Wild-type Epidermal Growth Factor Receptor Research Articles

Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.

GraphEGFR: Multi-task and transfer learning based on molecular graph attention mechanism and fingerprints improving inhibitor bioactivity prediction for EGFR family proteins on data scarcity.

The proteins within the human epidermal growth factor receptor (EGFR) family, members of the tyrosine kinase receptor family, play a pivotal role in the molecular mechanisms driving the development of various tumors. Tyrosine kinase inhibitors, key compounds in targeted therapy, encounter challenges in cancer treatment due to emerging drug resistance mutations. Consequently, machine learning has undergone significant evolution to address the challenges of cancer drug discovery related to EGFR family proteins. However, the application of deep learning in this area is hindered by inherent difficulties associated with small-scale data, particularly the risk of overfitting. Moreover, the design of a model architecture that facilitates learning through multi-task and transfer learning, coupled with appropriate molecular representation, poses substantial challenges. In this study, we introduce GraphEGFR, a deep learning regression model designed to enhance molecular representation and model architecture for predicting the bioactivity of inhibitors against both wild-type and mutant EGFR family proteins. GraphEGFR integrates a graph attention mechanism for molecular graphs with deep and convolutional neural networks for molecular fingerprints. We observed that GraphEGFR models employing multi-task and transfer learning strategies generally achieve predictive performance comparable to existing competitive methods. The integration of molecular graphs and fingerprints adeptly captures relationships between atoms and enables both global and local pattern recognition. We further validated potential multi-targeted inhibitors for wild-type and mutant HER1 kinases, exploring key amino acid residues through molecular dynamics simulations to understand molecular interactions. This predictive model offers a robust strategy that could significantly contribute to overcoming the challenges of developing deep learning models for drug discovery with limited data and exploring new frontiers in multi-targeted kinase drug discovery for EGFR family proteins.

Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features.

HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear. HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression. HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors. HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.

Abstract PO3-26-02: Identification of a novel, brain penetrant, EGFR sparing, ErbB2 inhibitor with activity against oncogenic ErbB2 mutations

Abstract Alterations in ErbB2 have an established role as oncogenic drivers in many solid tumors, including gastric and breast cancer. While ErbB2 amplification is well recognized, activating ErbB2 mutations including exon 20 YVMA insertions, S310F/Y, L755S, V777L, and V842I, have emerged as mutually exclusive oncogenic drivers. ErbB2 mutations are present in ~3-4% of breast cancers and are now recognized as known mechanisms of acquired resistance to ErbB2 targeted therapies. These alterations can also be found in metastastic breast cancer that has advanced to the brain, a major clinical challenge with few therapeutic options. Unfortunately, many approved ErbB2 inhibitors are limited in their utility due to the inhibition of wild type EGFR resulting in severe rash and diarrhea. Herein, we describe preclinical data on a brain penetrant, wild type EGFR sparing, ErbB2 inhibitor with activity against prevalent mutations including exon 20 YVMA insertions. Citation Format: Mark J. Chicarelli, Ben Arwood-Levine, Tanna Bettendorf, Karyn Bouhana, Richard Brizendine, LouAnn Cable, Michelle Crow, Brad Fell, John Fischer, Jennifer Fulton, Anna Guarnieri, Madison Hillman, Ravi Jalluri, Daniel Krischlunas, Keith Koch, Vijay Kumar, Cori Malinky, Rob Rieger, John Robinson, Lee Stunkard, Francis Sullivan, Roy Turton, Logan Vine, Shannon Winski, Yeyun Zhou. Identification of a novel, brain penetrant, EGFR sparing, ErbB2 inhibitor with activity against oncogenic ErbB2 mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-02.

Predicting epidermal growth factor receptor mutations in non-small cell lung cancer through dual-layer spectral CT: a prospective study

ObjectiveTo determine whether quantitative parameters of detector-derived dual-layer spectral computed tomography (DLCT) can reliably identify epidermal growth factor receptor (EGFR) mutation status in patients with non-small cell lung cancer (NSCLC).MethodsPatients with NSCLC who underwent arterial phase (AP) and venous phase (VP) DLCT between December 2021 and November 2022 were subdivided into the mutated and wild-type EGFR groups following EGFR mutation testing. Their baseline clinical data, conventional CT images, and spectral images were obtained. Iodine concentration (IC), iodine no water (INW), effective atomic number (Zeff), virtual monoenergetic images, the slope of the spectral attenuation curve (λHU), enhancement degree (ED), arterial enhancement fraction (AEF), and normalized AEF (NAEF) were measured for each lesion.ResultsNinety-two patients (median age, 61 years, interquartile range [51, 67]; 33 men) were evaluated. The univariate analysis indicated that IC, normalized IC (NIC), INW and ED for the AP and VP, as well as Zeff and λHU for the VP were significantly associated with EGFR mutation status (all p < 0.05). INW(VP) showed the best diagnostic performance (AUC, 0.892 [95% confidence interval {CI}: 0.823, 0.960]). However, neither AEF (p = 0.156) nor NAEF (p = 0.567) showed significant differences between the two groups. The multivariate analysis showed that INW(AP) and NIC(VP) were significant predictors of EGFR mutation status, with the latter showing better performance (p = 0.029; AUC, 0.897 [95% CI: 0.816, 0.951] vs. 0.774 [95% CI: 0.675, 0.855]).ConclusionQuantitative parameters of DLCT can help predict EGFR mutation status in patients with NSCLC.Critical relevance statementQuantitative parameters of DLCT, especially NIC(VP), can help predict EGFR mutation status in patients with NSCLC, facilitating appropriate and individualized treatment for them.Key PointsDetermining EGFR mutation status in patients with NSCLC before starting therapy is essential.Quantitative parameters of DLCT can predict EGFR mutation status in NSCLC patients.NIC in venous phase is an important parameter to guide individualized treatment selection for NSCLC patients.Graphical

