSCN1 is a target of miR-221-3p. Moreover, Gefitinib treatment resulted in an upregulation of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in MDA-MB-231 cells. Using Stattic, a small-molecule inhibitor of STAT3, we observed a significant enhancement in the inhibitory effect of Gefitinib on the growth, migration, and invasion of MDA-MB-231 cells. Additionally, Stattic treatment upregulated miR-221-3p expression and downregulated FSCN1 mRNA and protein expression. A strong positive correlation was noted between the expression of STAT3 and FSCN1 in breast cancer tissues. Furthermore, patients with high expression levels of both STAT3 and FSCN1 had a worse prognosis. Our findings suggest that elevated FSCN1 expression is linked to primary resistance to EGFR TKIs in TNBC. Moreover, we propose that STAT3 regulates the expression of miR-221-3p/FSCN1 and therefore modulates resistance to EGFR TKI therapy in TNBC. Combining EGFR TKI therapy with inhibition of FSCN1 or STAT3 may offer a promising new therapeutic option for TNBC.

Targeted Inhibitors of EGFR: Structure, Biology, Biomarkers, and Clinical Applications.

Members of the EGFR family of tyrosine kinase receptors are major regulators of cellular proliferation, differentiation, and survival. In humans, abnormal activation of EGFR is associated with the development and progression of many cancer types, which makes it an attractive target for molecular-guided therapy. Two classes of EGFR-targeted cancer therapeutics include monoclonal antibodies (mAbs), which bind to the extracellular domain of EGFR, and tyrosine kinase inhibitors (TKIs), which mostly target the intracellular part of EGFR and inhibit its activity in molecular signaling. While EGFR-specific mAbs and three generations of TKIs have demonstrated clinical efficacy in various settings, molecular evolution of tumors leads to apparent and sometimes inevitable resistance to current therapeutics, which highlights the need for deeper research in this field. Here, we tried to provide a comprehensive and systematic overview of the rationale, molecular mechanisms, and clinical significance of the current EGFR-targeting drugs, highlighting potential candidate molecules in development. We summarized the underlying mechanisms of resistance and available personalized predictive approaches that may lead to improved efficacy of EGFR-targeted therapies. We also discuss recent developments and the use of specific therapeutic strategies, such as multi-targeting agents and combination therapies, for overcoming cancer resistance to EGFR-specific drugs.

Splice-Switching Antisense Oligonucleotides Targeting Extra- and Intracellular Domains of Epidermal Growth Factor Receptor in Cancer Cells.

Cancer is one of the leading causes of death globally. Epidermal growth factor receptor is one of the proteins involved in cancer cell proliferation, differentiation, and invasion. Antisense oligonucleotides are chemical nucleic acids that bind to target messenger ribonucleic acid and modulate its expression. Herein, we demonstrate the efficacy of splice-modulating antisense oligonucleotides to target specific exons in the extracellular (exon 3) and intracellular (exon 18, 21) domains of epidermal growth factor receptor. These antisense oligonucleotides were synthesized as 25mer 2'-O methyl phosphorothioate-modified ribonucleic acids that bind to complementary specific regions in respective exons. We found that PNAT524, PNAT525, PNAT576, and PNAT578 effectively skipped exon 3, exon 18, and exon 21 in glioblastoma, liver cancer, and breast cancer cell lines. PNAT578 treatment also skipped partial exon 19, complete exon 20, and partial exon 21 in addition to complete exon 21 skipping. We also found that a cocktail of PNAT576 and PNAT578 antisense oligonucleotides performed better than their individual counterparts. The migration potential of glioblastoma cancer cells was reduced to a greater extent after treatment with these antisense oligonucleotides. We firmly believe that using these splice-modulating antisense oligonucleotides in combination with existing EGFR-targeted therapies could improve therapeutic outcomes.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

Progress of EGFR targeted therapy for oral cancer

Because of some uncertain factors such as eating habits and smoking, oral cancer has become one of the serious diseases of the oral and maxillofacial region. Especially in some Chinese cities where betel nut chewing is customary, the incidence of oral cancer has been high. Today, the main treatment methods are still traditional surgical excision, radiation and chemotherapy. However, with the development of medical technology, gene-targeted therapy has become the most important research object. Gene targeting technology is mainly targets the treatment of cancer organ cells, reducing the damage of traditional treatment methods on other areas without lesions. At present, the epidermal cytokine EGFR has been the most studied, and some research results have been put into clinical application. However, in terms of targeted therapy, the development of technology is still incomplete and immature. This article mainly discusses the pathogenic mechanism of EGFR in oral cancer, the synergistic effect of EGFR and other factors on the development of oral cancer, and the research results of EGFR-targeted therapy and combination therapy for oral cancer. The significance of this study is that based on these discussions, it provides reference opinions for treatment that can achieve a good prognosis for oral cancer patients and alleviate the pain of oral cancer patients, and evaluate the current development status of clinical application of EGFR targeted therapy for oral cancer. At present, the targeted therapy technology of oral cancer is still immature. In the future, the role of other factors in the development of oral cancer can be studied, and the influence of the combination of targeted therapy and other therapeutic methods on the treatment results of oral cancer can be studied.

