Abstract 183: High-content screening reveals kinase regulators of Dvl2 phase separation in Wnt signaling: Implications for targeted cancer therapy

Abstract Wnt signaling controls cell proliferation, differentiation, and migration, with critical roles in diseases such as cancer. It was proposed that Wnt signaling relies on the formation of dynamic biomolecular condensates, such as the “signalosome” and the “destruction complex”, with a potential to modify the spatial and temporal regulation of signaling outcomes. To investigate the role of phase separation in Wnt signaling, we generated a cell line with endogenously tagged Dvl2, a central Wnt scaffolding protein, using the mEos3.2 fluorophore. Applying live-cell and single-molecule localization microscopy, we could demonstrate that Dvl2 forms condensates at the centrosome in a Wnt- and cell-cycle-dependent manner in HEK293T cells (Schubert et al. PNAS, 2022). Here, we used image-enabled cell sorting to separate cell pools based on these endogenous Dvl2 condensates, followed by temporal and phosphoproteomic analysis to further understand their functional significance. Condensate-positive cells were enriched with proteins associated with planar cell polarity (PCP) Wnt signaling and proteins of the G2/M cell cycle phase were differentially phosphorylated, suggesting distinct signaling and cell-cycle states. We then conducted a high-throughput, image-based screen of more than 600 kinase inhibitors to identify modulators of Dvl2 condensate dynamics. Several kinases influenced Dvl2 phase separation, revealing a correlation of condensate formation and canonical Wnt signaling activation. Notably, a candidate inhibitor disrupted both Dvl2 condensates and Wnt signaling, affecting cell division, most likely through microtubule regulation. Further phosphoproteomic and chemical-genetic interaction analysis applying CRISPR/Cas9 targeting the Wnt scaffolds APC, Axin1, and MCC, alongside kinase inhibition, revealed distinct roles for these scaffolds in condensate regulation and, interestingly, pathway outcomes. This work underscores the regulatory complexity of Wnt condensates, highlighting post-translational modifications, cellular context, and scaffold roles in Wnt signaling. Our study introduces a high-content screening approach to intracellularly study phase-separated systems. Targeting condensates may represent a novel therapeutic strategy in cancers with aberrant Wnt signaling. Citation Format: Antonia Schubert, Florian Heigwer, Christian Scheeder, Oksana Voloshanenko, Dominique Kranz, Thilo Miersch, Barbara Schmitt, Melanie Kuhse, Franziska Ragaller, Nadine Winkler, Anastasija Paneva, Daniel Gimenes, Diana Ordoñez-Rueda, Jennifer Schwarz, Frank Stein, Dirk Jäger, Ulrike Engel, Michael Boutros. High-content screening reveals kinase regulators of Dvl2 phase separation in Wnt signaling: Implications for targeted cancer therapy [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 183.