Abstract CT247: Updated efficacy and safety of anti-TROP2 ADC SKB264 (MK-2870) for previously treated advanced NSCLC in Phase 2 study

Abstract Background: TROP2 (trophoblast cell surface antigen 2) is commonly over-expressed in non-small cell lung cancer (NSCLC) and associated with poor prognosis. SKB264 (MK-2870) is a TROP2 ADC developed with a novel linker to conjugate the payload, a belotecan-derivative topoisomerase I inhibitor. The linker is affected by both extracellular pH-sensitive cleavage and intracellular enzymatic cleavage within tumor cells, which leads to efficiently release payload inside tumor cells as well as within tumor microenvironment to exert its anti-tumor effects. Here, we present the updated data from a Phase 2 expansion cohort for patients (pts) with advanced NSCLC. Methods: Pts with previously treated advanced NSCLC were enrolled to receive SKB264 at 5 mg/kg Q2W until disease progression or unacceptable toxicity (KL264-01, NCT04152499). Tumor assessment was performed every 8 weeks per RECIST v1.1 assessed by investigator. Results: As of Nov 22, 2023, 43 NSCLC pts had been enrolled. Median follow-up was 17.2 months (mo). 21 pts with EGFR wild type had received median prior 3 regimens of therapy including anti-PD-1/L1 inhibitors. 22 pts with EGFR mutant had progressed on or after TKI therapy, 50% of whom also failed at least one line of chemotherapy. Updated efficacy results are shown in the Table. Overall, 30 pts (69.8%) experienced Grade ≥3 treatment-related adverse events (TRAEs). The most common Grade ≥3 TRAEs were neutrophil count decreased (34.9%), anemia (30.2%), WBC count decreased (25.6%), stomatitis (9.3%), and rash (7.0%). No TRAEs leading to treatment discontinuation or deaths occurred. No drug-related ILD/pneumonitis was reported. Conclusions: The updated data continues to demonstrate that SKB264 monotherapy delivers promising clinical efficacy with manageable toxicity in pretreated advanced NSCLC pts. A Phase 3 global study of SKB264 in pts with 3L+ EGFR mutant NSCLC (NCT06074588) and a Phase 3 study of SKB264 in China in pts with 2L EGFR mutant NSCLC (NCT05870319) are ongoing. TABLE 1: NAND Table Clinical outcomes by EGFR mutation status Overall (N=43) EGFR mutant (N=22) EGFR wild type Total (N=21) Non-squamous (N=9) Squamous (N=12) ORR*, % 43.6% 60.0% 26.3% 22.2% 30.0% Median DoR, mo (95%CI) 9.3 (3.7, 10.3) 8.7 (3.7, 10.3) 9.6 (3.5, NE) / / Median PFS, mo (95% CI) 7.2 (5.4, 11.3) 11.5 (5.7, 12.9) 5.3 (3.5, 6.2) 5.8 (1.5, 12.1) 5.1 (1.9, 9.3) Median OS, mo (95% CI) 22.6 (13.1, NE) 22.7 (19.7, NE) 14.1 (10.7, NE) 16.2 (5.8, NE) 12.8 (3.5, NE) 12-mo OS rate, % (95% CI) 69.0% (52.7, 80.7) 81.0% (56.9, 92.4) 57.1% (33.8, 74.9) 66.7% (28.2, 87.8) 50.0% (20.8, 73.6) 18-mo OS rate, % (95% CI) 56.5% (40.1, 70.0) 76.2% (51.9, 89.3) 35.9% (16.0, 56.4) 44.4% (13.6, 71.9) 30.0% (7.7, 56.9) *Including confirmed or unconfirmed response. Based on response evaluable pts (≥1 on-study scans) with 4 pts (2 EGFR mutant pts with non-squamous histology and 2 EGFR wild type pts with squamous histology) excluded. Citation Format: Wenfeng Fang, Ying Cheng, Zhendong Chen, Wei Wang, Yongsheng Li, Yongmei Yin, Xingya Li, Huiting Xu, Guohua Yu, Yanjun Mi, Zev A. Wainberg, Jordi Rodon, Xiang Wang, Xian Wang, Xiaoqing Zhang, Xiaoping Jin, Lian Lu, Junyou Ge, Jin Li, Li Zhang. Updated efficacy and safety of anti-TROP2 ADC SKB264 (MK-2870) for previously treated advanced NSCLC in Phase 2 study [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 CT247.

