EGFR-targeted Tyrosine Kinase Inhibitors Research Articles

Single molecule tracking based drug screening

The single-molecule tracking of transmembrane receptors in living cells has provided significant insights into signaling mechanisms, such as mobility and clustering upon their activation/inactivation, making it a potential screening method for drug discovery. Here we show that single-molecule tracking-based screening can be used to explore compounds both detectable and undetectable by conventional methods for disease-related receptors. Using an automated system for a fast large-scale single-molecule analysis, we screen for epidermal growth factor receptor (EGFR) from 1134 of FDA approved drugs. The 18 hit compounds include all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppress any phosphorylation-dependent mobility shift of EGFR, proving the concept of this approach. The remaining hit compounds are not reported as EGFR-targeted drugs and do not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affect the mobility and/or clustering of EGFR without EGF and induce EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides an alternative modality for discovering therapeutics on various receptor functions with previously untargeted mechanisms.

EGFR mutation based on lung laterality in adenocarcinoma type of non-small cell lung cancer

Targeted therapies have shown promise in improving survival rates for lung adenocarcinoma, a common and deadly malignancy. EGFR-targeting tyrosine kinase inhibitors (TKIs) are particularly effective among these therapies in cases with EGFR mutations. Detecting these mutations before TKI treatment is essential. Various radiological features have been linked to EGFR mutations. However, the relationship between tumor location and mutation types in Indonesian lung adenocarcinoma patients remains unexplored. This study aimed to identify the frequency of EGFR mutation in local lung adenocarcinoma cases based on the tumor location. Clinical data of lung adenocarcinoma patients (n = 272) diagnosed between 2018 and 2022 were retrospectively taken from the Department of Anatomical Pathology, Dr. Sardjito General Hospital, Yogyakarta. The qRT-PCR data of EGFR mutation status was obtained from the Department of Anatomical Pathology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta. Descriptive analysis was performed using STATA version 14.0. EGFR mutations were found in 60.7% of patients, with 58.2% having exon 19 mutations and 21.2% exhibiting exon 21 L858R mutations. Mutation status was found to be significantly different based on the patient's gender (p = 0.022) and age (p = 0.029) but not with lung laterality (p = 0.093). The proportion of exon 19, exon 21 L858R, and uncommon mutations in the right and left lung adenocarcinoma was similar across all samples. This study found no difference between specific EGFR mutation types and tumor location in lung adenocarcinoma.

Abstract 5868: Upregulation of HER3 and MET in non-small cell lung cancer cells resistant to EGFR tyrosine kinase inhibitor

Abstract Introduction: Prior research demonstrates the importance of studying epidermal growth factor receptor (EGFR) activating mutations for better understanding the biology of non-small cell lung cancer (NSCLC), which comprises the majority (~80%) of lung cancers. EGFR-targeted tyrosine kinase inhibitors (TKIs) have been used in the clinic to treat NSCLC with EGFR activating mutations. First and second-generation EGFR-TKIs are effective in NSCLC patients with the most common EGFR mutations (exon 19 deletion (Del19) and exon 21-point mutation (L858R)). A third generation EGFR-TKI, osimertinib (AZD9291), has been developed to target the T790M resistance mutation, but patients are starting to show resistance to this line of therapy as well. Our study aims to elucidate the underlying mechanism(s) of EGFR-TKI resistance in NSCLC cells. Methods: NSCLC cell lines, including PC-9 and HCC827, which have the Del19 EGFR activating mutation, were utilized for experiments. From the parental cell lines, several cell lines resistant to the EGFR-TKIs were previously established. Cell proliferation (MTS) assays were used to confirm the resistance phenotype of the established cell lines. Western blot assays and RT-qPCR were utilized to determine changes in the expression level of several membrane receptors and their downstream effectors. Results: Our preliminary analysis showed that both human epidermal growth factor 3 (HER3) and mesenchymal-epithelial transition factor (MET) were upregulated in the EGFR-TKI-resistant cell lines as compared to their parental counterparts. Further studies revealed an increased level of phosphorylated STAT3 (p-STAT3) and ERK1/2 (p-ERK1/2) in the resistant cell lines when compared to the parental cell lines, suggesting the possible involvement of HER3 and/or MET in the activation of downstream signaling pathways. Upregulation of HER3 and MET was found at both the protein and mRNA level. Conclusion: Based on our initial findings, membrane receptors, including HER3 and MET, could be working together to continue activating downstream signaling pathways to evade the effects of osimertinib, resulting in resistance. Additional work is underway to define the role of HER3 and MET in the NSCLC cells resistant to EGFR-TKIs. Citation Format: Margaret Larsen, Hui Lyu, CongCong Tan, Yakun Wu, Bolin Liu. Upregulation of HER3 and MET in non-small cell lung cancer cells resistant to EGFR tyrosine kinase inhibitor [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 5868.

