Table_4_Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.xlsx

IntroductionAcute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined.
MethodsWe studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry.
ResultsWe found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation.
DiscussionOur study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

## 引言
急性髓系白血病（Acute myeloid leukemia, AML）是成人最常见的急性白血病，总体预后不良且复发率居高不下。遗传异常、分化缺陷及细胞代谢重塑等多种因素共同参与AML的发生与进展。目前氧化磷酸化与糖酵解在AML中的作用已得到明确阐释，但调控细胞质与细胞核蛋白质O-连接β-N-乙酰葡糖胺修饰（O-GlcNAcylation）的己糖胺生物合成通路（hexosamine biosynthetic pathway, HBP），其在AML中的功能仍未被充分阐明。

## 方法
本研究通过单细胞及批量水平的RNA测序，分析AML原始细胞与干细胞中HBP关键酶的表达谱；采用流式细胞术检测O-连接β-N-乙酰葡糖胺转移酶（O-GlcNAc Transferase, OGT）的蛋白表达水平及整体O-连接β-N-乙酰葡糖胺修饰状态。通过蛋白质免疫印迹（Western blotting）与实时荧光定量PCR（real time PCR），探究抑制O-连接β-N-乙酰葡糖胺修饰对AML细胞转录激活的影响，并借助流式细胞术分析其对细胞周期的调控作用。

## 结果
相较于健康供者的全血样本，AML样本中HBP关键酶的表达水平显著升高。与正常造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）相比，AML干细胞与批量细胞中OGT及O-连接β-N-乙酰葡糖胺酶（O-GlcNAcase, OGA）的表达水平均显著上调；同时AML批量细胞与干细胞的OGT蛋白表达及整体O-连接β-N-乙酰葡糖胺修饰水平均显著高于正常HSPCs，验证了本研究前期的生物信息学分析结果。基因集分析显示，在OGT高表达的AML细胞中，核因子κB（NF-κB）通路存在显著富集。抑制O-连接β-N-乙酰葡糖胺修饰可降低NF-κB的核转位效率，并下调部分调控细胞周期的NF-κB靶基因的表达；同时可阻断细胞周期进程，提示AML细胞中增强的O-连接β-N-乙酰葡糖胺修饰与NF-κB激活之间存在紧密关联，进而影响AML细胞的存活与增殖能力。

## 讨论
本研究提示，HBP可作为潜在治疗靶点，单独或联合其他治疗策略同时作用于AML原始细胞与干细胞。此外，传统化疗难以有效靶向清除AML干细胞被认为是疾病复发的核心诱因之一，因此靶向抑制AML干细胞中的HBP及O-连接β-N-乙酰葡糖胺修饰，或可成为控制AML复发的新型有前景的治疗靶点。