Abstract CT154: Phase 1/2 clinical trial of JIN-A02, a 4th generation EGFR-TKI, in patients with 3rd generation EGFR-TKI resistance in EGFR mutated advanced/metastatic non-small cell lung cancer (NSCLC)

Abstract Background: Epidermal growth factor receptor (EGFR) mutations are the most common driver mutations in NSCLC. EGFR tyrosine kinase inhibitors (TKIs) are the recommended front-line option for EGFR-mutant advanced NSCLC patients, but patients invariably develop acquired resistance and face disease progression. JIN-A02, a 4th generation EGFR-TKI intended for oral administration, selectively and reversibly binds to EGFR mutations, including the C797S and/or T790M mutation that causes resistance to 3rd generation of EGFR-TKIs. Pre-clinical studies with EGFR C797S and/or T790M mutated cell lines showed that JIN-A02 can inhibit cell growth in a dose dependent manner and has a high degree of selectivity over wild-type EGFR. In vivo studies showed that administration of JIN-A02 dose-dependently inhibited tumor growth in the C797S+ xenograft mice model. Moreover, JIN-A02 shown to penetrate blood-brain barrier and exhibit anti-tumor activity in an intracranial tumor model. The strong potency and high selectivity of JIN-A02 for mutant EGFR may offer improved therapeutic window in the clinical setting vs available EGFR-TKIs. Methods: JIN-A02 is being evaluated in this Phase 1/2, multicenter, an open-label trial (NCT05394831) for subjects with advanced NSCLC harboring C797S and/or T790M mutation as a monotherapy. The primary object of this study is to assess safety, tolerability, pharmacokinetics, and anti-tumor effect for determining the recommended phase 2 dose (RP2D) of JIN-A02. The inclusion criteria are that the subject (≥ 18 years) must have an advanced or metastatic NSCLC showed progress disease (PD) after receiving approved standard EGFR-TKI therapies and/or platinum-based anticancer chemotherapy is allowed up to 1 time after using EGFR-TKI treatment with ECOG status 0 or 1. Before enrollment in the study, the EGFR mutation status is determined using either tumor tissue and/or plasma ctDNA. This study comprises three parts, dose escalation (Part A), dose exploration (Part B), and dose expansion (Part C). In Part A, dose escalation, comprised of 5 cohorts ranged from a starting dose of 12.5 mg P.O, QD to a top dose of 150 mg P.O, QD with every three subjects, is carried out where the maximum tolerated dose (MTD) is evaluated in subjects with 28-day cycles. Dose-limiting toxicities (DLT) are evaluated for 21 days. In Part B, dose exploration is carried out to evaluate the safety of JIN-A02 further and to determine the RP2D using 2 preliminary effective dose levels from Part A in subjects. In Part C dose-expansion study, subjects (n=36 for each cohort) are divided into 5 different cohorts based on the EGFR mutation (both or single positive of C797S and T790M), and the anti-tumor activity of JIN-A02 is evaluated according to RECIST v1.1 at RP2D. Citation Format: Sun Min Lim, Byoung Chul Cho, Ji Youn Han, Sang We Kim, Ki Hyeong Lee, Misako Nagasaka, Anna Jo, Ethan Seah, Choonok Kim, Thanyanan Reungwetwattana. Phase 1/2 clinical trial of JIN-A02, a 4th generation EGFR-TKI, in patients with 3rd generation EGFR-TKI resistance in EGFR mutated advanced/metastatic non-small cell lung cancer (NSCLC) [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 CT154.

Real-World Outcomes of Patients with Advanced Epidermal Growth Factor Receptor-Mutated Non-Small Cell Lung Cancer in Canada Using Data Extracted by Large Language Model-Based Artificial Intelligence.

Real-world evidence for patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC) in Canada is limited. This study's objective was to use previously validated DARWENTM artificial intelligence (AI) to extract data from electronic heath records of patients with non-squamous NSCLC at University Health Network (UHN) to describe EGFR mutation prevalence, treatment patterns, and outcomes. Of 2154 patients with NSCLC, 613 had advanced disease. Of these, 136 (22%) had common sensitizing EGFR mutations (cEGFRm; ex19del, L858R), 8 (1%) had exon 20 insertions (ex20ins), and 338 (55%) had EGFR wild type. One-year overall survival (OS) (95% CI) for patients with cEGFRm, ex20ins, and EGFR wild type tumours was 88% (83, 94), 100% (100, 100), and 59% (53, 65), respectively. In total, 38% patients with ex20ins received experimental ex20ins targeting treatment as their first-line therapy. A total of 57 patients (36%) with cEGFRm received osimertinib as their first-line treatment, and 61 (39%) received it as their second-line treatment. One-year OS (95% CI) following the discontinuation of osimertinib was 35% (17, 75) post-first-line and 20% (9, 44) post-second-line. In this real-world AI-generated dataset, survival post-osimertinib was poor in patients with cEGFR mutations. Patients with ex20ins in this cohort had improved outcomes, possibly due to ex20ins targeting treatment, highlighting the need for more effective treatments for patients with advanced EGFRm NSCLC.