Radiomics of metastatic brain tumor as a predictive image biomarker of progression-free survival in patients with non-small-cell lung cancer with brain metastasis receiving tyrosine kinase inhibitors

Background and objectiveEpidermal growth factor receptor (EGFR)–targeted tyrosine kinase inhibitors (TKIs) are the first-line therapy for EGFR-mutant non-small-cell lung cancer (NSCLC). Early prediction of treatment failure in patients with brain metastases treated with EGFR–TKIs may help in making decisions for systemic drug therapy or local brain tumor control. This study examined the predictive power of the radiomics of both brain metastasis tumors and primary lung tumors. We propose a deep learning based CoxCC model based on quantitative brain magnetic resonance imaging (MRI), a prognostic index and clinical data; the model can be used to predict progression-free survival (PFS) after EGFR–TKI therapy in advanced EGFR-mutant NSCLC. MethodsThis retrospective single-center study included 271 patients receiving first-line EGFR–TKI targeted therapy in 2018–2019. Among them, 72 patients who had brain metastases before receiving first-line EGFR–TKI treatment. Three radiomic features were extracted from pretreatment brain MRI images. A CoxCC model for the progression risk stratification of EGFR–TKI treatment was proposed on the basis of MRI radiomics, clinical features, and a prognostic index. We performed time-dependent PFS predictions to evaluate the performance of the CoxCC model. ResultsThe CoxCC model based on a prognostic index, clinical features, and radiomic features of brain metastasis exhibited higher performance than clinical features combined with indexes previously proposed for determining the prognosis of brain metastasis, including recursive partitioning analysis, diagnostic-specific graded prognostic assessment, graded prognostic assessment for lung cancer using molecular markers (lung-molGPA), and modified lung-molGPA, with c-index values of 0.75, 0.67, 0.66, 0.65, and 0.65, respectively. The model achieved areas under the curve of 0.88, 0.73, 0.92, and 0.90 for predicting PFS at 3, 6, 9 and 12 months, respectively. PFS significantly differed between the high- and low-risk groups (p < 0.001). ConclusionsFor patients with advanced-stage NSCLC with brain metastasis, MRI radiomics of brain metastases may predict PFS. The CoxCC model integrating brain metastasis radiomics, clinical features, and a prognostic index provided reliable multi-time-point PFS predictions for patients with advanced NSCLC and brain metastases receiving EGFR–TKI treatment.

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

FDA analysis of toxicity profiles of oral TKIs recently approved for non-small cell lung cancer based on receipt of prior immune checkpoint inhibitor therapy.

9119 Background: Immune checkpoint inhibitors (ICIs) targeting PD-(L)1 are standard front-line therapy for advanced non-small cell lung cancer (NSCLC). Molecularly targeted tyrosine kinase inhibitors (TKIs) are standard of care for subsets of oncogene-driven NSCLC. Published literature suggests the possibility of increased toxicity in patients who receive ICIs followed by EGFR or ALK TKI treatment. However, the toxicity profile in patients who receive ICIs followed by TKIs with other genomic targets has not been well described. Methods: Marketing applications for TKIs in patients with NSCLC submitted to the Office of Oncologic Diseases between Jan 2019 to Dec 2022 were reviewed. Datasets were analyzed for demographics, treatment-emergent adverse events (TEAE), and prior anti-cancer therapies for TKI-treated patients in the primary efficacy population. Drug classes for which &lt; 5% of patients received prior ICI were excluded. Grade 3-4 TEAE, serious adverse events (SAE), discontinuation TEAE and TEAE of interest (hepatotoxicity, rash, and interstitial lung disease [ILD]) were summarized. Results: 9 applications were identified and 2 were excluded. The remaining applications involved TKIs targeting EGFR exon 20 insertion, KRAS G12C, MET exon 14 skipping, and RET fusion alterations. Analyses included 867 patients; 61% received prior ICI therapy. Patient characteristics and results of analyses are presented in the table. In our pooled analysis, the incidence of Grade 3-4 AEs, SAEs, and hepatotoxicity were slightly higher in the prior ICI group, while the incidence of discontinuation TEAE, ILD, and rash were similar between groups. Analysis for hepatotoxicity was limited to reported AEs (did not include analysis of laboratory data). Review of toxicity data across individual drug classes did not show major differences compared to the overall results. Conclusions: Our analysis of safety data for newer TKIs with varied genomic targets does not appear to show excessive toxicity in patients with NSCLC who received prior ICI compared to those who did not. In future clinical trials evaluating TKIs in this population, inclusion of patients who have received prior ICI therapy should be considered when appropriate. [Table: see text]

