口服阿扎胞苷（Oral-Aza；CC-486）治疗可延长强化化疗后缓解期的急性髓系白血病（AML）患者的中位总生存期（OS）（安慰剂为 24.7 个月，安慰剂为 14.8 个月）。 Oral-Aza 的给药方案（14 天/28 天周期）允许长时间低阿扎胞苷暴露，从而促进持续的治疗效果。然而，支持 Oral-Aza 在维持治疗中临床影响的潜在机制仍有待充分了解。在这项临床前工作中，我们通过体外和体内建模探索 Oral-Aza/长期暴露于阿扎胞苷的机制基础。在细胞系中，长时间暴露于阿扎胞苷会导致 DNMT1 持续丢失，从而导致持久的低甲基化和基因表达变化。在小鼠模型中，延长暴露于阿扎胞苷，优先针对未成熟的白血病细胞。在白血病干细胞 (LSC) 模型中，延长剂量的阿扎胞苷可诱导分化并耗尽 CD34 CD38-LSC。从机制上讲，LSC 分化部分是由髓过氧化物酶 (MPO) 表达增加驱动的。通过使用 MPO 特异性抑制剂或阻断氧化应激（MPO 的已知机制）来抑制 MPO 活性，可部分逆转 LSC 的分化。总体而言，我们的临床前工作揭示了对口服 Aza 及其靶向白血病干细胞的能力的新机制见解。

Oral azacitidine (Oral-Aza; CC-486) treatment results in longer median overall survival (OS) (24.7 vs 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy. The dosing schedule of Oral-Aza (14 days/28-day cycle) allows for low exposure of azacitidine for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of Oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of Oral-Aza/extended exposure to azacitidine through in vitro and in vivo modeling. In cell lines, extended exposure to azacitidine results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to azacitidine, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of azacitidine induces differentiation and depletes CD34+CD38- LSCs. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSCs. Overall, our pre-clinical work reveals novel mechanistic insights into oral-Aza and its ability to target leukemic stem cells.