STING介导了DNMT3A突变的造血干细胞/祖细胞中自我更新能力增强和谱系偏移-动脉网

STING mediates increased self-renewal and lineage skewing in DNMT3A-mutated hematopoietic stem/progenitor cells

Nature 等信源发布 2025-02-22 13:27

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Abstract

摘要

Somatic mutations in DNA methyltransferase 3 A (

DNA甲基转移酶3A中的体细胞突变（

DNMT3A

) are frequently observed in patients with hematological malignancies. Hematopoietic stem/progenitor cells (HSPCs) with mutated

) 在血液系统恶性肿瘤患者中经常被观察到。带有突变的造血干细胞/祖细胞 (HSPCs)

DNMT3A

demonstrate increased self-renewal activity and skewed lineage differentiation. However, the molecular mechanisms underlying these changes remain largely unexplored. In this study, we show that

表现出增强的自我更新活性和偏向的谱系分化。然而，这些变化背后的分子机制在很大程度上仍未被探索。在这项研究中，我们展示了

Dnmt3a

loss leads to the upregulation of endogenous retroviruses (ERVs) in HSPCs, subsequently activating the cGAS-STING pathway and triggering inflammatory responses in these cells. Both genetic and pharmacological inhibition of STING effectively corrects the increased self-renewal activity and differentiation skewing induced by .

损失导致造血干细胞和祖细胞（HSPCs）中内源性逆转录病毒（ERVs）的上调，随后激活cGAS-STING通路并触发这些细胞的炎症反应。对STING的基因和药理学抑制均能有效纠正由其引起的自我更新活性增加和分化偏向。

Dnmt3a

deficiency in mice. Notably, targeting STING showed inhibited acute myeloid leukemia (AML) development in a

小鼠的缺陷。值得注意的是，靶向STING显示抑制了急性髓系白血病（AML）的发展。

Dnmt3a

-KO; Flt3-ITD AML model, comparable to AC220, an FDA-approved FLT3-ITD inhibitor. A patient-derived xenograft (PDX) model further demonstrated that targeting STING effectively alleviates the leukemic burden of

-KO；Flt3-ITD AML模型，与FDA批准的FLT3-ITD抑制剂AC220相当。患者来源的异种移植（PDX）模型进一步证明，靶向STING能有效减轻白血病负担。

DNMT3A

-mutant AML. Collectively, our findings highlight a critical role for STING in hematopoietic disorders induced by

-突变型AML。总的来说，我们的研究结果强调了STING在由以下因素诱导的血液疾病中的关键作用

DNMT3A

mutations and propose STING as a potential therapeutic target for preventing the progression of

突变并提出STING作为预防进展的潜在治疗靶点

DNMT3A

mutation-associated leukemia.

突变相关性白血病。

Introduction

介绍

DNA methylation plays a critical role in embryogenesis and adult tissue homeostasis by influencing gene regulation, chromatin structure and stability, gene imprinting, X chromosome inactivation, and more. In mammalian cells, DNMT3A is a de novo DNA methyltransferase that adds a methyl group to the C5 position of cytosine, producing 5-methylcytosine (5mC) on unmethylated DNA [.

DNA甲基化通过影响基因调控、染色质结构和稳定性、基因印记、X染色体失活等，在胚胎发生和成人组织稳态中发挥关键作用。在哺乳动物细胞中，DNMT3A是一种从头DNA甲基转移酶，它将甲基添加到胞嘧啶的C5位置，在未甲基化的DNA上产生5-甲基胞嘧啶（5mC）。

]. This enzyme plays a crucial role in normal development, as evidenced by

]. 这种酶在正常发育中起着至关重要的作用，这一点由以下事实证明：

Dnmt3a

knockout mice, which die within one month after birth [

敲除小鼠，出生后一个月内死亡 [

], and is particularly significant in hematopoietic differentiation [

】，在造血分化中尤为重要【

]. For example, in clonal hematopoiesis (CH), a condition characterized by the clonal expansion of hematopoietic stem cells (HSCs) in the absence of hematologic disease,

