急性髓系白血病(AML)细胞抵抗分化刺激,尽管先天免疫受体的高表达,例如Toll样受体9(TLR9)。我们先前证明了使用TLR9靶向的诱饵寡脱氧核苷酸(CpG-STAT3d)靶向信号转导和转录激活因子3(STAT3)会增加人和小鼠AML细胞的免疫原性。这里,我们阐明了体内由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,从静脉内给予CpG-STAT3d或白血病靶向的Stat3沉默和TLR9共刺激后的小鼠分离的AML细胞,显示类似的髓样细胞分化上调(Irf8,Cebpa,Itgam)和抗原呈递(Ciita,Il12a,B2m)相关基因伴随白血病促进Runx1的减少。单细胞转录组学显示CpG-STAT3d诱导AML细胞多向分化为单核细胞/巨噬细胞,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控制的甲基化调节剂而实现的。如DNMT1和DNMT3。事实上,使用阿扎胞苷和CpG寡核苷酸的DNA甲基转移酶(DNMT)抑制的组合模拟CpG-STAT3d效应,导致AML细胞分化,T细胞活化,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。
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.