293 The Impact of Tumor Mutational Status on Protoporphyrin IX Accumulation in Tumor-Associated Macrophages in Glioblastoma Patients

INTRODUCTION: Immunotherapies provide promising new treatment options for lung cancer and melanoma, however success in glioblastoma (GBM) patients has been much rarer and uneven. Identifying candidates with GBM that are most likely to benefit from immunotherapy remains a challenge. The abundance of tumor-associated macrophages (TAMs) correlates response to immunotherapy in GBM patients. Here we leverage a new imaging technique for quantifying TAMs via tandem stimulated Raman histology (SRH) and two-photon excitation fluorescence microscopy (TPEF) to assess the impact of genetic classification on the abundance of TAMs. METHODS: We conducted tandem SRH and TPEF on specimens from 79 patients undergoing high-grade glioma resection with 5-ALA. We trained a MaskR-CNN deep neural network to identify TAMs and compute their density in each specimen. Corresponding clinical, demographic, genomic and methylomic data were collected for each patient. Independent sample t-tests were used to identify significant correlations between genetic markers, treatment, and TAM density. RESULTS: We analyzed 3,121,524 300 x 300 micron fields of view, from 151 fresh specimens, enabling segmentation of 611,789 TAMs. IDH-mutant tumors had significantly lower TAM density than IDH-wildtype tumors (p &lt; 0.01). The absence of MGMT promoter hypermethylation was associated with significantly higher TAM density (p = 0.03). Tumors with EGFR amplification averaged significantly lower TAM density than EGFR wildtype specimens (p &lt; 0.01). Recurrent tumors possessed 31.5% lower TAM density than primary resections. CONCLUSIONS: Tandem SRH and TPEF can estimate variations in TAM density in human GBMs. We found TAM density to be associated with IDH status, MGMT promoter methylation, and EGFR amplification. This imaging technique creates a new avenue for forecasting response to immunotherapy in GBM patients.

Prediction performance comparison of biomarkers for response to immune checkpoint inhibitors in advanced non-small cell lung cancer.

The aim of the present study was to compare the predictive accuracy of PD-L1 immunohistochemistry (IHC), tissue or blood tumor mutation burden (tTMB, bTMB), gene expression profile (GEP), driver gene mutation, and combined biomarkers for immunotherapy response of advanced non-small cell lung cancer (NSCLC). In part 1, clinical trials involved with predictive biomarker exploration for immunotherapy in advanced NSCLC were included. The area under the curve (AUC) of the summary receiver operating characteristic (SROC), sensitivity, specificity, likelihood ratio and predictive value of the biomarkers were evaluated. In part 2, public datasets of immune checkpoint inhibitor (ICI)-treated NSCLC involved with biomarkers were curated (N = 871). Odds ratio (OR) of the positive versus negative biomarker group for objective response rate (ORR) was measured. In part 1, the AUC of combined biomarkers (0.75) was higher than PD-L1 (0.64), tTMB (0.64), bTMB (0.68), GEP (0.67), and driver gene mutation (0.51). Combined biomarkers also had higher specificity, positive likelihood ratio and positive predictive value than single biomarkers. In part 2, the OR of combined biomarkers of PD-L1 plus TMB (PD-L1 cutoff 1%, 0.14; cutoff 50% 0.13) was lower than that of PD-L1 (cutoff 1%, 0.33; cutoff 50% 0.24), tTMB (0.28), bTMB (0.48), EGFR mutation (0.17) and KRAS mutation (0.47), for distinguishing ORR of patients after immunotherapy. Furthermore, positive PD-L1, tTMB-high, wild-type EGFR, and positive PD-L1 plus TMB were associated with prolonged progression-free survival (PFS). Combined biomarkers have superior predictive accuracy than single biomarkers for immunotherapy response of NSCLC. Further investigation is warranted to select optimal biomarkers for various clinical settings.

The radiological characteristics, tertiary lymphoid structures, and survival status associated with EGFR mutation in patients with subsolid nodules like stage I-II LUAD.

BackgroundEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) recommended for the patients with subsolid nodule in early lung cancer stage is not routinely. The clinical value and impact in patients with EGFR mutation on survival outcomes is further needed to be elucidated to decide whether the application of EGFR-TKIs was appropriate in early lung adenocarcinoma (LUAD) stage appearing as subsolid nodules.Materials and methodsThe inclusion of patients exhibiting clinical staging of IA-IIB subsolid nodules. Clinical information, computed tomography (CT) features before surgical resection and pathological characteristics including tertiary lymphoid structures of the tumors were recorded for further exploration of correlation with EGFR mutation and prognosis.ResultsFinally, 325 patients were enrolled into this study, with an average age of 56.8 ± 9.8 years. There are 173 patients (53.2%) harboring EGFR mutation. Logistic regression model analysis showed that female (OR = 1.944, p = 0.015), mix ground glass nodule (OR = 2.071, p = 0.003, bubble-like lucency (OR = 1.991, p = 0.003) were significant risk factors of EGFR mutations. Additionally, EGFR mutations were negatively correlated with TLS presence and density. Prognosis analysis showed that the presence of TLS was associated with better recurrence-free survival (RFS)(p = 0.03) while EGFR mutations were associated with worse RFS(p = 0.01). The RFS in patients with TLS was considerably excel those without TLS within EGFR wild type group(p = 0.018). Multivariate analyses confirmed that EGFR mutation was an independent prognostic predictor for RFS (HR = 3.205, p = 0.037).ConclusionsIn early-phase LUADs, subsolid nodules with EGFR mutation had specific clinical and radiological signatures. EGFR mutation was associated with worse survival outcomes and negatively correlated with TLS, which might weaken the positive impact of TLS on prognosis. Highly attention should be paid to the use of EGFR-TKI for further treatment as agents in early LUAD patients who carrying EGFR mutation.