Abstract 605: Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer

Abstract Background: Somatic alterations in the Epidermal Growth Factor Receptor (EGFR) gene represent a common subset of oncogene-driven lung cancer. Patients who receive EGFR-targeted tyrosine kinase inhibitor (TKI) therapy inevitably develop treatment resistance with limited options after disease progression, with immune checkpoint inhibitor therapy (ICI) typically ineffective. Tumor Immune Microenvironment (TME) characteristics, including T-cell receptor (TCR) sequencing and clonality have been incompletely explored in this population. Methods: We further analyzed our group’s available data from a previous study on therapy-induced evolution of human lung cancer (Maynard et. al, PMID: 32822576). Tumor samples were sorted and subjected to smartseq-based single-cell RNA sequencing. A total of 7495 cells were derived from 17 samples from 10 patients with tumors that harbored EGFR driver gene mutations. This included 1322 gene marker-annotated T-cells. Samples were grouped into three treatment conditions: treatment naïve (TN), residual disease (RD), and progressive disease (PD). We then used the TraCeR tool to infer the TCR α, β, and the complementarity-determining region 3 (CDR3) sequences. Results: T-cells were enriched in RD (55.2%) relative to TN (12.8%) and PD (31.9%) (p&amp;lt;0.0001, Chi-square test) with cytotoxic T-cells as the predominant phenotype across treatment conditions. Increased expression of CTLA4, and LAG3 occurred in PD (p&amp;lt;0.0001, Kruskal-Wallis test). Single-cell TCR analysis revealed increased TCR clonal diversity in PD (Shannon Diversity Index: 0.261 PD &amp;gt; 0.255 TN &amp;gt; 0.205 RD) while increased clonal expansion occurred in RD (32 clones from 4 patients) as compared to TN (17 clones from 1 patient) and PD (14 clones from 1 patient) (p&amp;lt;0.0001, Chi-square test). Conclusion: T-cell infiltration is greatest in RD, while immunosuppressive genes were significantly expressed in PD. Increased TCR clonal expansion with less clonal diversity in RD suggests the presence of tumor-specific TCR clones and that this may be the optimal time to initiate ICI treatment. Future directions will include the identification of clonally expanded TCR and matched neoantigens to provide potential tailored immunotherapies for patients with TKI-resistant tumors. Citation Format: Turja Chakrabarti, Wei Wu, Daniel L. Kerr, Paul Allegakoen, Trever G. Bivona, Collin M. Blakely. Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 605.

Novel EGFR-mutant mouse models of lung adenocarcinoma reveal adaptive immunity requirement for durable osimertinib response

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer patient biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficient nu/nu or Rag1−/− mice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral T cell content and decreased neutrophil content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

Cyy-287, a novel pyrimidine-2,4-diamine derivative, inhibits tumor growth of EGFR-driven non-small cell lung cancer via the ERK pathway.

In recent decades, EGFR-targeted tyrosine kinase inhibitors (TKIs) have been proven to be an effective therapy for EGFR-mutant non-small cell lung cancer (NSCLC). However, resistance to EGFR-TKIs limits their clinical application. In the present study, we investigate the antitumor effect and underlying mechanism of a novel pyrimidine-2,4-diamine derivative, cyy-287, in NSCLC. We find that cyy-287 has a high affinity for lung tissue and inhibits the proliferation of NSCLC cells. Interestingly, the significant suppression of migration and induction of apoptosis by cyy-287 are only observed in EGFR-driven but not in EGFR-wild-type (wt) cells. According to the RNA sequencing and KEGG enrichment analysis results, cyy-287 markedly inhibits the MAPK pathway in EGFR-driven PC9 cells, and western blot analysis results further indicate that cyy-287 selectively blocks the ERK pathway in EGFR-driven cells. Meanwhile, apoptosis induced by cyy-287 could be partially reversed by ERK pathway inhibition. Further experiment indicates that cyy-287 inhibits the EGFR pathway in both EGFR-driven and EGFR-overexpressing cells. Interestingly, it only induces apoptosis in EGFR-driven cells, not in EGFR-overexpressing cells. The growth of EGFR-driven cells is suppressed by cyy-287 in vivo, with fewer side effects. Our results suggest that cyy-287 may be a potential therapeutic drug with promising antitumor effects against NSCLC.

