Abstract 1876: Distinct kinase signatures define therapeutically relevant subtypes in colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is heterogenous disease and second leading cause of cancer-related mortality worldwide. Despite advances in understanding its pathophysiology, molecular mechanisms underlying this heterogeneity remain poorly understood. Identifying actionable molecular markers is crucial to improve patient stratification and therapeutic decision-making. Recent studies have highlighted the kinome—a dynamic network of kinase signaling pathways—as a promising avenue to uncover molecular signatures associated with cancer progression and treatment response. We hypothesized that profiling global kinase activity in CRC patient-derived xenografts (PDXs) could provide valuable insights into subtype-specific signaling alterations. Methods: Kinase activity profiling was conducted using PamGene’s KinomePro platform on 12 CRC-PDX models. Frozen tissue sample were lysed with M-PER Mammalian Extraction Buffer supplemented with Halt Phosphatase and Protease Inhibitor Cocktail and protein quantification was determined using Pierce Coomassie Plus (Bradford) Assay Kit. Tyrosine and Serine/threonine kinase activity profiles were generated using PamChip® comprising 340 different phosphosites that are the known downstream substrates. Peptide phosphorylation and Upstream Kinase Analysis was performed with PamGene’s Tercen analysis pipeline. Results: Kinase activity profiling revealed two distinct molecular subtypes of CRC based on EPH and SRC kinase activity. The EPH-high subgroup showed elevated JAK kinase activity, whereas EPH-low tumors exhibited heightened SRC kinase activity. Functional validation was conducted using dose-response assays with specific inhibitors: Ruxolitinib (JAK inhibitor) and Dasatinib (SRC inhibitor). These experiments demonstrated strong correlation between kinase activity status and inhibitor sensitivity, with the EPH/JAK-high group exhibiting a higher IC50 for Ruxolitinib compared to the low subgroup. To further elucidate signaling perturbations in these subtypes, we integrated transcriptomic data with kinase activity profiles. This in silico analysis confirmed their distinct RNA expression signatures, underscoring the heterogeneity in molecular signaling across the cohort. Conclusions: This study highlights the utility of kinase activity profiling as a robust approach for molecular stratification of colorectal cancer, identifying EPH and SRC kinase activity as potential biomarkers. The validation of differential inhibitor sensitivity supports the potential clinical utility of these markers in guiding personalized therapies. By integrating kinome analysis with transcriptomics, we provide a comprehensive framework for understanding the molecular underpinnings of CRC heterogeneity. PamGene’s KinomePro technology emerges as a promising tool for biomarker discovery and patient stratification in CRC. Citation Format: Gitanjali Dharmadhikari, Simar Pal Singh, Monique Mommersteeg, Liesbeth Houkes, Martijn Dankers. Distinct kinase signatures define therapeutically relevant subtypes in colorectal cancer [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 1876.