Abstract 4668: Characterization of fourth-generation EGFR inhibitors in binding experiments with C797S mutant EGFR and cell-based assays with osimertinib-resistant non-small cell lung cancer cell lines

Abstract Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that inhibits the EGFR kinase domain by irreversible binding of the cysteine-797 residue (C797). Osimertinib was originally designed to target the T790M mutation, which is clinically the most frequently observed resistance mechanism to treatment with first- or second-generation EGFR inhibitors. Osimertinib has a high selectivity for the classical EGFR driver mutations (exon 19 deletion and L858R) compared to wild-type EGFR. Due to this favorable selectivity profile, osimertinib has become the standard first-line therapy for non-small cell lung cancer (NSCLC). Common resistance mechanisms following osimertinib treatment are MET oncogene amplification, and cysteine-to-serine substitution at position 797 (C797S) of EGFR, which precludes binding of osimertinib. Fourth-generation EGFR inhibitors and combinations with other targeted agents are potential strategies to overcome osimertinib resistance or to increase its efficacy. Three fourth-generation EGFR inhibitors were profiled in biochemical and cell-based assays: BDTX-1535, BLU-945, and JBJ-09-063. The binding kinetics for wild-type and C797S mutant EGFR were determined in surface plasmon resonance binding experiments on a Biacore 1S+ (Cytiva). The effect on osimertinib-resistant cells, generated using two different approaches, was studied in cell viability assays. In the first model, subclones of the NSCLC cell line PC-9, harboring an exon 19 deletion activating EGFR mutation, were selected for osimertinib resistance by in vitro drug selection [1]. In the second model, the EGFR C797S mutation was introduced into the NSCLC cell line NCI-H1975, which expresses both the L858R activating mutation and the T790M resistance mutation, by gene-editing with CRISPR-Cas9. Synergies with fourth-generation EGFR inhibitors were determined by profiling combinations in a 6-by-6 matrix, and quantifying synergistic effects using the Excess over Bliss score. We will present a detailed comparison of the binding kinetics of the three fourth-generation EGFR inhibitors on wild-type and C797S-mutant EGFR, and their ability to compete with osimertinib for binding to the kinase domain of EGFR. The different fourth-generation inhibitors show various selectivities for the various EGFR mutants and differences in their ability to spare wild-type EGFR. Profiling results of the three fourth-generation EGFR inhibitors in the osimertinib-resistant cell lines and on a panel of more than one hundred human cancer cell lines will be presented. Reference: [1] Bertran-Alamillo et al. Nature Communications 10, 1812; 2019 Citation Format: Janneke J. Melis, Kirsten Kevenaar, Jeffrey J. Kooijman, Yvonne Grobben, Jacob Ytsma, Jordi Bertran-Alamillo, Miguel-Angel Molina-Vila, Nicole Willemsen-Seegers, Guido J. Zaman. Characterization of fourth-generation EGFR inhibitors in binding experiments with C797S mutant EGFR and cell-based assays with osimertinib-resistant non-small cell lung cancer cell lines [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 4668.

Abstract 6868: Exploring myeloid landscapes according to EGFR mutated NSCLC tumor microenvironment

Abstract Introduction: Although the use of immune checkpoint inhibitors (ICIs) has improved the survival rate of some patients, EGFR mutant (MT) NSCLC patients exhibit a poor response to ICIs compared to EGFR wild-type (WT) patients. It has been demonstrated that EGFR MT NSCLC cells contribute to immunosuppressive tumor microenvironments (TMEs) by secreting inhibitory cytokines and metabolites and recruiting pro-tumor immunogenic cells, thereby diminishing ICI efficacy. In particular, myeloid cells represent a major component of the TME and are involved in regulating tumor progression and metastasis, indicating anti- and pro-inflammatory heterogeneous functions. While single-cell based analyses have been conducted in various cell types, studies on the diversity of myeloid cells between EGFR WT and EGFR MT NSCLCs are insufficient. Therefore, we explored to reveal the presence of multifaceted myeloid cell populations and to modulate its functions to augment ICI response in NSCLC patients with EGFR MT using single-cell RNA analysis. Methods: Using the 10x Genomics Chromium system, we have generated single-cell RNA expression of a total of 904,174 cells from primary lung tissues of 95 patients with EGFR-WT (n=35), EGFR-MT TKI naïve (n=55), TKI post (n=5) and matched normal (n=22). Nine distinct cell lineages were annotated with canonical marker gene expression, focusing particularly on the myeloid (n=212,673 cells). Results: We characterized 22 transcriptional states of myeloid cells: 4 clusters of alveolar macrophages (AMs; PPARG+), 4 clusters of interstitial macrophages (IMs; SPP1+, FOLR2+, CCL2+), 4 clusters of conventional DCs (cDCs; CLEC9A+, CD1C+, LAMP3+), 2 clusters of mast cells (KIT+), 1 cluster of plasmacytoid DCs (pDCs; LILRA4+), proliferating cells (MKI67+), monocytes (FCN1+), neutrophil like cells (CSF3R+) and 4 other unidentified clusters. In tumor tissues, IMs constituted a significantly higher proportion compared to normal tissues (p&amp;lt;0.001), with EGFR-WT and EGFR-MT IMs exhibiting distinct characteristics. EGFR-WT IMs had a high proportion of cluster expressing FOLR2, a marker of tissue resident macrophages associated with CD8 T cell infiltration and activation. On the other hand, a major component of IMs from EGFR-MT was a cluster expressing SPP1 and its regulator MIF. SPP1 is well known to be involved in cancer cell progression, epithelial-mesenchymal transition, autophagy, and epigenetic alterations. The abundance of SPP1 IMs in the EGFR-MT TME refers to the formation of an immunosuppressive TME, potentially compromising the effectiveness of ICIs. Conclusions: We found that EGFR-MT TME comprised a high proportion of immunosuppressive IM cluster expressing SPP1, implying that it contributes to an immunosuppressive TME. Our findings suggest the possibility of developing strategies to enhance ICI responses by targeting SPP1-induced immunosuppressive TME in EGFR-mutant NSCLC patients. Citation Format: Eun Ji Lee, Juwon Ahn, You Won Lee, Minyeop Kim, Seung Yeon Oh, Yong Joon Park, Su-Jin Choi, JuHyeon Lee, Kyumin Lim, Kyoung-Ho Pyo, Jae Hwan Kim, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Mi Ran Yun, Byoung Chul Cho. Exploring myeloid landscapes according to EGFR mutated NSCLC tumor microenvironment [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 6868.

