Abstract LB450: Dissecting the role of condensation in signaling from RTK fusion oncoproteins

Abstract Receptor tyrosine kinase (RTK) fusions are a diverse class of oncoproteins that have been identified in ∼5% of cancers. RTK fusions are chimeric proteins where the intracellular domain of an RTK is fused to an oligomeric domain from an unrelated protein. Such fusions can drive oncogenic signaling from the RTK fragment despite their cytoplasmic localization, away from the plasma membrane where normal transmembrane RTK signaling occurs, thus posing fundamental mechanistic questions of how RTK fusions transmit oncogenic signals. Recent studies have suggested that signaling might result from the formation of large cytoplasmic granules or condensates, which allow organization of important signaling machinery. In our study we tested this hypothesis using live cell fluorescent imaging, quantitative analysis of condensation, single-cell analysis, and synthetic biology, focusing on condensation of EML4-ALK, a granule-forming RTK fusion that is found in ∼5% of non-small cell lung cancer. Surprisingly, we found weak correspondence between condensate formation and downstream signaling. Although signaling (Erk activation) was a direct function of oncogene expression, condensation was maximal only at intermediate expression levels, resulting in high-expressing cells that had the strongest signaling but no condensation as assessed under high magnification confocal microscopy. Furthermore, increasing the magnitude of condensation through multiple methods did not result in increase in downstream signaling. We then constructed synthetic fusions to test the lower limit of multimerization that could trigger signaling. While monomers of the ALK domain did not signal, dimers triggered signaling to approximately the same magnitudes as EML4-ALK but did not form condensates. Finally, a survey of 9 distinct oncogenic RTK fusions found marked heterogeneity in the propensity of condensate formation. Nevertheless, all fusions could strongly activate signaling, independent of whether or not they formed condensates. Our work thus suggests that condensation is not essential for RTK fusion signaling and that strong signaling can result from fusions that appear diffuse under fluorescence microscopy, from both synthetic and natural fusions. These results suggest a more nuanced role of condensation in RTK fusion signaling and call for further mechanistic studies to understand how cytoplasmic fusions can productively trigger oncogenic signals. Citation Format: Lukasz Bugaj, David Gonzalez-Martinez, Thomas Mumford. Dissecting the role of condensation in signaling from RTK fusion oncoproteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB450.