Abstract 248: Dissecting therapeutic mechanisms in EGFR and HER2 signaling pathways using Lumit cellular immunoassays

Due to the importance of aberrant receptor tyrosine kinase (RTK) regulation in many cancers, we sought to employ the Lumit Immunoassay Cellular System (Lumit ICS) in order to characterize signaling nodes within important RTK signaling pathways. Lumit ICS is a homogenous immunoassay for detecting targets of interest in cellular lysate. Phosphorylated or total protein can be detected in as little as two hours with a simple add and read format. In this study, we generated several Lumit assays to explore whether Lumit was capable of teasing out mechanistic differences between various drug treatment modalities. These assays include p-EGFR, p-HER2, p-cMET, and various downstream nodes such as p-AKT and p-H2AX. In EGFR WT cell lines, we observed increased phosphorylation of EGFR when treating cells with the ligand, EGF. This increased phosphorylation was inhibited with both small molecule inhibitors, such as erlotinib, and monoclonal antibodies, such as cetuximab. However, in mutant EGFR cell lines that have a level of basal EGFR phosphorylation, only small molecule inhibitors could completely inhibit EGFR phosphorylation. Cetuximab was only capable of inhibiting EGFR phosphorylation stemming from extracellular stimulation (EGF) and not basal p-EGFR. Within the HER2 overexpressed cell line BT474, we detected a high level of basal HER2 phosphorylation. This increased phosphorylation could be inhibited by the small molecule inhibitor CP-724714. However, when treating BT474 cells with monoclonal therapies such as trastuzumab and pertuzumab, we saw a robust increase in HER2 phosphorylation at early timepoints and a more modest increase at longer treatment timepoints. We also noted differences in other cell signaling nodes when treating HER2 overexpressing cells with varying treatment modalities. When treating cells with the antibody drug conjugate (ADC) DS-8201a, we noted increased HER2 phosphorylation, as well as increased phosphorylation of the DNA damage response (DDR) mark, p-H2AX. Increased p-H2AX was not seen when treating the same cell line with trastuzumab. An increase in DNA damage response with DS-8201a treatment fits within the context of DS-8201a delivering a topoisomerase inhibitor as its payload. Finally, we noted a loss of AKT phosphorylation when treating BT474 cells with trastuzumab, but not DS-8201a. While we have yet to explore the reason for this difference in AKT phosphorylation, this result highlights the divergent effects of varying treatment modalities on downstream signaling. Overall, these data exemplify the complexity of cell signaling pathway inhibition and demonstrate Lumit ICS as being an assay capable of teasing out some of that complexity. Citation Format: Matthew R. Swiatnicki, Mindy Lin, Hicham Zegzouti. Dissecting therapeutic mechanisms in EGFR and HER2 signaling pathways using Lumit cellular immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 248.