Abstract LB096: EGFR-dependent inflammation as a driver of breast cancer

Abstract Triple Negative Breast Cancer (TNBC) is one of the most aggressive and metastatic subtypes of hormone receptor negative breast cancers. Evidence supports that most patients with TNBC overexpress the Epidermal Growth Factor Receptor (EGFR), yet its role in driving the disease remains unclear. Therapies have been developed to target EGFR, but EGFR-targeted antibodies and tyrosine kinase inhibitors fail to work effectively in breast cancer patients. A possible explanation is that in addition to its canonical function at the membrane, EGFR has a non-canonical function where it can undergo retrograde trafficking to the nucleus, making it resistant to antibody-based therapeutics. Once in the nucleus, EGFR is known to function as a transcriptional cofactor for STAT3, STAT5, and E2F1, thereby regulating the expression of genes that promote inflammation and cell proliferation. Via RNA-Seq analysis, we demonstrate that EGFR can regulate different subsets of genes depending on its canonical signaling activity or nuclear role. Our goal is to understand and distinguish this difference in regulation to assess how EGFR affects inflammation. We hypothesize that EGFR is modifying inflammation differently depending on its canonical or non-canonical role, thereby driving disease progression in multiple ways. We have found that canonical EGFR can induce interleukin 8 while nuclear EGFR can repress interleukin 6 through the use of either kinase inhibitors (IL8) or blocking nuclear translocation of EGFR (IL6). Correlated with these changes are alterations in growth and inflammatory profiles in both a triple negative inflammatory mouse model and an EGFR-driven breast cancer mouse model. We are now investigating the role of nuclear-EGFR-dependent inflammation in driving tumor progression. Understanding the different mechanisms by which EGFR modifies inflammation and the tumor microenvironment would allow us to develop more specific targets for patients with TNBC. Citation Format: Angelica Escoto. EGFR-dependent inflammation as a driver of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB096.

Abstract 3260: Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC

Abstract Background: Resistance to targeted tyrosine kinase inhibitors (TKI) inevitably develops in metastatic EGFR-mutant non-small cell lung cancer (NSCLC). Resistance mechanisms are diverse, and mechanisms beyond receptor tyrosine kinase (RTK) pathway mutations are poorly understood. We hypothesized that the use of osimertinib as first-line therapy is increasing the prevalence of RTK-independent resistance mechanisms, and that RTK-independent resistant tumors undergo enhanced tumor cell lineage plasticity as an escape mechanism to EGFR TKI therapy. Methods: We identified patients who developed osimertinib resistance (OR) after first line (1L, n=54) and second line (2L, n=42) treatments and determined the resistance mechanisms based on clinical sequencing and histopathology. We also performed single-cell RNA-seq of 24 samples from 13 patients with EGFRm NSCLC at TKI treatment-naïve (TN, n=2), residual disease (RD, n=4), and progression disease (PD, n=7) stages. Results: Compared to 2L OR tumors, 1L OR tumors had increased RTK-independent mechanisms of resistance (76% vs. 46%, p=0.002), including 8% with small cell transformation (n=4), 2% with squamous transformation (n=1) and 66% with unknown mechanisms (n=34). To understand inter- and intra-tumor heterogeneity, we analyzed transcriptomic profiles of 76,266 single cells. Lung developmental lineages were assigned to 10,250 EpCAM+ cells, including 4,735 cells classified as malignant cells by inferCNV and RTK signaling analysis. In the two EGFRm TN tumors, the malignant cells demonstrated bronchoalveolar lineage and moderate EGFR expression. In the TKI resistant cases (PD, n=7), both RTK-dependent and RTK-independent resistance were observed. The RTK-dependent tumors (EGFR T790M n=1; ERBB2 amplification n=1) demonstrated preserved bronchoalveolar lineage identity. In the RTK-independent resistant tumors (n=5), one had complete lineage switch from epithelial to small cell neuroendocrine and very low expression level of EGFR. The remaining 4 PD tumors displayed varying expression of epithelial-to-mesenchymal transformation (EMT) features. One tumor had sarcomatoid histology and a high proportion of cells having positive VIM expression (84%) and 92% of cells having complete loss of NAPSA expression; 3 tumors had partial EMT demonstrated by heterogeneous proportion of cells having VIM expression (18-56%) and loss of NAPSA (26-67%). Interestingly, some of the cells with EMT and partial-EMT had moderate levels of EGFR expression, similar to the levels in the TN tumors. Conclusion: With osimertinib use at 1L, the incidence of RTK-independent resistance has increased to become the dominant mechanism, whereas RTK-dependent resistance has decreased. Increased lineage plasticity (small cell neuroendocrine, squamous and EMT) potentially serves as an RTK-independent TKI-resistance mechanism in EGFRm NSCLC. Citation Format: Xiuning Le, Ruiping Wang, Natalie Vokes, Yasir Elamin, Neda Kalhor, Daniel McGrail, Yuanxin Xi, Santiago Treviño III, Lingzhi Hong, Robyn Du, George Blumenschein, Carl Gay, Marcelo Negrao, Mehmet Altan, Hai Tran, Limei Hu, Jing Wang, Simon Heeke, Monique Nilsson, Jacqulyne Robichaux, Minghao Dang, Guangchun Han, Lauren Byers, Anne Tsao, Boris Sepesi, Chantale Bernatchez, Jianjun Zhang, Linghua Wang, John Heymach. Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC [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 3260.

