二甲双胍抑制骨髓微环境中基质细胞向AML细胞传递线粒体以增强化疗敏感性的作用及机制研究

In the bone marrow (BM), acquisition of mitochondria from BM stromal cells and increase in oxidative phosphorylation（OXPHOS）renders acute myeloid leukemia (AML) cells resistant to chemotherapy. Metformin (Met) can direct anti-tumor effects. However, whether Met can alter chemosensitivity of AML cells has not been documented. Our prior work has demonstrated that low concentrations of Met did not obviously affect AML cells cultured alone, but significantly enhanced chemosensitivity of AML cells when they were co-cultured with BM stromal cells, and inhibited the mitochondrial transfer from stromal cells to AML cells. Thus, we hypothesize that the low concentration of Met enhanced AML chemosensitivity through inhibition of mitochondrial transfer. To verify this hypothesis, we will employ the technics including lentiviral vector fluorescent labeling of mitochondria, laser confocal microscopy, energy metabolism measurements, mass spectrometry, over-expression and Crispr/cas9 gene editing to explore the role of mitochondrial transfer in mediating AML resistance to chemotherapy, and how Met enhances chemosensitivity of AML cells by blocking mitochondrial transfer, reducing the level of OXPHOX, activating AMPK and suppressing mTORC1. we will also establish a human AML model with NCG mice to confirm that Met can enhance chemosensitivity of AML cells in vivo. This study will provide new ideas for the establishment of new treatments for AML.

骨髓微环境中，急性髓系白血病（AML）细胞从基质细胞摄取线粒体、增强氧化磷酸化（OXPHOS）是其逃逸化疗杀伤的重要机制；二甲双胍（Met）可直接抗肿瘤，但其对AML细胞化疗增敏作用未见报道。我们前期研究发现低浓度Met对单独培养的AML细胞生存无明显影响，但能增强与骨髓基质细胞粘附共培养的AML细胞的化疗敏感性，且抑制基质细胞向AML细胞传递线粒体。基于此，我们推测：低浓度Met可抑制线粒体传递以增强AML细胞的化疗敏感性。为验证该假说，我们拟采用慢病毒荧光标记线粒体、激光共聚焦、能量代谢仪、质谱流式、过表达与CRISPR/Cas9等手段探讨共培养体系中AML细胞从基质细胞获取线粒体后发生化疗抵抗，以及低浓度Met通过抑制线粒体传递、降低OXPHOS、升高AMPK、降低mTORC1以增敏化疗的分子机制；并构建人AML小鼠模型探讨低剂量Met的化疗增敏作用及机制，为AML治疗拓宽新思路。

化疗耐药是AML复发的关键因素。骨髓微环境为AML细胞提供各种信号支持，促使AML细胞逃逸化疗杀伤。因此，探寻有效策略阻断AML细胞和骨髓微环境间的相互交联，将有助于提高化疗效果。基于此，本项目从干扰骨髓微环境-AML细胞关联以增强AML细胞化疗敏感性这一全新角度探寻积极有效的AML治疗新方案，一方面，验证对AML细胞无明显直接影响的二甲双胍（Met）可通过阻断骨髓基质细胞向AML细胞传递线粒体增强AML细胞化疗敏感性；另一方面，从机制上深入探讨OXPHOS–AMPK-mTORC1轴在线粒体传递影响骨髓微环境中AML细胞化疗敏感性中的重要作用。我们的体外研究结果表明Met对单独培养的AML细胞生存无明显影响，但能增强与骨髓基质细胞粘附共培养的AML细胞的化疗敏感性，且抑制基质细胞向AML细胞传递线粒体；同时，体内动物实验结果也表明，所采用的Met剂量对AML肿瘤负荷无明显影响，但明显增强了Ara-C的治疗效果。为了进一步阐明基质细胞向AML细胞传递线粒体后，AML细胞内部的关键分子机制，我们利用线粒体ATP合酶抑制剂Oligomycin A抑制AML细胞的ATP产生验证了共培养后的AML细胞的OXPHOS水平和ATP产量增加与AMPK-mTORC1轴及化疗敏感性的相关性；同时，利用分子生物学手段（敲除/过表达AMPK基因）及药理手段（AMPK激动剂和抑制剂）进一步明确AMPK-mTORC1轴在微环境诱导AML细胞化疗耐药中的作用。这些研究结果阐释了AML微环境性化疗耐药的分子机制，为将Met联合常规化疗药物应用于AML治疗提供了坚实的实验依据和理论基础，为探寻积极有效的AML治疗新方案拓宽了思路。