目的

急性髓系白血病（acute myeloid leukemia，AML）作为一种血液系统恶性肿瘤，具有高度异质性，尽管强化化疗可诱导缓解，但复发后预后较差。白血病干细胞（leukemia stem cells，LSCs）被认为是AML的复发根源，其代谢特征与正常造血干细胞（hematological stem cells，HSCs）显著不同：LSCs依赖线粒体中氧化磷酸化（oxidative phosphorylation，OXPHOS）供能，而HSCs可通过糖酵解代偿OXPHOS抑制。因此，靶向OXPHOS通路可能选择性清除LSCs，同时保留正常造血功能。ATP合成酶是OXPHOS中的最后一个限速酶，靶向ATP合成酶清除LSCs具有理论可行性。因此本研究旨在验证ATP合成酶抑制剂（ATP synthase inhibitor，ASI）对AML的治疗效果及机制，包括对LSCs的杀伤作用和对正常造血的影响，探索ASI联合阿糖胞苷（Cytosine Arabinoside，Ara-C）的协同效应，以克服化疗耐药性，并且阐明ASI通过干扰线粒体功能及代谢重编程促进LSCs分化的分子机制。

方法

1. 体外实验：选用两种AML细胞系（Molm13和THP1），通过CCK-8检测ASI的半数抑制浓度，Annexin V/PI双染法评估凋亡，ATP检测分析细胞内ATP水平，JC-1荧光探针检测线粒体膜电位。

2. 体内实验：构建MLL-AF9突变AML小鼠模型，分为ASI单药组（10 mg/kg，静脉注射）、Ara-C单药组（50 mg/kg，腹腔注射）、ASI+Ara-C联合组及对照组。

3. 疗效评估：监测生存期、外周血肿瘤负荷、脾脏重量及骨髓/脾脏中LSCs比例。

4. 安全性分析：检测野生型小鼠长期给药后体重、血常规、脾脏重量及骨髓造血功能（移植实验评估HSCs长期重建能力）。

5. 代谢机制研究：NAD+/NADH比值检测：通过酶标法分析ASI处理后LSCs的代谢状态。体外添加NADH观察CD11b表达及髓系分化基因变化。

结果

1. ASI单药显著抑制AML细胞活性：

ASI显著抑制Molm13和THP1细胞增殖，并呈现浓度依赖性凋亡诱导效应。

ASI降低线粒体膜电位及细胞内ATP水平，表明其直接干扰线粒体功能。

2. 体内疗效验证：

ASI单药显著延长AML小鼠生存期，降低肿瘤负荷及脾脏重量。

ASI与Ara-C联合治疗进一步减少肿瘤细胞及LSCs比例，疗效优于单药组。

3. 代谢与分化机制：

ASI抑制电子传递链导致NADH蓄积，体外添加NADH可增强CD11b表达并加速髓系分化。

4. 安全性评估：

ASI对正常HSCs无显著毒性：野生型小鼠给药30天后，外周血细胞计数、脾脏重量及骨髓移植后造血重建能力均与对照组无差异。

结论

1. 治疗潜力：ASI通过选择性抑制LSCs的OXPHOS通路中的ATP合成酶，有效清除白血病细胞并延缓复发，且与Ara-C联用可显著增强疗效，为克服化疗耐药提供新策略。

2. 机制创新：揭示了ASI通过NADH蓄积促进LSCs分化的代谢重编程机制，为靶向代谢脆弱性治疗AML奠定理论基础。

3. 临床转化价值：ASI对正常造血功能影响轻微，安全性良好，具备临床开发潜力。未来需进一步优化给药方案，并探索其与其他靶向药物的协同效应。

Objective

1.Background and Research Question: Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy with a high relapse rate (40%~70%) despite intensive chemotherapy. Leukemic stem cells (LSCs) are considered the root of relapse, exhibiting distinct metabolic features from normal hematopoietic stem cells (HSCs): LSCs rely on mitochondrial oxidative phosphorylation (OXPHOS), while HSCs compensate via glycolysis. Targeting OXPHOS may selectively eliminate LSCs while sparing normal hematopoiesis.

2. Aims:

To evaluate the therapeutic efficacy and mechanisms of ATP synthase inhibitor (ASI) in AML, focusing on LSCs eradication and safety in normal hematopoiesis.

To explore the synergistic effect of ASI combined with cytarabine (Ara-C) to overcome chemoresistance.

To elucidate the molecular mechanism by which ASI promotes LSCs differentiation through mitochondrial dysfunction and metabolic reprogramming.

Methods

1. In Vitro Studies:

Cell Models: AML cell lines (Molm13 and THP1) were used for IC50 determination (CCK-8 assay), apoptosis analysis (Annexin V/PI staining), mitochondrial membrane potential measurement (JC-1 probe), and ATP level detection.

2. In Vivo Studies:

Animal Models: MLL-AF9-mutated AML mice were established via viral transduction of LSK cells and secondary transplantation. Groups included ASI monotherapy (10 mg/kg, IV), Ara-C monotherapy (50 mg/kg, IP), ASI+Ara-C combination, and controls.

Efficacy Evaluation: Survival analysis, tumor burden (CD45.2+ cells by flow cytometry), spleen weight, and LSCs frequency (Lineage⁻c-Kit⁺Sca-1⁺CD34⁺CD16/32⁺) were assessed.

Safety Profiling: Wild-type mice received ASI for 30 days; parameters included body weight, blood counts, spleen weight, and hematopoietic reconstitution capacity post-transplantation.

3. Metabolic Mechanism Investigation:

NAD+/NADH Ratio: Enzymatic assays evaluated metabolic shifts in LSCs.

Differentiation Induction: Exogenous NADH was added to assess CD11b expression and myeloid differentiation genes.

Results

1. ASI Monotherapy Suppresses AML Cell Viability:

ASI suppressed proliferation and induced apoptosis in a dose-dependent manner.

Mitochondrial membrane potential and ATP levels were significantly reduced, indicating OXPHOS disruption.

2. In Vivo Efficacy:

ASI monotherapy prolonged survival, reduced tumor burden, and decreased spleen weight in AML mice.

ASI+Ara-C combination further reduced tumor cells and LSCs compared to monotherapy.

3. Metabolic and Differentiation Mechanisms:

ASI caused NADH accumulation by inhibiting the electron transport chain, and exogenous NADH enhanced myeloid differentiation.

4. Safety Assessment:

ASI showed minimal toxicity to normal HSCs: No significant differences in blood counts, spleen weight, or post-transplant reconstitution were observed in wild-type mice.

Mild hepatic hemorrhage was noted pathologically without functional impairment.

Conclusion

1. Therapeutic Potential: ASI selectively targets LSCs via OXPHOS inhibition, effectively eradicating leukemia cells and delaying relapse. Its synergy with Ara-C offers a novel strategy to overcome chemoresistance.

2. Mechanistic Insight: ASI promotes LSC differentiation through NADH-driven metabolic reprogramming, providing a theoretical foundation for targeting metabolic vulnerabilities in AML.

3. Clinical Translation: ASI exhibits favorable safety profiles, supporting its clinical development. Future studies should optimize dosing regimens and explore synergies with other targeted therapies.