STAT3 inhibition permits epigenetic reprogramming and TLR9/IRF8-driven differentiation of inv(16) acute myeloid leukemia [RRBS]. STAT3 inhibition permits epigenetic reprogramming and TLR9/IRF8-driven differentiation of inv(16) acute myeloid leukemia [RRBS]

Cultured acute myeloid leukemia (AML) blasts can differentiate into antigen-presenting cells but achieving leukemic cell immunogenicity proved challenging in clinical setting. Despite expressing multiple immunostimulatory receptors, such as Toll-like Receptor 9 (TLR9), AML cells resist immunostimulation. We previously demonstrated that eliminating Signal Transducer and Activator of Transcription 1 and 3 (STAT1/3) signaling in AML cells using decoy DNA strategy (CpG-STAT3dODN) synergized with TLR9 stimulation and resulted in T cell-mediated leukemia regression. Here, we interrogated the molecular underpinnings of CpG-STAT3dODN-driven differentiation and immunogenicity of mouse Cbfb/MYH11/Mpl AML. The transcriptional profiling of AML cells isolated from mice after inducible Stat3 silencing plus TLR9 stimulation or CpG-STAT3dODN treatment, revealed comparable upregulation of genes related to myeloid cell differentiation (Irf8, Cebpa) and immune stimulation (Gadd45a, Ciita, Il12a, Ifng) but decreased expression of leukemia-promoting Runx1 and Run1t1. The conditional Irf8 silencing in AML cells almost completely abrogated TLR9-driven leukemia cell differentiation. The AML cell reprogramming by CpG-STAT3dODN was likely regulated epigenetically as revealed by reduced global promoter hypomethylation of crucial myeloid cell differentiation and immune activation genes. These changes were associated with reduced expression of known STAT3 targets, DNMT1 and DNMT3a/b. Finally, we provide initial evidence of anti-leukemic effects of CpG-STAT3 inhibitor against human AML model in humanized mice. Our studies suggest that, beyond oncogenic functions, STAT3 in AML cells may be a primary checkpoint in control of immune-stimulatory and differentiation driving effects. These findings support further development of CpG-STAT3 inhibitors as a new bi-functional agents for AML immunotherapy. Overall design: We assessed whether levels of DNMTs present in CMM cells will change specifically in sorted CpG-STAT3dODN-induced CD11b+ CMM cell subset compared to the bulk CD11b-CMM cells or to the untreated CMM cells in control mice.

体外培养的急性髓系白血病（acute myeloid leukemia, AML）母细胞可分化为抗原呈递细胞，但在临床场景中实现白血病细胞的免疫原性仍颇具挑战。尽管AML细胞可表达多种免疫刺激受体，例如Toll样受体9（Toll-like Receptor 9, TLR9），但其仍可抵抗免疫刺激作用。我们此前已证实，采用诱饵DNA（decoy DNA）策略（CpG-STAT3dODN）阻断AML细胞中的信号转导与转录激活因子1和3（Signal Transducer and Activator of Transcription 1 and 3, STAT1/3）信号通路，并联合TLR9刺激，可诱导T细胞介导的白血病消退。本研究旨在阐明CpG-STAT3dODN诱导小鼠Cbfb/MYH11/Mpl型AML细胞分化与免疫原性的分子机制。我们对经可诱导Stat3沉默联合TLR9刺激，或经CpG-STAT3dODN处理后从小鼠体内分离的AML细胞进行转录组分析，结果显示：髓系细胞分化相关基因（Irf8、Cebpa）与免疫刺激相关基因（Gadd45a、Ciita、Il12a、Ifng）的表达上调水平相当，但白血病促发基因Runx1与Run1t1的表达水平降低。在AML细胞中条件性沉默Irf8，可几乎完全阻断TLR9诱导的白血病细胞分化。CpG-STAT3dODN介导的AML细胞重编程可能受表观遗传调控：关键髓系细胞分化与免疫激活基因的全基因组启动子低甲基化水平降低，这一结果证实了这一点。上述变化与已知STAT3靶基因DNMT1、DNMT3a/b的表达下调相关。最后，我们首次证实了CpG-STAT3抑制剂对人源化小鼠（humanized mice）体内的人AML模型具有抗白血病作用。本研究表明，除致癌功能外，AML细胞中的STAT3可能是调控免疫刺激与分化驱动效应的核心检查点。上述研究结果支持将CpG-STAT3抑制剂进一步开发为用于AML免疫治疗的新型双功能制剂。实验整体设计：本研究旨在评估，与对照小鼠中未分选的CD11b- CMM细胞或未经处理的CMM细胞相比，经CpG-STAT3dODN诱导分选获得的CD11b+ CMM细胞亚群内的DNA甲基转移酶（DNMTs）表达水平是否会发生特异性改变。