Organelle proteomics reveals novel metabolic vulnerabilities in FLT3-ITD cells

Leukemia ARTICLE www.nature.com/leu OPEN Organelle proteomics reveals novel metabolic vulnerabilities in FLT3-ITD cells Valeria Bica 1,2,6, Anna Francesca Pacilè1,6, Martin Boettcher 3,4, Valentina Marano 2, Veronica Marabitti1, Francesca Nazio1, ✉ Mirko Cortese 2, Thomas Fischer3,4, Livia Perfetto5, Dimitrios Mougiakakos 3,4, Giorgia Massacci 1 and Francesca Sacco 1,2 ✉ 1234567890();,: © The Author(s) 2026 In acute myeloid leukemia (AML), the insertion site of internal tandem duplications (ITDs) within the FLT3 gene critically determines the sensitivity to tyrosine kinase inhibitors (TKIs). Despite recent advances, patients harboring ITDs in the tyrosine kinase domain (TKD) still lack effective therapeutic options. To elucidate the molecular basis underlying the differential TKI sensitivity of FLT3-ITD cells, we integrated high-resolution mass spectrometry–based (phospho)proteomics with subcellular fractionation. Our analysis revealed that midostaurin induces the subcellular redistribution of approximately 2500 proteins involved in crucial biological processes, including cell cycle control, autophagy, and metabolism. Functional analyses further demonstrated that the ITD insertion site determines the autophagy response to midostaurin and modulates mitochondrial metabolism, influencing organelle architecture and ATP production, even at steady state. Importantly, by integrating subcellular proteomic dataset with functional metabolic assays, we uncovered a lipid-dependent vulnerability of FLT3-ITD cells: lipid restriction enhances FLT3 trafficking to the plasma membrane, and markedly reduces cell viability, restoring midostaurin sensitivity of resistant FLT3-ITD cells. Together, our findings reveal that the FLT3-ITD insertion site orchestrates a coordinated remodeling of subcellular protein organization, autophagy, and metabolism, and identify lipid-mediated control of FLT3 compartmentalization as a therapeutically actionable mechanism to overcome TKI resistance in FLT3-ITD AML. Leukemia; https://doi.org/10.1038/s41375-026-03000-6 INTRODUCTION Internal tandem duplications (ITDs) in the FLT3 gene are detected in approximately 25–30% of younger adults with newly diagnosed acute myeloid leukemia (AML) and are associated with an adverse prognosis [1]. These mutations most commonly involve exons 14–15, affecting the juxtamembrane domain (JMD) or the first tyrosine kinase domain (TKD1), and resulting in constitutive FLT3 activation through disruption of its autoinhibitory conformation [2]. The implementation of the multikinase inhibitor midostaurin to standard induction and consolidation chemotherapy has improved outcomes for patients with FLT3-mutated AML [3, 4]. However, an ever-growing number of clinical and experimental evidence indicates that the prognostic and therapeutic impact of FLT3-ITD is influenced by the ITD insertion site [5]. While patients harboring JMD insertions (FLT3ITD-JMD) benefit from FLT3 inhibition, ITDs within the TKD region (FLT3ITD-TKD) are associated with reduced sensitivity to both chemotherapy and FLT3 tyrosine kinase inhibitors (TKIs), as well as lower relapse-free and overall survival. Consistent with these observations, experimental models demonstrate that FLT3ITD-TKD mutations confer resistance to apoptosis in response to multiple TKIs, including gilteritinib and quizartinib, and to cytarabine, compared with FLT3ITD-JMD cells [6–9]. We recently demonstrated that the impact of ITD insertion site on TKI response is mediated by widespread phosphorylation changes, occurring independently of alterations in mRNA expression or protein abundance [6]. Based on these findings, we hypothesized that differential sensitivity of FLT3ITD-JMD and FLT3ITD-TKD cells to midostaurin is driven by phosphorylationdependent redistribution of the proteome across subcellular compartments. To test this, we performed high-resolution mass spectrometry (MS)–based proteomics combined with subcellular fractionation. This approach identified approximately 1800 proteins exhibiting genotype-dependent changes in localization following midostaurin treatment. Notably, protein complexes involved in cell-cycle progression, DNA replication, and mitosis translocated from the nucleus to the cytosol exclusively in FLT3ITD-JMD cells, underscoring the profound impact of ITD location on TKI-mediated cell-cycle control. Integration with phosphoproteomic data further revealed ITD-dependent differences in protein phosphorylation and trafficking, highlighting a different regulation of autophagic processes and distinct metabolic shifts in response to midostaurin. Specifically, we found that midostaurin induced autophagy and reduced oxidative phosphorylation and glycolysis in FLT3ITD-JMD but not in FLT3ITD-TKD cells. Furthermore, 1 Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy. 2Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, Pozzuoli 80078, Italy. 3Health Campus for Inflammation, Immunity and Infection (GCI3), Otto-von-Guericke University of Magdeburg, Magdeburg, Germany. 4Department of Hematology, Oncology and Cell Therapy, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany. 5Department of Biology and Biotechnologies “Charles Darwin”, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy. 6These authors contributed equally: Valeria Bica, Anna Francesca Pacilè. ✉email: ; Received: 20 January 2026 Revised: 24 April 2026 Accepted: 22 May 2026 V. Bica et al. 2 pharmacological inhibition of key metabolic pathways revealed that midostaurin-treated FLT3ITD-TKD cells retain a partial dependence on lipid metabolism. Notably, lipid deprivation sensitized FLT3ITD-TKD cells to TKI treatment, uncovering a novel metabolic vulnerability in FLT3-ITD AML cells. Overall, our work highlights the importance of integrating proteomic, phosphoproteomic, and metabolic analyses to uncover context-specific vulnerabilities in AML and offers a rationale for developing strategies aimed at overcoming resistance in high-risk FLT3-ITD patients. MATERIALS AND METHODS Cell culture Mouse Ba/F3 cells expressing ITD-JMD and ITD-TKD constructs were provided by courtesy of T. Fischer. The cells were cultured in RPMI 1640 medium (Hyclone, Thermo Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (ECS0090D Euroclone, Italy, MI), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco 15140122), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, Missouri, United States, S8636) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma H0887). Midostaurin (Selleck chemical, S8064) was used at 100 nM for 24 h. RESULTS The experimental strategy To investigate how the FLT3-ITD insertion site shapes midostaurindependent proteome reorganization, we applied a streamlined spatial proteomics workflow [10] combining chemical fractionation with MS-based proteomics (Fig. 1A). This approa (...truncated)