Abstract 4373: Molecular interactions of human receptor tyrosine kinase oncogenic fusions
Authors: Giovanna Dashi, Markku Varjosalo
Journal: Cancer Research (2024)

Abstract Oncogenic fusions (OFs) play a critical role in cancer development. Despite the exponential increase in identifications, only a small number have been extensively characterized functionally. In this study, we are focused on receptor tyrosine kinase OFs (RTK-OFs) prevalent in aggressive cancers. Our primary objective is to elucidate the underlying molecular mechanisms of 182 in-frame RTK-OFs identified in The Cancer Genome Atlas. An automated high-throughput PCR technique will be used to clone individual gene fusions using the sequences obtained from RNA-Seq data sourced from the TCGA dataset. These clones will be used to establish an RTK-OF-expressing isogenic cell line library. Multiple Approaches Combined (MAC)-tag workflow combining Affinity Purification (AP) and proximity biotin-dependent (BioID) purification within a single construct will be used. Subsequent high-throughput EvoSep One Liquid Chromatography coupled to ultra-fast TimsTOF Pro 2 Mass Spectrometry sample processing enables sensitive and robust analysis. These methods enable the capture of stable protein complexes and transient interactors associated with RTK-OFs. In our proof of concept study, we have identified four in-frame variants of FGFR3::TACC3 oncogenic fusions.FGFR3, part of the fibroblast growth factor (FGF) RTK family, activates cellular proliferation and growth through FGF binding and receptor dimerization. In FGFR3-TACC3 OFs, diverse isoforms are observed, with prevalent rearrangements linking FGFR3 exon 17 to various TACC3 exons. TACC3 is vital for mitotic microtubule organization and stabilizes kinetochore fibers and the mitotic spindle through its conserved C-terminal coiled-coil domain (TACC domain). TACC3 contributes to the fusion with varying sizes of the C-terminal coiled-coil domain which all constitutively activate the FGFR3 tyrosine kinase. In our studies, we show that FGFR3::TACC3 variants have a different localization to their wild-type counterparts. While FGFR3 and TACC3 are found in the cytoplasm all the OF variants strongly localize around the nucleus. Our identified high-confidence interactors represent a comprehensive FGFR3::TACC3 interaction network and reveal central pathways and key interactors related to microtubule assembly and possible deregulation of the cell cycle. The resultant data from these advanced technologies enable the construction of comprehensive interaction networks, shedding light on critical signaling pathways and subnetworks relevant to RTK-OFs. Further in-depth analysis of 182 RTK-OFs may uncover actionable pathways and druggable interactors within these networks. Our multi-faceted research strategy holds significant promise for advancing targeted drug discovery, ultimately contributing to improved cancer treatment strategies. Citation Format: Giovanna Dashi, Markku Varjosalo. Molecular interactions of human receptor tyrosine kinase oncogenic fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4373.