Abstract 6511: BPI-520105, a highly potent, CNS-penetrant 4th generation EGFR inhibitor overcoming major EGFR resistance mutations in NSCLC

Abstract EGFR activating mutations are prevalent in 10-50% of NSCLC patients. The most common EGFR mutations, L858R (L) and Del19 (D), initially respond to first-, second-, or third-generation (1G, 2G and 3G) EGFR-TKIs. However, on-target resistance, primarily by T790M (T) and C797X(C), inevitably emerges post-treatment and leads to disease progression. Different treatment sequence give rise to different complex mutations. In patients resistant to the 3G TKI osimertinib, double mutations L858R/C797X or Del19/C797X (LC or DC) were observed at a frequency of up to 12% after first-line osimertinib, and triple mutations L858R/T790M/C797S or Del19/T790M/C797S (LTC or DTC) were up to 20% after second-line osimertinib. There are no approved targeted therapy options for these complex resistant mutations. In this study, we developed BPI-520105, a 4th generation EGFR inhibitor capable of targeting all aforementioned major forms of EGFR activating and resistance mutations in NSCLC. It might be an optimal way to address this urgent medical need. In vitro, BPI-520105 potently inhibited the activity of a variety of EGFR kinases, as well as inhibited the proliferation and EGFR phosphorylation in cell lines bearing relative mutations, including single (D, L), double (DT, LT, and DC, LC) and triple (LTC, DTC) mutations. Meanwhile, BPI-520105 exhibited relative weakness towards wild type EGFR and IGF1R, demonstrating good selectivity in kinase and cell-based assays. Despite being a reversible EGFR inhibitor, BPI-520105 sustained the inhibition of pEGFR and pERK for up to 24 hours in the western blot assay. In vivo, oral administration of BPI-520105 displayed significant dose-dependent antitumor effect in multiple EGFR mutant xenograft models, including HCC827 (D), NCI-H1975 (LT), BaF3-EGFR-LC/DC, and BaF3-EGFR-LTC models. Remarkably, BPI-520105 significantly prolonged the lifespan of mice with brain orthotropic BaF3-EGFR-DTC-Luc2 allografts, demonstrating its activity towards brain metastasis. Additionally, BPI-520105 exhibited favorable ADME properties, with high oral exposure across various preclinical species. In summary, BPI-520105 is a highly potent, CNS-penetrant, wide type sparing, and orally bioavailable 4th generation EGFR inhibitor, holding promise for the treatment of NSCLC patients with resistance mutations post 3G progression or even at front line. Citation Format: Jing Guo, Lijia Liu, Xiangyong Liu, Bang Fu, Guangcan Bai, Changyong Qiu, Wei Ren, Xiaodong Song, Guolong Du, Xiao Sun, Yongchun Zhang, Weidong Cai, Pingping Wang, Haibo Chen, Xiaoyun Liu, Zhengyao Zou, Jiayu Zhao, Xuepeng Jv, Hong Lan, Lieming Ding, Jiabing Wang. BPI-520105, a highly potent, CNS-penetrant 4th generation EGFR inhibitor overcoming major EGFR resistance mutations in NSCLC [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 6511.

Abstract 1230: MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance

Abstract Adaptive resistance mechanisms compromise the long-term effectiveness of kinase-targeted agents dictating the need for strategic combination approaches. MTX-531 was computationally designed to selectively target both PI3K and wild type EGFR. MTX-531 exhibits low nanomolar potency against the PI3K isoform family and EGFR with a high degree of specificity predicted by co-crystal structural analyses. Full kinome screening confirmed that MTX-531 is exquisitely selective for its intended targets. We evaluated the pharmacological profile of MTX-531, whereupon single agent treatment with this lead molecule elicited a high incidence of durable tumor regressions in a broad panel of PIK3CA mutant CDX and PDX squamous head and neck (HNSCC) models. The combination of MTX-531 with RAS pathway inhibitors, including agents directed against KRAS G12C, led to durable regressions of KRAS mutant colorectal and pancreatic xenografts, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to hyperglycemia and hyperinsulinemia commonly seen with predecessor pan PI3K inhibitors. MTX-531 acts as an agonist of PPARγ, a structural feature of the molecule that is thought to mitigate hyperglycemia induced by PI3K inhibition. This unique attribute of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors. To the best of our knowledge, MTX-531 is the first reported pan-PI3K inhibitor that does not lead to hyperglycemia. The pharmaceutical profile of MTX-531 includes favorable cross-species oral bioavailability, microsomal stability, and a facile 3 step chemical synthesis. MTX-531, which has the unique capability of concurrently and selectively inhibiting PI3K and EGFR, illustrates the power of rational computational drug design to target multiple adaptive resistance mechanisms in a single molecule. The versatility of MTX-531 in both the single agent and combination settings offers a breadth of development strategies that continue to evolve as new combination candidates become available. MTX-531 is currently undergoing advanced preclinical development for anticipated first in human clinical trials in late 2024. Citation Format: Christopher E. Whitehead, Elizabeth K. Ziemke, Christy L. Frankowski-McGregor, Rachel A. Mumby, June Chung, Jinju Li, Nathaniel Osher, Oluwadara Coker, Michelle Norris, Scott Kopetz, Veera Baladandayuthapani, Melinda Hollingshead, Sharad Verma, Judith S. Sebolt-Leopold. MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance [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 1230.