]. 例如，在克隆性造血（CH）中，这种状况的特点是在没有血液疾病的情况下造血干细胞（HSCs）的克隆扩增，

DNMT3A

mutations are the most common genetic event, occurring in up to 40% of all CH cases [

突变是最常见的遗传事件，在所有CH病例中发生率高达40% [

，

]. While in patients with acute myeloid leukemia (AML), mutations in

]. 而在急性髓系白血病（AML）患者中，突变发生在

DNMT3A

occur at frequencies of up to 22% [

发生的频率高达22% [

Dnmt3a

deficiency in the mouse hematopoietic system results in increased self-renewal of HSPCs and eventually malignant transformation [

小鼠造血系统中的缺陷导致HSPCs自我更新增加，并最终发生恶性转化[

，

]. Patient data indicate that

]. 患者数据表明

DNMT3A

mutations often serve as the ‘first-hit’ event in hematologic malignancies, requiring additional genetic alterations such as

突变通常作为血液恶性肿瘤中的“首次打击”事件，需要额外的遗传改变，例如

FLT3-ITDs

，

NPM1

mutations, and others to initiate leukemia [

突变，以及其他导致白血病的[

，

]. This finding aligns with the phenotypes observed in mouse models where the combined loss of

]. 这一发现与在小鼠模型中观察到的表型一致，在这些模型中，

Dnmt3a

and expression of

和表达

FLT3-ITD

led to the development of AML, with a median survival time of 5-7 months. In contrast, mice with either a

导致了AML的发展，中位生存时间为5-7个月。相比之下，具有以下特征的小鼠

Dnmt3a

deletion or FLT3-ITD alone survived for at least one year [

单独的FLT3-ITD缺失至少存活一年 [

，

]. As DNMT3A is an epigenetic regulator, mutations

]. 由于DNMT3A是一种表观遗传调控因子，突变

DNMT3A

lead to the reshaping of the epigenomic landscape in HSPCs and altered gene expression. Studies on the loss of

导致HSPCs的表观基因组景观重塑和基因表达改变。关于丢失的研究

Dnmt3a

in the hematopoietic system of mice have shown that hypomethylation of genes related to stemness leads to enhanced self-renewal at the expense of differentiation [

在小鼠的造血系统中，与干性相关的基因低甲基化会导致自我更新增强，而分化能力减弱 [

，

], even when combined with other cooperating genetic mutations [

], 即使与其他协同遗传突变结合时 [

，

]. This results in a competitive advantage of

]. 这带来了竞争优势

Dnmt3a

mutated HSCs over normal HSCs and likely increase the chance of acquiring additional oncogenic mutations in the expanded clone [

突变的HSCs超过正常的HSCs，并可能增加扩增克隆中获得额外致癌突变的机会 [

The transformation of

转型

DNMT3A

mutation-associated CH into leukemia takes time, and the detailed mechanisms are still not fully understood. However, several laboratories have uncovered some of the effectors that are involved in this process. For example, inflammatory signaling during infections can promote CH in

突变相关的克隆性造血转化为白血病需要时间，其详细机制仍未完全了解。然而，一些实验室已经揭示了参与这一过程的部分效应因子。例如，感染期间的炎症信号可以促进克隆性造血。

Dnmt3a

mutants. Treatment with recombinant interferon-gamma (IFNγ) alone has been sufficient to mimic the effects of infection on

突变体。单独使用重组干扰素-γ（IFNγ）治疗足以模拟感染对

Dnmt3a

mutation-associated CH [

突变相关的CH [

]. This finding aligns with cohort analyses of patients with ulcerative colitis, an autoimmune disease characterized by elevated levels of IFNγ, where there is notably a positive selection of clones harboring

]. 这一发现与溃疡性结肠炎患者的队列分析一致，溃疡性结肠炎是一种以IFNγ水平升高为特征的自身免疫性疾病，其中明显存在对携带特定克隆的正向选择。

DNMT3A

mutations [

突变 [

]. Additionally, epidemiological studies have demonstrated that the development of CH is associated with smoking and conditions related to chronic lung disease [

