PKM2在AML细胞外泌体重塑微血管促进白血病化疗抵抗中的作用

Vascular endothelial cells (VECs) and acute myeloid leukemia (AML) cells reprogram each other, contributing to the development of disease in the bone marrow microenvironment. Aerobic glycolysis is the main source of energy for VECs. The glycolytic rate-limiting enzyme pyruvate kinase M2 (PKM2) is rich in exosomes and can be transferred through exosomes to promote microangiogenesis and tumor progression. However, PKM2 in AML exosomes (AML-Exo) has not been reported to be involved in vascular remodelling in the microenvironment. Our previous study showed that AML-Exo enhanced aerobic glycolysis in human umbilical vein endothelial cells (HUVECs) and facilitated vascular formation. Furthermore, AML-Exo transfered PKM2 to HUVECs. Based on these findings, we hypothesized that AML-Exo can deliver PKM2 to VECs, stimulate the remodeling of microvasculature and enhance the chemotherapeutic resistance of AML cells. To test this hypothesis, we will use the technics including Seahorse energy metabolism analyser, flow cytometer, laser confocal microscopy, gene knock-down and over- expression to explore the role of PKM2 in AML-Exo-mediated remodelling of microvessels and enhancement of the chemotherapeutic resistance of AML cells in vitro. We will also confirm the findings by subcutaneously transplanting human AML cells in mice. This study provides a foundation for identifying new targets for AML therapy from the perspective of blocking vascular remodelling.

骨髓微环境中，血管内皮细胞（VECs）与急性髓系白血病（AML）细胞相互重塑促进疾病发展。有氧糖酵解是VECs主要供能方式，糖酵解限速酶丙酮酸激酶2（PKM2）是外泌体常见蛋白且可经外泌体传递，促进微血管生成与肿瘤发展。但AML细胞外泌体（AML-Exo）中PKM2对微环境中血管重塑未见报道。我们前期研究发现AML-Exo能增强脐静脉内皮细胞（HUVECs）有氧糖酵解，促进血管形成；且AML-Exo含有PKM2，可传递至HUVECs。基于此，我们推测：AML-Exo可传递PKM2给VECs，促进微血管重塑，进而促进AML细胞化疗抗性。为验证该假说，我们将采用能量代谢仪、流式细胞仪、激光共聚焦、基因敲减与过表达技术、建立人AML小鼠皮下移植瘤等方法在体内外探讨PKM2在AML-Exo重塑微血管增强AML抵抗化疗杀伤中的作用。该研究将为从阻扰微血管重塑的角度寻找AML治疗新靶点奠定实验基础。

骨髓微环境中残存的白血病细胞是AML耐药与复发的关键因素。因此，深入了解微环境重塑机制将有利于更好地理解与解决AML耐药与复发的问题。骨髓微环境中的血管内皮细胞（VECs）与AML细胞相互重塑促进疾病发展。有氧糖酵解是VECs主要供能方式，糖酵解限速酶丙酮酸激酶2(PKM2)是外泌体常见蛋白且可经外泌体传递，促进微血管生成与肿瘤发展。本项目组前期发现，在体外，AML细胞来源的外泌体（AML-Exo）可促进HUVECs的增殖、迁移和血管形成能力；且AML-Exo含有PKM2，可传递至HUVECs。基于此，我们采用能量代谢仪、流式细胞仪、激光共聚焦显微镜、基因敲减与过表达技术、建立人AML小鼠皮下移植瘤等方法在体内外探讨PKM2在AML-Exo重塑微血管增强AML抵抗化疗杀伤中的作用。结果表明，AML-Exo增强HUVECs生存活力、迁移能力、血管形成能力与糖酵解水平，且与HUVECs中PKM2蛋白上调相关；为了更严谨验证这一结果，我们分别对HUVECs中PKM2进行过表达与敲减，得到了与AML-Exo处理效应相似与相反的结果。我们还对U937细胞中PKM2进行敲减与过表达，U937-Exoshpkm2与U937-Exo oepkm2分别作用于HUVECs，进一步在体外全面深入探讨AML-Exo可传递PKM2给HUVECs，促进微血管重塑。同时，在NOD/SCID小鼠体内建立人AML细胞皮下移植瘤模型，在体内证实AML-Exo中PKM2对微血管的重塑作用及促进AML细胞化疗抗性。该研究从阻扰微血管重塑的角度寻找AML治疗新靶点奠定实验基础，具有较好的潜在临床应用价值。

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