Abstract 1974: Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Various types of EGFR-activating mutations are one of the most important oncogenic drivers in non-small cell lung cancer (NSCLC). Among NSCLC patients with EGFR mutations, 5-10% of patients have EGFR exon 20 insertion (ex20ins) mutations that are insensitive to clinically available EGFR tyrosine kinase inhibitors (TKIs). Recently, as mobocertinib (EXKIVITY®) failed to improve progression-free survival in Phase 3 clinical trial, there remains a high unmet medical need to develop new drugs with wider therapeutic window to treat NSCLC harboring EGFR ex20ins mutations. Herein, we report a novel EGFR-TKI, TRX-211-399, specifically designed to target ex20ins mutations. TRX-211-399 potently inhibited the proliferation of not only Ba/F3 cells overexpressing near-loop (A767dupASV, S768dupSVD) and far-loop (H773insNPH, H773dupH) mutations but also patient-derived NSCLC cell line. In particular, TRX-211-399 demonstrated high selectivity against wild-type EGFR. With favorable DMPK profiles, once-daily oral administration of TRX-211-399 showed significant tumor regression in multiple Ba/F3 xenograft models carrying different EGFR exon20ins mutations. Furthermore, in a PC-9-luc cell intracranial xenograft model, TRX-211-399 induced substantial anti-tumor effects and prolonged survival without body weight loss. TRX-211-399 is currently undergoing further preclinical evaluation as a potential candidate for clinical development for patients with EGFR exon20ins mutations. Citation Format: Kwangwoo Chun, Sumin Lim, Joohyun Lee, Seulgi Choi, Seongrak Kim, Youngil Choi, Areum Park, Bohee Kim, Yongseo Park, Eunhyun Choi, Sungwon Lee, Koo Lee. Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations [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 1974.

A prognostic model for overall survival in recurrent glioma patients treated with bevacizumab-containing therapy

Predictive markers and prognostic models are useful for the individualization of cancer treatment. In this study, we sought to identify clinical and molecular factors to predict overall survival in recurrent glioma patients receiving bevacizumab-containing regimens. A cohort of 102 patients was retrospectively collected from June 2011 to January 2022 at our institution. A nomogram was generated by Cox regression and feature selection algorithms based on 19 clinicopathological and 60 molecular variables. The model's performance was internally evaluated by bootstrapping in terms of discrimination and calibration. The median overall survival from the initiation of bevacizumab administration to death or last follow-up was 11.6 months (95% CI: 9.2–13.8 months) for all 102 patients, 10.2 months (95% CI: 6.4–13.3 months) for 66 patients with grade 4 tumors, and 13.8 months (lower limit of 95% CI: 11.5 months) for 36 patients with tumors of grade lower or not available. In the final model, a lower WHO 2021 grade (Grade lower or not available vs. Grade 4, HR: 0.398, 95% CI: 0.223–0.708, p = 0.00172), having received adjuvant radiochemotherapy (Yes vs. No, HR: 0.488, 95% CI: 0.268–0.888, p = 0.0189), and wildtype EGFR (Wildtype vs. Altered, HR: 0.193, 95% CI: 0.0506–0.733, p = 0.0157; Not available vs. Altered, HR: 0.386, 95% CI: 0.184–0.810, p = 0.0118) were significantly associated with longer overall survival in multivariate Cox regression. The overall concordance index was 0.652 (95% CI: 0.566–0.714), and the areas under the time-dependent curves for 6-, 12-, and 18-month overall survival were 0.677 (95% CI: 0.516–0.816), 0.654 (95% CI: 0.470–0.823), and 0.675 (95% CI: 0.491–0.860), respectively. A prognostic model for overall survival in recurrent glioma patients treated with bevacizumab-based therapy was established and internally validated. It could serve as a reference tool for clinicians to assess the extent the patients may benefit from bevacizumab and stratify their treatment response.

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [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 3706.