]. 此外，流行病学研究表明，CH 的发展与吸烟和慢性肺病相关的疾病有关 [

]. Meanwhile, murine models have shown that cells with

]. 同时，鼠模型显示具有

DNMT3A

mutations gain a selective advantage in the fatty bone marrow (BM) environment. This advantage may be mediated by the release of IL-6 from both BM fluid and BM-derived adipocytes [

突变在脂肪骨髓（BM）环境中获得选择性优势。这种优势可能是由BM液和BM衍生的脂肪细胞释放的IL-6介导的[

]. Consequently, environmental selection pressures are considered to play a significant role in the emergence of CH.

]. 因此，环境选择压力被认为在CH的出现中起着重要作用。

In this study, we demonstrated that loss of

在本研究中，我们证明了损失

Sting

刺痛

significantly inhibits the expansion of HSPCs with

显著抑制了HSPCs的扩增

Dnmt3a

deficiency. Mechanistically, the deletion of

不足。从机制上讲，删除

Dnmt3a

in HSPCs led to the hypomethylation and activation of endogenous retroviruses (ERVs) in the genome. The RNA from these ERVs was reverse transcribed into cDNA, which activated the STING pathway and triggered endogenous inflammatory responses. Both inhibition of STING and blocking reverse transcriptase with small molecules effectively reduced the enhanced repopulating capability of .

在HSPCs中导致基因组内源性逆转录病毒（ERVs）的低甲基化和激活。这些ERVs的RNA被逆转录为cDNA，激活了STING通路并触发了内源性炎症反应。抑制STING以及用小分子阻断逆转录酶都能有效降低增强的重建能力。

Dnmt3a

-deficient HSPCs in

-缺陷的HSPCs在

vitro

体外

. Furthermore, targeting STING in a

。此外，针对STING的

Dnmt3a

-deficient murine model of AML and in a DNMT3A-mutated PDX model inhibited disease progression. Therefore, our findings propose a novel mechanism by which

在AML的缺陷小鼠模型和DNMT3A突变的PDX模型中抑制了疾病进展。因此，我们的研究提出了一种新的机制，通过这种机制

Dnmt3a

deficiency induces intrinsic inflammation in the hematopoietic system and disrupts HSPC homeostasis through the activation of the STING pathway. Moreover, this suggests that targeting STING could be a potential strategy to prevent the development of hematopoietic malignancies in patients with

缺陷会诱导造血系统内的固有炎症，并通过激活STING通路破坏HSPC的稳态。此外，这表明靶向STING可能是一种预防患者发生造血系统恶性肿瘤的潜在策略。

DNMT3A

mutations.

突变。

Results

结果

Despite the role of extrinsic stimulus in promoting the development of hematopoietic diseases associated with

尽管外部刺激在促进与造血疾病相关的发展方面发挥了作用，但

DNMT3A

mutations, an intriguing question remains: are there any intrinsic factors in

突变，一个有趣的问题仍然存在：是否在

DNMT3A

mutated cells required for this process? To address this question, we initiated our study by performing a transcriptome analysis to examine the differences in gene expression between

这个过程需要突变细胞吗？为了解决这个问题，我们通过进行转录组分析来研究基因表达之间的差异。

Dnmt3a

f/f

，

Mx1-Cre

(hereafter named

（以下简称

Dnmt3a

-KO) and wild type (WT) HSPCs (Supplementary Fig.

-KO) 和野生型 (WT) HSPCs（补充图）。

A, B).

Activated STING pathway mediates intrinsic inflammatory response in

激活的STING通路介导内在的炎症反应

Dnmt3a

-deficient HSPCs

-缺陷的HSPCs

Gene ontology (GO) analysis revealed that mitosis-related genes were the most upregulated in

基因本体（GO）分析显示，与有丝分裂相关的基因是最显著上调的。

Dnmt3a

-KO HSCs. In contrast, in the common myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP), and common lymphoid progenitor (CLP) cells, there was a significant enrichment of pathways related to inflammatory responses in the

-KO HSCs。相反，在常见的髓系祖细胞（CMP）、粒细胞/单核细胞祖细胞（GMP）和常见的淋巴系祖细胞（CLP）中，与炎症反应相关的通路显著富集。

Dnmt3a

-KO cells (Supplementary Fig.

