Abstract 244: Identification of proteins in the oncogenic FLT3-ITD signaling pathway using the APEX2 system
Authors: Özge Tatli, Magnus E. Jakobsson, Georgia Mitsa, Julhash U. Kazi, Lars Rönnstrand
Journal: Cancer Research (2025)

Abstract The FLT3-ITD oncogenic mutation is commonly found in acute myeloid leukemia (AML) and correlated with a poor prognosis. In order to identify proteins within the FLT3-ITD pathway, we employed the engineered ascorbate peroxidase 2 system (APEX2). Monoclonal Ba/F3 cells stably expressing the APEX2-FLT3-ITD fusion protein were incubated with biotin-phenol and treated with H2O2, enabling the APEX2 enzyme to catalyze the biotinylation of proteins in close proximity to FLT3-ITD. Given that quizartinib, an FLT3 inhibitor, alters the localization of FLT3-ITD protein to the cell surface, a quizartinib-treated group was used to mimic wild-type FLT3 and designated as pseudo-wild-type FLT3. Biotinylated proteins were selectively enriched using streptavidin beads to obtain proximity proteome of FLT3-ITD. Enriched proteins were analyzed by state-of-the-art data independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS/MS), uncovering differential associations between wild-type and oncogenic FLT3-ITD. After quantitation by mass spectrometry, the proteins were classified by KEGG enrichment analysis. Interestingly, the protein export group (mmu03060) was enriched in only FLT3-ITD protein interactors. Given that activation of differential signaling cascades relies upon the subcellular localization of FLT3, this set of proteins suggests a possible nuclear localization of FLT3. Upon categorizing the identified proteins based on their molecular function, a significant number of FLT3-ITD interactor proteins were found to be engaged in nuclear events, supporting that FLT3-ITD protein is translocated to the nucleus. Subsequently, we grouped the identified FLT3-ITD interactors based on the domains that they harbor. We selected some specific candidates of particular interest for validation by reciprocal co-immunoprecipitation (co-IP) assays. Protein kinases CDK5 and CHEK1, small GTP binding protein GSPT1, and the nuclear export protein XPO1 were tested for their interaction with FLT3-ITD. Plasmids encoding these proteins with a C-terminal FLAG-tag were co-transfected into human kidney cells with FLT3-ITD. Of these, CDK5 displayed a robust interaction with FLT3-ITD in reciprocal co-IP experiments. Besides, the CDK5 interaction of FLT3-ITD was significantly greater than that of wild-type FLT3. Thereof, we surveyed the phosphorylation dependence of FLT3/CDK5 interaction by suppressing FLT3 phosphorylation in both wild-type FLT3 and oncogenic FLT3-ITD. The inhibition of FLT3 kinase activity with quizartinib drastically reduced CDK5 interaction, revealing that CDK5/FLT3 interaction is dependent of FLT3 phosphorylation. To summarize, we leveraged exploratory proteomics to uncover a panel of candidate FLT3-ITD effectors based on their association or proximity with FLT3-ITD. Future studies will investigate the role of these proteins in FLT3-ITD-induced transformation. Citation Format: Özge Tatli, Magnus E. Jakobsson, Georgia Mitsa, Julhash U. Kazi, Lars Rönnstrand. Identification of proteins in the oncogenic FLT3-ITD signaling pathway using the APEX2 system [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 244.