Abstract 417: Identification of next-generation EGFR degraders to treat non-small cell lung cancer (NSCLC) patients

Abstract Although EGFR-targeted tyrosine kinase inhibitors (TKIs) developed over a couple of decades have made great progress in the treatment of EGFR-mutant driven NSCLC, patients are at great risk to their lives due to drug resistance caused by constantly emerging EGFR mutations. Targeted protein degraders (TPDs), also known as heterobifunctional protein degraders or PROteolysis TArgeting Chimeras (PROTACs), have drawn great attention as a new modality to overcome drug resistance by achieving selective degradation of disease-causing proteins, like an EGFR mutant protein, which are required for the survival and proliferation of various malignant tumor cells. Therefore, we have developed a series of EGFR mutant degraders using BBT-176, a 4th generation EGFR TKI developed by Bridge BioTherapeutics, and novel EGFR inhibitors as warheads which are expected not only to treat various EGFR mutated NSCLC including C797S but also to delay drug resistant-mechanisms by further mutations in the EGFR TKI target site. We synthesized a set of heterobifunctional degraders applying Cyrus BiFx Degrader (CBD) library platform containing different types of linkers and E3 ligase binders. DC50 (half-maximal degradation concentrations) values of the EGFR degraders were obtained in multiple human EGFR mutant NSCLC cell lines, where C-2051, C-2482, and C-4383 showed excellent EGFR degradation potency (DC50 = 7-25 nM, Dmax at 100 nM = 80-97%) with high selectivity against WT EGFR. Among those compounds, C-4383 was potent in cell viability assays with EGFR mutant HCC827 (Del19) and NCI-H1975 (L858R, T790M) cell lines showing a range of GI50 values from 77 to 81 nM at 72 h of compound exposure. By contrast, the compound was much less potent in A549 cells harboring wild-type EGFR (GI50 value at 72 h of treatment, 4.2 μM). We also performed proteomic analysis of C-4383 in H1975 cells resulting that EGFR was the most degraded protein with no off-target events at 100 nM (Dmax dose). A PK study of C-4383 in rats showed good exposure with moderate CL when administered intravenously (CL = 8.51 mL/min/kg, AUC = 5911 ng•h/mL). In summary, we identified heterobifunctional degraders using a 4th generation EGFR inhibitor, BBT-176, that are active in EGFR mutant NSCLC cells harboring single or C797S-containing multiple mutations. We are currently making our efforts on the improvement of physicochemical properties of EGFR mutant targeted heterobifunctional compounds. Citation Format: Dong Hyuk Ki, Joonwoo Nam, Eunjung Kim, Hunmi Choi, Chulwon Kim, Jiyeon Kim, Jimmy Jin, Sang-Uk Kang, Wooseok Han. Identification of next-generation EGFR degraders to treat non-small cell lung cancer (NSCLC) patients [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 417.

A phase 1/2 study of the highly selective EGFR inhibitor, BLU-701, in patients with EGFR-mutant non–small cell lung cancer (NSCLC).

