尽管 Toll 样受体 9 (TLR9​​) 等先天免疫受体高表达，但急性髓系白血病 (AML) 细胞仍能抵抗分化刺激。我们之前证明，使用 TLR9 靶向诱饵寡脱氧核苷酸 (CpG-STAT3d) 靶向信号转导器和转录激活剂 3 (STAT3) 可增加人和小鼠 AML 细胞的免疫原性。在这里，我们阐明了体内 STAT3 抑制/TLR9 激活驱动的 inv(16) AML 重编程的分子机制。在转录水平上，静脉注射 CpG-STAT3d 或白血病靶向 Stat3 沉默和 TLR9 共刺激后从小鼠中分离出的 AML 细胞，显示出类似的骨髓细胞分化（Irf8、Cebpa、Itgam）和抗原呈递（Ciita、 Il12a、B2m) 相关基因，同时减少促进白血病的 Runx1。单细胞转录组学显示，CpG-STAT3d 诱导 AML 细胞多谱系分化为单核细胞/巨噬细胞、成红细胞和 B 细胞亚群。如体内诱导型 Irf8 沉默所示，IRF8 上调对于白血病细胞的单核细胞-巨噬细胞分化至关重要。 TLR9 驱动的 AML 细胞重编程可能是通过下调 STAT3 控制的甲基化调节因子（例如 DNMT1 和 DNMT3）来实现的。事实上，使用阿扎胞苷与单独的 CpG 寡核苷酸组合抑制 DNA 甲基转移酶 (DNMT) 可以模拟 CpG-STAT3d 效应，从而导致 AML 细胞分化、T 细胞激活和系统性白血病消退。这些发现强调了双功能寡核苷酸通过同时抑制 STAT3 和/或 DNMT 释放 TLR9 驱动的白血病细胞分化的免疫治疗潜力。© 2024 作者。

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.© 2024 The Author(s).