Abstract C104: Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC

Abstract Aberrant expression of the transcription factor MYC is found in most cancers including non-small cell lung cancer (NSCLC) and has critical roles in a wide range of oncogenic processes including immune evasion and EGFR inhibitor resistance. Despite MYC’s therapeutic attractiveness, potent inhibitors of MYC activity have been elusive, due to its intrinsically disordered protein structure and tightly autoregulated expression. We have developed NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC Omega epigenomic controller (NSCLC MYC-OEC) that is designed to target the insulated genomic domain of MYC. We have previously shown that NSCLC MYC-OEC effectively downregulates MYC expression pre-transcriptionally and have demonstrated that NSCLC MYC-OEC inhibits NSCLC patient-derived organoid viability in vitro and abrogates xenograft tumor growth in vivo. To identify combination treatments with NSCLC MYC-OEC, we retrospectively evaluated baseline MYC expression and real-world progression-free survival (PFS) in a cohort of 283 NSCLC patients treated with pembrolizumab, an anti-PD1 monoclonal antibody. This analysis revealed that NSCLC patients with MYC-overexpressing tumors exhibited shorter PFS when treated with pembrolizumab. Moreover, analysis of PD1 or PD-L1 transcript levels in tumors from 13,230 lung adenocarcinoma patients revealed a significant positive correlation between PD1 or PD-L1 and MYC mRNA levels, consistent with a role for MYC in regulation of the immune response. In vivo evaluation of NSCLC MYC-OEC activity and immune checkpoint immunotherapy (ICI) treatment of preclinical Lewis lung carcinoma syngeneic tumor models demonstrated that the combination of NSCLC MYC-OEC with anti-PD-L1 antibody enhanced inhibition of tumor growth and prolonged survival over administration when compared to either NSCLC MYC-OEC or anti-PD-L1 antibody monotherapy. Retrospective analysis of a cohort of 301 NSCLC patients also showed that patients with MYC-overexpressing tumors exhibited significantly shorter PFS when treated with osimertinib, a third generation EGFR inhibitor. To further understand this, we investigated the effects of NSCLC MYC-OEC and osimertinib treatment alone or in combination in EGFR mutant cells, H1975 and PC9. While NSCLC MYC-OEC and osimertinib monotherapy treatments resulted in downregulation of MYC protein expression in both cell lines, combination treatment led to a significantly greater decrease in MYC expression. Furthermore, the combination synergistically reduced H1975 and PC9 cell viability in vitro and led to a significantly greater inhibition of H1975 xenograft growth relative to each single agent in vivo. Together, these data provide preclinical proof-of-concept in NSCLC for development of OEC-mediated pre-transcriptional epigenomic inhibition of MYC expression in combination with immune checkpoint and EGFR inhibitors. Furthermore, these findings suggest that NSCLC MYC-OEC combination with ICI or EGFR-targeted therapy may improve patient outcomes. Citation Format: Defne Yarar, Eugine Lee, Padraich Flahardy, Cameron Vergato, Graeme Hodgson, Thomas McCauley. Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C104.

Epidermal growth factor potentiates EGFR(Y992/1173)-mediated therapeutic response of triple negative breast cancer cells to cold atmospheric plasma-activated medium

Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills various types of malignant cells. We used CAP-activated media (PAM) to utilize a range of the generated short- and long-lived reactive species. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in up-regulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells. The proposed approach based on the synergy of CAP and EGFR-targeted therapy may provide new opportunities to improve the clinical management of TNBC.

Prognostic and predictive biomarkers for anti-EGFR monoclonal antibody therapy in RAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis

BackgroundRAS mutations affect prognosis in patients with metastatic colorectal cancer (mCRC) and have been identified as strong negative predictive markers for anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy, but many tumors containing wild-type RAS genes still do not respond to these therapies. Some additional biomarkers may have prognostic or predictive roles, but conclusions remain controversial.MethodsWe performed a meta-analysis and systematic review of randomized controlled trials comparing anti-EGFR mAb therapy with alternative therapy that investigated the prognostic and predictive impact of additional biomarkers in RAS wild-type (wt) mCRC patients. Hazard ratios (HRs) and 95% confidence intervals (CIs) for progression-free survival (PFS) and overall survival (OS) and odds ratios (ORs) for objective response rate (ORR) were calculated. The prognostic value of biomarkers was investigated by separately pooling HR and OR for different treatment groups in an individual study. The predictive value was assessed by pooling study interactions between treatment effects and biomarker subgroups.ResultsThirty publications reporting on eighteen trials were selected, including a total of 13,507 patients. In prognostic analysis, BRAF mutations were associated with poorer PFS [HRs = 3.76 (2.47–5.73) and 2.69 (1.82–3.98)] and OS [HRs = 2.66 (1.95–3.65) and 2.45 (1.55–3.88)] in both the experimental and control arms; low miR-31-3p expression appeared to have longer PFS and OS. In terms of predictive effect, a lack of response to anti-EGFR therapy was observed in patients with BRAF mutant tumors (Pinteraction < 0.01 for PFS). Patients with tumors with any mutation in the KRAS/NRAS/BRAF/PIK3CA gene also showed similar results compared with all wild-type tumors (Pinteraction for PFS, OS, and ORR were < 0.01, < 0.01 and 0.01, respectively). While low miR-31-3p expression could predict PFS (Pinteraction = 0.01) and OS (Pinteraction = 0.04) benefit. The prognostic and predictive value regarding PIK3CA mutations, PTEN mutations or deletions, EGFR, EREG/AREG, HER2, HER3, and HER4 expression remains uncertain.ConclusionsIn RAS wt mCRC patients receiving EGFR-targeted therapy, BRAF mutation is a powerful prognostic and therapy-predictive biomarker, with no effect found for PIK3CA mutation, PTEN mutation or deletion, but the combined biomarker KRAS/NRAS/BRAF/PIK3CA mutations predict resistance to anti-EGFR therapy. Low miR-31-3p expression may have positive prognostic and therapy predictive effects. Evidence on the prognostic and predictive roles of EGFR and its ligands, and HER2/3/4 is insufficient.

MODL-05. ELUCIDATING THE EPIGENETIC DRIVEN TRANSCRIPTIONAL PROGRAM UNDERLYING THE INVASIVE PHENOTYPE OF THE NOVEL EGFRA289VARIANT OF GBM

Abstract Glioblastoma (GBM) is a devastating malignant brain tumor with a dismal 15-month median survival. Current treatments have not been successful at preventing recurrence, thus novel therapeutic modalities are urgently needed. EGFR is a tyrosine kinase receptor that is commonly mutated in ~60% of GBM and is an attractive target for GBM precision oncology. Aberrant EGFR signaling drives tumor proliferation and maintenance, thus further study of common EGFR alterations in GBM, including interstitial deletions (EGFRvIII) and missense mutations (EGFRA289), is needed to develop effective EGFR-targeted therapies. This is especially important in the context of a retrospective study that showed EGFRA289 driven GBM was more clinically aggressive. EGFRA289 positive GBM had a unique, invasive radiographic signature on magnetic resonance imaging (MRI) and patients had a significantly shorter median overall survival of 6-months. We hypothesize that the malignant clinical phenotypes of EGFRA289 GBM are driven by unique epigenomic and transcriptomic, collectively referred to as (epi)genomic, programs linked to EGFRA289 signaling. Prior studies using established cell lines show that EGFRvIII overexpression drives malignant (epi)genomic programs mediated by transcription factor (TF) activation. To better understand variant specific EGFR biology in GBM, we have developed novel genetically engineered mouse astrocyte (mAc) models that overexpress wtEGFR (MA-WT), vIII (MA-v3), and EGFRA289V (MA-A289V). Phospho-immunoblots show that these mAc models have differential EGFR ligand dependance, which match published studies. Despite this difference in EGFR signaling, all mAc models have similar sensitivities to EGFR tyrosine kinase inhibitor (TKI) treatment. However, MA-A289V is more proliferative and migratory compared to the other mAc variants. Preliminary RNA-Seq on our mAc models has identified similar and unique TFs differentially expressed (DE) between MA-v3 and MA-A289V when compared to parental MA-C. Integration of RNA-Seq and CUT&amp;RUN followed by mechanistic genetic interrogation will elucidate the relationship between EGFR variant-specific (epi)genomic mechanisms and their unique malignant phenotype.

Adjuvant Effect of Cinnamon Polyphenolic Components in Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is the second-leading cause of cancer death, with a worldwide incidence rate constantly increasing; thus, new strategies for its prevention or treatment are needed. Here, we describe the adjuvant effect of the polyphenol-enriched fractions of cinnamon, from cinnamon bark and buds, when co-administered with a potent anticancer drug, cetuximab, used for CRC therapy. The co-administration significantly reduces the cetuximab dose required for the antiproliferative activity against colorectal cancer cell line E705, which is sensitive to EGFR-targeted therapy. The anticancer activity of these cinnamon-derived fractions, whose major components (as assessed by UPLC-HRMS analysis) are procyanidins and other flavonoids, strictly correlates with their ability to induce apoptosis in cancer cell lines through ERK activation and the mitochondrial membrane potential impairment. Due to the severe side effects of cetuximab administration, our results suggest the use of nutraceuticals based on the polyphenolic fractions of cinnamon extracts as adjuvants in the therapy of CRC.