TPS9142 Background: Although 3rd-generation tyrosine kinase inhibitors (TKIs), such as osimertinib, are highly effective in front-line metastatic EGFR-mutated ( EGFRm) NSCLC, treatment resistance ultimately occurs, including the emergence of the on-target C797X mutation for which there are no approved TKIs. BLU-701 is an investigational, reversible, brain-penetrant, wildtype-sparing oral TKI with nanomolar potency on common activating (exon 19 deletion and L858R) and C797X resistance mutations (Tavera L et al. AACR 2022). BLU-701 has shown promising preclinical data, including antitumor central nervous system (CNS) activity that may improve patient outcomes. Additionally, combining BLU-701 with standard of care therapies may provide enhanced disease control across multiple lines of treatment, including against heterogenous tumors, in patients with EGFRm NSCLC. Methods: HARMONY (NCT05153408) is an ongoing, global phase 1/2, open-label, first-in-human study designed to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of BLU-701 as a monotherapy or in combination with osimertinib or platinum-based chemotherapy in patients with EGFRm NSCLC. Key inclusion criteria include patients ≥18 years of age with metastatic EGFRm NSCLC; Eastern Cooperative Oncology Group performance status 0–1; and previous treatment with ≥1 EGFR-targeted TKI. Patients in the phase 2 monotherapy part must harbor an EGFR C797X resistance mutation (locally assessed). Key exclusion criteria are tumors harboring EGFR T790M mutations, EGFR exon 20 insertions, or other known driver alterations, including KRAS, BRAF V600E, NTRK1/2/3, HER2, ALK, ROS1, MET, or RET. Phase 1 primary endpoints are maximum tolerated dose, recommended phase 2 dose (RP2D), and safety. The phase 2 primary endpoint is overall response rate (ORR) by RECIST 1.1. Secondary endpoints include ORR (phase 1), duration of response, and PK/PD (phase 1 and phase 2); disease control rate, progression-free survival, overall survival, antitumor CNS activity, and safety (phase 2). The phase 1 dose escalation will adopt a Bayesian optimal interval design. Patients will be enrolled into 3 treatment cohorts: part 1A (n≈40–80; BLU-701), part 1B (n≈35; BLU-701 + osimertinib), and part 1C (n≈18; BLU-701 + carboplatin and pemetrexed). Patients in the phase 2 dose expansion (n≈24) will be treated at the RP2D of BLU-701 as monotherapy. Patients may receive treatment until disease progression, unacceptable toxicity, or other discontinuation criteria are met. Enrollment in this study has started, and sites will be open across North America, Europe, and Asia. Clinical trial information: NCT05153408.

A phase 1/2 study of BLU-945 in patients with common activating EGFR-mutant non–small cell lung cancer (NSCLC): SYMPHONY trial in progress.

TPS9156 Background: Although EGFR-targeted therapies have improved outcomes in patients with EGFR-mutant ( EGFRm) NSCLC, such as EGFR ex19del and L858R, resistance to these drugs is inevitable. BLU-945 is an investigational next-generation EGFR tyrosine kinase inhibitor (TKI) designed to suppress common activating and on-target resistance EGFR mutations, such as C797S and T790M. Preclinically it has shown activity as monotherapy in osimertinib-resistant patient-derived xenograft (PDX) models. In addition, BLU-945 has &gt; 450-fold selectivity for C797S and T790M mutants over wildtype, advantageous for combinations with complementary EGFR-targeting agents, such as osimertinib. These combinations have shown enhanced activity in PDX models. The SYMPHONY trial (BLU-945-1101; NCT04862780) is an international, open-label, first-in-human, phase 1/2 study designed to evaluate safety, tolerability, and antitumor activity of BLU-945 as monotherapy and in combination with osimertinib in patients with EGFRm NSCLC. Methods: Key eligibility criteria include adults with pathologically confirmed metastatic NSCLC with an activating EGFR mutation, Eastern Cooperative Oncology Group performance status 0–1, and previous treatment with ≥1 EGFR-targeted TKI. Patients with asymptomatic brain metastases who are on stable doses of corticosteroids are eligible. Tumors with additional known driver alterations, including EGFR exon 20 insertions and other kinase drivers, are excluded. Primary endpoints are maximum tolerated dose, recommended phase 2 dose (RP2D) and safety (phase 1); and overall response rate (ORR) by RECIST 1.1 (phase 2). Key secondary endpoints include ORR, duration of response (DOR), pharmacokinetics (PK), and pharmacodynamics (PD; phase 1); and DOR, progression-free survival, overall survival, CNS efficacy, PK, and safety (phase 2). Phase 1 dose escalation will be done using Bayesian optimal interval design; ̃85 patients will receive BLU-945 monotherapy and ̃18 will receive combination BLU-945 and osimertinib. In the phase 2 dose expansion, patients will receive BLU-945 at the RP2D of the monotherapy in 3 groups based on EGFR mutational profile: T790M and C797S (n≈37), T790M, but not C797S (n≈12), and C797S, but not T790M (n≈12). In a combination group, BLU-945 plus osimertinib will be administered at RP2D of the combination to n≈24 patients, ≥12 with both T790M and C797S mutations. Patients may receive treatment until disease progression or unacceptable toxicity. Recruitment has started and approximately 30 sites will be open for enrollment across North America, Europe, and Asia. Clinical trial information: NCT04862780.

Antitumor Efficacy of EGFR-Targeted Recombinant Immunotoxin in Human Head and Neck Squamous Cell Carcinoma.