Abstract 2650: IGFBP2 assists EGFRvIII-positive glioblastoma to evade immune elimination

Abstract In recent years, immunotherapy has achieved significant success in the treatment of a variety of cancer types, including melanoma, non-small cell lung cancer, and colorectal cancer. However, in stark contrast, patients with glioblastoma (GBM) show remarkably poor responses to immunotherapy, failing to derive any therapeutic benefit. Thus, an understanding of the immunosuppressive microenvironment of GBM, which is considered an immunotherapy-cold tumor, is of paramount importance. One common and distinctive genetic mutation found in gliomas is that of EGFRvIII, which is linked with enhanced cell proliferation, invasion, migration, and angiogenesis in GBM. Intriguingly, we found that this mutation induced a significantly more severe immunosuppressive microenvironment compared with wild-type EGFR. To unravel the mechanisms underlying EGFRvIII mutation-induced immunosuppression in GBM, we posited that the mutant variant secretes an array of immunosuppression-related proteins into the glioma microenvironment. To investigate this, we conducted proteomic analysis of conditioned media from EGFRvIII mutant cell lines and discovered significant changes in the expression of insulin-like growth factor-binding protein-2 (IGFBP2), a well-documented oncogenic protein. Although IGFBP2 is known to be upregulated in various tumor types and is associated with malignant behaviors such as proliferation and invasion in gliomas, its role in glioma immunology remains largely unexplored. In line with our earlier bioinformatic analyses, our study revealed significant enrichment of IGFBP2 in pathways related to immunosuppression in GBM. Thus, in the present investigation, we analyzed the mechanisms by which IGFBP2 induces an immunosuppressive microenvironment. Using co-culture models, we found that IGFBP2-overexpressing glioma cells as well as glioma cells treated with exogenous IGFBP2 recruited immune cells through binding to integrin αvβ5. Furthermore, we demonstrated that IGFBP2 promoted the secretion of the classical macrophage polarization factor IL-10 by phosphorylation of STAT3. These findings indicate the significant influence of IGFBP2 in steering glioma-associated macrophages toward a pro-tumor M2-like phenotype. We also observed that IGFBP2 upregulated the expression of PD-L1 on tumor cell surfaces. These results were verified in animal experiments. Intriguingly, our preliminary data indicated that exogenous IGFBP2 translocated to the cell nucleus within hours, and immunoprecipitation assays confirmed its interaction with STAT3 in vitro. These findings collectively underscore the pivotal role of IGFBP2 in the creation of the immunosuppressive microenvironment induced by EGFRvIII mutation. Consequently, targeting IGFBP2 holds great promise for advancing immunotherapeutic approaches in EGFRvIII-mutant glioblastomas, offering an exciting avenue for potential treatments of GBM. Citation Format: Jikang Fan, Xisen Wang, Tao Li, Xuejun Yang, Wei Zhang. IGFBP2 assists EGFRvIII-positive glioblastoma to evade immune elimination [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 2650.

Abstract 4371: ERBB4 heterodimers appear to drive BRAFWT melanoma cell lines

Abstract Purpose of The Study: To determine whether heterodimerization of ERBB4 with EGFR or ERBB2 drives the proliferation of BRAF WT melanoma cell lines. Introduction: Metastatic melanomas that possess wild-type (WT) BRAF alleles are just as aggressive as melanomas that harbor gain-of-function (e.g., V600E) BRAF alleles. Moreover, no targets for effective chemotherapeutic intervention (other than immune checkpoint inhibitors) have been identified for these tumors. Thus, our goal is to identify candidate targets for therapeutic intervention in BRAF WT melanomas. ERBB4 (HER4) is a receptor tyrosine kinase that is closely related to the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). We have previously demonstrated that ERBB4 is both sufficient and necessary for the proliferation of BRAF WT melanoma cell lines. However, the constitutively homodimerized ERBB4 Q646C mutant inhibits the proliferation of BRAF WT melanoma cell lines. We have also previously demonstrated that ERBB4-EGFR and ERBB4-ERBB2 heterodimers stimulate proliferation and other oncogenic phenotypes in a variety of tumor model systems. Hence, here we test the hypothesis that ERBB4-EGFR or ERBB4-ERBB2 heterodimers drive BRAF WT melanomas. Experimental Approach: We are testing whether wild-type EGFR or ERBB2 alleles stimulate the proliferation of four different human BRAF WT melanoma cell lines. We are testing whether the dominant-negative EGFR (K721A) or ERBB2 (K753A) mutant alleles inhibit the proliferation of these cell lines. Likewise, we are testing whether EGFR or ERBB2 shRNAs inhibit the proliferation of these cell lines. Finally, we are testing whether EGFR or ERBB2 enables ERBB4 mutant alleles found in BRAF WT melanoma samples to drive greater levels of proliferation in 32D cells than wild-type ERBB4. Unpublished Results: Wild-type ERBB2 causes a significant increase in the proliferation of the MEL-JUSO and IPC-298 BRAF WT female melanoma cell lines. The ERBB2 dominant-negative allele causes a significant decrease in the proliferation of these cell lines. Finally, preliminary data suggest that the EGFR and ERBB2 dominant-negative alleles inhibit the proliferation of the MeWo BRAF WT male melanoma cell line. Preliminary Conclusions: Our data suggest that ERBB2-ERBB4 heterodimers drive two female BRAF WT melanoma cell lines, whereas ERBB2-ERBB4 and EGFR-ERBB4 heterodimers drive a male BRAF WT melanoma cell line. Thus, we predict that FDA-approved ERBB2 inhibitors (and in some cases, EGFR inhibitors) may be effective in ERBB4-dependent, BRAF WT melanomas. Moreover, ERBB4 heterodimerization with ERBB2 or EGFR may account for the gender disparities observed in BRAF WT melanomas. Citation Format: Vipasha Dwivedi, Lauren M. Lucas, Jen Davis, Kaitlyn O'Daniel, Rees H. Cooke, Joelle N. Woggerman, Ella N. Wilson, Maddy N. Ingrao, David J. Riese. ERBB4 heterodimers appear to drive BRAFWT melanoma cell lines [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 4371.