Acute myeloid leukaemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) frequently relapses after FLT3 tyrosine kinase inhibitor (TKI) therapy, highlighting the need to define mechanisms of drug tolerance. Here, we identify the mitochondrial complex I core subunit NDUFS2 as a determinant of FLT3 TKI tolerance in FLT3-ITD AML. A metabolism-focused CRISPR-Cas9 sensitiser screen, integrated with transcriptomic profiling of gilteritinib-resistant cells and primary samples, identified NDUFS2 as upregulated in acquired resistance and preferentially expressed in FLT3-ITD AML. NDUFS2 knockdown impaired complex I activity, oxidative phosphorylation, mitochondrial membrane potential and ATP production, thereby promoting apoptosis and sensitising FLT3-ITD AML cells to gilteritinib. Pharmacologic complex I inhibition with IACS-010759 similarly enhanced gilteritinib activity in FLT3-ITD AML cell lines and primary FLT3-ITD samples, activated AMPK-PERK-ATF4-CHOP stress signalling, promoted apoptosis, and reduced leukaemia burden in a patient-derived xenograft model. Genetic NDUFS2 knockdown also prolonged survival in a xenograft model. A preliminary murine safety assessment showed no overt toxicity under the tested short-term dosing schedule. These findings identify NDUFS2-dependent complex I activity as an adaptive metabolic dependency supporting FLT3 inhibitor tolerance and provide mechanistic insight for further preclinical evaluation.
