Abstract B007: Low-order assemblies drive RTK fusion signaling without condensation

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 mechanistic questions of how RTK fusions transmit oncogenic signals. Recent studies have suggested that condensation is essential for signaling from RTK fusions because condensates can organize the necessary signaling machinery in the cytoplasm. In our study we tested this hypothesis using live cell fluorescent imaging, quantitative analysis of condensation, single-cell analysis, and synthetic biology, focusing on condensates of EML4-ALK, a granule-forming RTK fusion that is found in ∼5% of non-small cell lung cancer. 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 and promoted colony formation in soft agar and tumor formation in xenograft models, but notably did not form condensates. Finally, a survey of 9 distinct oncogenic RTK fusions found that only few other fusions formed condensates, while most appeared diffuse. Nevertheless, all fusions could strongly activate signaling, independent of condensate formation. Our work thus suggests that condensation is not essential for RTK fusion signaling and that strong signaling and cell transformation 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: David Gonzalez-Martinez, Thomas R. Mumford, Delaney Wilde, Sofia Wissert, Yuzhi (Carol) Gao, Emily Brackhahn, Richard Kriwacki, Elizabeth Rhoades, Lukasz J. Bugaj. Low-order assemblies drive RTK fusion signaling without condensation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B007.