The Role and Mechanism of NDUFS2 in FLT3-Mutated Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by poor prognosis and high recurrence rates. Disease relapse and the lack of precise treatment tailored to each patient pose challenges in AML treatment. The mitochondria, crucial for cellular energy center and signaling, are essential for maintaining the metabolic flexibility of tumor cells. However, their complex and elusive functions in cancer pose challenges for targeted mitochondrial therapies. Therefore, there is an urgent need to discover new mitochondrial-targeted drugs and novel targeted therapeutic strategies for AML. In this study, we explored the expression of NDUFS2 in AML using real-time quantitative PCR and immunohistochemistry. Subsequently, we performed knockdown and overexpression experiments of NDUFS2 in MOLM-13 and MV4-11 cell lines and assessed its effects on AML cell proliferation, apoptosis, cell cycle, reactive oxygen species (ROS) levels, and mitochondrial membrane potential using flow cytometry. Additionally, we evaluated the impact of NDUFS2 on mitochondrial morphology using transmission electron microscopy and confocal microscopy. Seahorse energy metabolism assay kits were utilized to examine the influence of NDUFS2 on AML cell energy metabolism, while mitochondrial complex I and II assay kits were employed to assess its effect on mitochondrial enzyme activity. Furthermore, Western blot analysis was conducted to evaluate the effects of NDUFS2 knockdown on the AMPK pathway, endoplasmic reticulum stress pathway, and apoptosis pathway in AML cells, with reverse validation performed using AMPK and PERK inhibitors. Finally, we established xenograft models of FLT3-mutated AML tumor cells to investigate the impact of NDUFS2 on tumor growth and the synergistic effect of NDUFS2 with FLT3 inhibitor Gilteritinib against FLT3-mutated AML. Concurrently, we developed xenograft models of relapsed/refractory FLT3-mutated AML patients to explore the synergistic effect of mitochondrial complex I inhibitor IACS-010759 with Gilteritinib against relapsed/refractory FLT3-mutated AML. We validated that NDUFS2 exhibits the highest expression level in AML, especially in FLT3-mutated AML cells. Knockdown of NDUFS2 inhibited the proliferation of FLT3-mutated AML cell lines, caused cell cycle arrest, induced mitochondrial fission, decreased mitochondrial membrane potential, increased ROS levels, impaired the function of mitochondrial complex I, altered cellular energy metabolism by reducing oxidative phosphorylation (OXPHOS) levels, and activated the AMPK/PERK pathways, leading to apoptosis. Knockdown of NDUFS2 enhanced the cytotoxicity of Gilteritinib against FLT3-mutated AML cells, and the combination of NDUFS2 knockdown and Gilteritinib reduced mitochondrial membrane potential, increased ROS levels, and activated the AMPK/PERK pathways to induce cell apoptosis. In vivo experiments confirmed that knockdown of NDUFS2 enhanced the antitumor effect of Gilteritinib. Furthermore, IACS-010759 and Gilteritinib exhibited synergistic activity against FLT3-mutated AML by synergistically inducing cell apoptosis, inhibiting cell proliferation, affecting mitochondrial function, activating the AMPK/PERK pathways. In vivo experiments in mice also confirmed the synergistic killing effect on FLT3-mutated AML tumor cells without affecting normal hematopoiesis or the normal function of other organs. We found that NDUFS2 is associated with the prognosis of AML and is highly expressed in FLT3-mutated AML. Knockdown of NDUFS2 suppresses the proliferation of FLT3-mutated AML cells, induces excessive ROS production, disrupts mitochondrial membrane potential, inhibits the enzyme activity of mitochondrial complex I, thereby suppressing OXPHOS, decreasing ATP production, and activating the AMPK/PERK pathways, ultimately leading to cell apoptosis. Targeting NDUFS2 can exert synergistic anti-FLT3-mutated AML effects with FLT3 inhibitors by activating the AMPK/PERK pathways. Additionally, the inhibition of mitochondrial complex I by the inhibitor IACS-010759 can synergize with Gilteritinib to combat FLT3-mutated AML. This indicates that NDUFS2 may serve as a potential target for the treatment of FLT3-mutated AML and enhance the sensitivity of targeted drugs to FLT3-mutated AML.