Over 90% of head and neck squamous cell carcinoma (HNSCC) overexpresses the epidermal growth factor receptor (EGFR). However, the EGFR-targeted monotherapy response rate only achieves 10-30% in HNSCC. Recombinant immunotoxin (RIT) often consists of an antibody targeting a tumor antigen and a toxin (e.g., diphtheria toxin [DT]) that kills cancer cells. We produced a humanized RIT, designated as hDT806, targeting overexpressed EGFR and investigated its effects in HNSCC. Distinct from the EGFR-targeted tyrosine kinase inhibitor erlotinib or antibody cetuximab, hDT806 effectively suppressed cell proliferation in the four HNSCC lines tested (JHU-011, -013, -022, and -029). In JHU-029 mouse xenograft models, hDT806 substantially reduced tumor growth. hDT806 decreased EGFR protein levels and disrupted the EGFR signaling downstream effectors, including MAPK/ERK1/2 and AKT, while increased proapoptotic proteins, such as p53, caspase-9, caspase-3, and the cleaved PAPR. The hDT806-induced apoptosis of HNSCC cells was corroborated by flow cytometric analysis. Furthermore, hDT806 resulted in a drastic inhibition in RNA polymerase II carboxy-terminal domain phosphorylation critical for transcription and a significant increase in the γH2A.X level, a DNA damage marker. Thus, the direct disruption of EGFR signaling, transcription inhibition, DNA damage, as well as apoptosis induced by hDT806 may contribute to its antitumor efficacy in HNSCC.

Uncommon single and compound EGFR mutations: clinical outcomes of a heterogeneous subgroup of NSCLC

Molecular characterization of non-small-cell lung cancer (NSCLC) is essential to define the correct therapeutic algorithm in metastatic disease. Approximately 90% of epidermal growth factor receptor (EGFR) mutations are usually associated with sensitivity to EGFR tyrosine kinase inhibitors (TKIs). The remaining 10% defines a small, extremely heterogeneous subgroup of mutations, with a varied profile of sensitivity and response to target therapies.This retrospective observational study includes 47 patients affected by metastatic NSCLC harboring uncommon EGFR mutations (single or compound mutation). Patients were treated with EGFR-targeting TKIs or platinum-based chemotherapy as first-line treatment.Median OS resulted longer in the compound mutation group when compared to single rare mutations (33.6 vs 12 months; P = 0.473); a similar result was observed for PFS (16 vs 7.6 months; P = 0.281), although statistical significance was not reached. ORR, PFS and OS resulted similar for patients treated with first-line EGFR TKIs or chemotherapy. No difference in terms of PFS and OS was found according to the TKI administered.Compound mutations seem to be a good prognostic indicator for OS; they are also predictive of response to 1st and 2nd generation EGFR TKIs, as well as exon 19 insertions and mutations in codon 719 of exon 18. For mutations in exon 18 (not in codon 719) and exon 20 insertions, chemotherapy seems the most effective available option. The addition of immunotherapy to chemotherapy could change this approach in the next future.

Abstract 2453: Resistance of triple-negative breast cancer cells to EGFR-targeted tyrosine kinase inhibitors is associated with Annexin A6-dependent metabolic reprogramming

Abstract The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in multiple tumor types, including triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) remains a hard-to-treat breast cancer subtype. Significant effort has thus far been devoted to epidermal growth factor receptor (EGFR) targeted therapies as 70% of TNBC tumors overexpress EGFR. However, clinical trials utilizing anti-EGFR therapies have shown great success in various cancer types including non-small cell lung cancer but have been largely unsuccessful in TNBC due to the lack of understanding of TKI-acquired resistance. We have shown that the expression status of annexin-A6 (AnxA6), a calcium-dependent membrane binding tumor suppressor, stabilizes activated EGFR on the cell surface. We have also shown that chronic treatment of AnxA6-low TNBC cells with Lapatinib induces AnxA6 expression and accumulation of cholesterol in late endosomes suggesting a novel mechanism for resistance of TNBC to EGFR-TKIs. Other studies have revealed that AnxA6 expression contributes to impaired fat storage and adiponectin release in adipose tissues, and that lack of AnxA6 compromises alanine-dependent gluconeogenesis and liver regeneration in mice. However, whether the EGFR-TKI induced upregulation of AnxA6 is associated with metabolic reprogramming in TNBC cells remains unclear. Using the Seahorse XF Analyzer, we assessed the live-cell oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TNBC cells. We demonstrate that the basal mitochondrial respiration and glycolytic rates classified TNBC cells into two distinct metabolic phenotypes. Those with higher OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 high cells (energetic phenotype), while those with lower OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 low cells (quiescentphenotype). Induction of AnxA6 expression following chronic treatment of AnxA6-low TNBC cells with Lapatinib (LapR) reverted these otherwise quiescent metabolic phenotypes to the energetic phenotype associated with higher OCR/ECAR ratios and cellular ATP production capacity. However, downregulation of AnxA6 in AnxA6 high TNBC cells attenuated the mitochondrial and glycolytic function with reduced OCR/ECAR ratios and decreased cellular ATP production capacity. NMR-based metabolomics revealed that AnxA6 depleted and/or lapatinib-resistant TNBC cells have a greater dependency on gluconeogenic amino acids including alanine, cysteine, glycine, and proline. These data suggest that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and provide novel insights into the failure of EGFR-targeted therapies as therapeutic options for TNBC. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Resistance of triple-negative breast cancer cells to EGFR-targeted tyrosine kinase inhibitors is associated with Annexin A6-dependent metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2453.

