During acute myeloid leukemia (AML) growth, the bone marrow (BM) niche acquires significant vascular changes that can be offset by therapeutic blast cytoreduction. The molecular mechanisms of this vascular plasticity remain to be fully elucidated. Herein, we report on the changes that occur in the vascular compartment of the FLT3-ITD+ AML BM niche pre and post treatment and their impact on leukemic stem cells (LSCs). BM vasculature was evaluated in FLT3-ITD+ AML models (MllPTD/WT/Flt3ITD/ITD mouse and patient-derived xenograft) by 3D confocal imaging of long bones, calvarium vascular permeability assays, and flow cytometry analysis. Cytokine levels were measured by Luminex assay and miR-126 levels evaluated by Q-RT-PCR and miRNA staining. Wild-type (wt) and MllPTD/WT/Flt3ITD/ITD mice with endothelial cell (EC) miR-126 knockout or overexpression served as controls. The impact of treatment-induced BM vascular changes on LSC activity was evaluated by secondary transplantation of BM cells after administration of tyrosine kinase inhibitors (TKIs) to MllPTD/WT/Flt3ITD/ITD mice with/without either EC miR-126 KO or co-treatment with tumor necrosis factor alpha (TNFα) or anti-miR-126 miRisten. In the normal BM niche, CD31+Sca-1high ECs lining arterioles have miR-126 levels higher than CD31+Sca-1low ECs lining sinusoids. We noted that during FLT3-ITD+ AML growth, the BM niche lost arterioles and gained sinusoids. These changes were mediated by TNFα, a cytokine produced by AML blasts, which induced EC miR-126 downregulation and caused depletion of CD31+Sca-1high ECs and gain in CD31+Sca-1low ECs. Loss of miR-126high ECs led to a decreased EC miR-126 supply to LSCs, which then entered the cell cycle and promoted leukemia growth. Accordingly, antileukemic treatment with TKI decreased the BM blast-produced TNFα and increased miR-126high ECs and the EC miR-126 supply to LSCs. High miR-126 levels safeguarded LSCs, as shown by more severe disease in secondary transplanted mice. Conversely, EC miR-126 deprivation via genetic or pharmacological EC miR-126 knock-down prevented treatment-induced BM miR-126high EC expansion and in turn LSC protection. Treatment-induced CD31+Sca-1high EC re-vascularization of the leukemic BM niche may represent a LSC extrinsic mechanism of treatment resistance that can be overcome with therapeutic EC miR-126 deprivation.

中文翻译：

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治疗诱导的小动脉血运重建和骨髓微环境中 miR-126 的增强可保护 AML 中的白血病干细胞

在急性髓系白血病 (AML) 生长过程中，骨髓 (BM) 生态位会发生显着的血管变化，这些变化可以通过治疗性母细胞减灭术来抵消。这种血管可塑性的分子机制仍有待充分阐明。在此，我们报告了治疗前后 FLT3-ITD+ AML BM 微环境血管室中发生的变化及其对白血病干细胞 (LSC) 的影响。通过长骨 3D 共聚焦成像、颅骨血管通透性测定和流式细胞术分析，在 FLT3-ITD+ AML 模型（MllPTD/WT/Flt3ITD/ITD 小鼠和患者来源的异种移植物）中评估 BM 脉管系统。通过 Luminex 测定测量细胞因子水平，并通过 Q-RT-PCR 和 miRNA 染色评估 miR-126 水平。内皮细胞 (EC) miR-126 敲除或过表达的野生型 (wt) 和 MllPTD/WT/Flt3ITD/ITD 小鼠作为对照。治疗诱导的 BM 血管变化对 LSC 活性的影响通过给 MllPTD/WT/Flt3ITD/ITD 小鼠施用酪氨酸激酶抑制剂 (TKI) 后二次移植 BM 细胞来评估，有/无 EC miR-126 KO 或 co-用肿瘤坏死因子 α (TNFα) 或抗 miR-126 MiRisten 治疗。在正常的 BM 生态位中，小动脉内衬的 CD31+Sca-1high ECs 的 miR-126 水平高于血窦内衬的 CD31+Sca-1low ECs。我们注意到，在 FLT3-ITD+ AML 生长过程中，BM 生态位失去了小动脉并获得了血窦。这些变化是由 TNFα 介导的，TNFα 是 AML 母细胞产生的一种细胞因子，可诱导 EC miR-126 下调，并导致 CD31+Sca-1high ECs 耗尽，CD31+Sca-1low ECs 增加。miR-126high ECs 的缺失会导致 LSCs 的 EC miR-126 供应减少，然后 LSCs 进入细胞周期并促进白血病生长。因此，TKI 抗白血病治疗减少了 BM 母细胞产生的 TNFα，增加了 miR-126high ECs 和 EC miR-126 对 LSCs 的供应。高 miR-126 水平可以保护 LSC，正如二次移植小鼠中更严重的疾病所表明的那样。相反，通过遗传或药理学 EC miR-126 敲低来剥夺 EC miR-126 可防止治疗诱导的 BM miR-126high EC 扩张，进而防止 LSC 保护。治疗诱导的 CD31+Sca-1high EC 对白血病 BM 生态位的血运重建可能代表 LSC 治疗抵抗的外在机制，可以通过治疗性 EC miR-126 剥夺来克服。