Impact of EGFR mutation on outcomes following SRS for brain metastases in non-small cell lung cancer

IntroductionPatients with EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC) are at particularly high risk of developing brain metastases (BrM). In addition to EGFR targeting tyrosine kinase inhibitors (TKI), radiosurgery (SRS) has an important role in the management of EGFRm BrM. However, data specific to the response and toxicity of EGFRm BrM to SRS are sparse. We evaluated the incidence of local failure (LF) and toxicity of EGFRm and EGFR-wild-type (EGFRwt) BrM treated with SRS. MethodsWe analyzed a prospective registry of BrM patients treated at our centre between 2008 and 2017 and identified EGFRm and EGFRwt NSCLC patients treated with SRS ± systemic therapy for BrM. Incidences of local failure (LF) and radionecrosis (RN) were determined, and Cox regression was performed for univariate and multivariate analyses (MVAs). ResultsWe analyzed data from 218 patients (615 lesions - 225 EGFRm and 390 EGFRwt). Median imaging follow-up per patient was 14.5 months (0.5–96.3). Prior to or concomitant with SRS, 62 % of EGFRm patients received TKI and 93 % received TKI post SRS. The 24-month incidence of LF was 6% and 16 % for EGFRm BrM and EGFRwt, respectively (0.43(0.19−0.95); p = 0.037). The 24-month incidence of RN was 4% and 6% for EGFRm and EGFRwt BrM, respectively (0.8(0.32−1.98) p = 0.63). On MVA, BrM size and prescription dose (PD) significantly correlated with a higher risk of LF and BrM size correlated with a higher risk of RN. ConclusionWe observed excellent rates of response and toxicity following SRS in EGFRm compared to EGFRwt NSCLC, suggesting that EGFRm BrM have a favourable risk benefit ratio compared to EGFRwt NSCLC.

Moving beyond epidermal growth factor receptor resistance in metastatic non-small cell lung cancer - a drug development perspective.

Epidermal Growth Factor Receptor (EGFR) mutations are the most common targetable oncogenic driver mutation in metastatic non-small lung cancer (NSCLC). There have been significant advances in the management of metastatic EGFR-mutant NSCLC from the advent of first and second generation EGFR inhibitors to, more recently, the third-generation inhibitor osimertinib. Osimeritinib is now established as first-line therapy on the basis of improved outcomes compared to first and second generation agents. However, despite excellent initial response rates, responses may not be durable due to the development of acquired resistance. Understanding these mechanisms of resistance is critical to the development of rational drug and drug combinations capable of circumventing them. We discuss the major mechanisms of resistance to first, second and third generation EGFR TKIs. The potential of drug combinations utilising chemotherapy, immunotherapy and anti-angiogenic drugs are explored. We examine strategies to aid drug development, including circulating tumour DNA and novel trial designs.

Downregulation of miR-506-3p Facilitates EGFR-TKI Resistance through Induction of Sonic Hedgehog Signaling in Non-Small-Cell Lung Cancer Cell Lines.

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs). Treatment resistance remains the primary obstacle to the successful treatment of NSCLC. Although drug resistance mechanisms have been studied extensively in NSCLC, the regulation of these mechanisms has not been completely understood. Recently, increasing numbers of microRNAs (miRNAs) are implicated in EGFR-TKI resistance, indicating that miRNAs may serve as novel targets and may hold promise as predictive biomarkers for anti-EGFR therapy. MicroRNA-506 (miR-506) has been identified as a tumor suppressor in many cancers, including lung cancer; however, the role of miR-506 in lung cancer chemoresistance has not yet been addressed. Here we report that miR-506-3p expression was markedly reduced in erlotinib-resistant (ER) cells. We identified Sonic Hedgehog (SHH) as a novel target of miR-506-3p, aberrantly activated in ER cells. The ectopic overexpression of miR-506-3p in ER cells downregulates SHH signaling, increases E-cadherin expression, and inhibits the expression of vimentin, thus counteracting the epithelial–mesenchymal transition (EMT)-mediated chemoresistance. Our results advanced our understanding of the molecular mechanisms underlying EGFR-TKI resistance and indicated that the miR-506/SHH axis might represent a novel therapeutic target for future EGFR mutated lung cancer treatment.