-KO细胞（补充图）。

). Furthermore, the heatmap illustrating distinctly upregulated inflammatory-related genes in

）。此外，热图展示了显著上调的炎症相关基因，

Dnmt3a

-KO cells compared to WT cells (Supplementary Fig.

与野生型细胞相比，敲除细胞（补充图）。

), confirmed that the inflammatory responses were mainly contributed by progenitor cell populations. We validated these results through quantitative reverse transcription PCR (qRT-PCR) assays to measure the expression levels of inflammatory cytokines in

），证实了炎症反应主要由祖细胞群体引起。我们通过定量逆转录PCR（qRT-PCR）检测验证了这些结果，以测量炎症因子的表达水平在

Dnmt3a

-KO and WT c-Kit

-KO 和 WT c-Kit

加号

cells. Key inflammatory cytokines, including IL-6, ISG15, and CXCL10, were significantly elevated in

细胞。关键的炎症细胞因子，包括IL-6、ISG15和CXCL10，在其中显著升高

Dnmt3a

-KO cells compared to WT cells (Supplementary Fig.

与野生型细胞相比，敲除细胞（参见补充图）。

). We also measured the secreted levels of cytokines such as CXCL10, S100A9, and IFNβ using the enzyme-linked immunosorbent assay (ELISA). A significant increase in these cytokines was observed in the peripheral blood (PB) of

我们还使用酶联免疫吸附试验（ELISA）测量了细胞因子如CXCL10、S100A9和IFNβ的分泌水平。在周围血液（PB）中观察到这些细胞因子显著增加。

Dnmt3a

-KO mice compared to WT mice (Supplementary Fig.

与野生型（WT）小鼠相比，基因敲除（KO）小鼠（补充图）。

1楼

). Together, these data suggest that

). 总之，这些数据表明

Dnmt3a

deficiency triggers an intrinsic inflammatory response in mouse BM progenitor cells.

缺陷会触发小鼠骨髓祖细胞的内在炎症反应。

Recently, we have demonstrated that STING plays a crucial role in the expansion of clones with mutations in TET2—a dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine—in a murine model of CH [

最近，我们已经证明，在CH的小鼠模型中，STING在具有TET2（一种将5-甲基胞嘧啶转化为5-羟甲基胞嘧啶的双加氧酶）突变的克隆扩增中起着关键作用。[

]. Given the similar phenotypes observed in the murine hematopoietic system with deficiencies in either

]. 鉴于在小鼠造血系统中观察到的相似表型，无论是哪种缺陷均使其

Dnmt3a

或

Tet2

, such as increased BM inflammatory signals and skewed differentiation [

，例如骨髓炎症信号增加和分化偏斜 [

], we hypothesize that the activated STING pathway also acts as an intrinsic factor inducing the inflammatory responses and mediating the development of hematopoietic disorders associated with

我们假设激活的STING通路也是一种诱导炎症反应和介导与之相关的造血障碍发展的内在因素。

Dnmt3a

deficiency. We first measured the level of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), a secondary messenger that activates STING [

缺乏。我们首先测量了环鸟苷酸单磷酸-腺苷酸单磷酸（cGAMP）的水平，这是一种激活STING的次级信使[

], in both WT and

], 在野生型和

Dnmt3a

-KO BM cells. As a comparison, we measured cGAMP levels in

-KO BM细胞。作为比较，我们测量了cGAMP的水平。

Tet2

-KO bone marrow (BM) cells, which served as a positive control [

-KO骨髓（BM）细胞，作为阳性对照[

]. The

]. 这个

Dnmt3a

-KO BM cells exhibited approximately 4 fmol of cGAMP per 10⁷ cells, while

-KO BM细胞每10⁷个细胞大约表现出4 fmol的cGAMP，而

Tet2

-KO BM cells showed around 3 fmol per 10⁷ cells. In contrast, no signal was detected in the WT BM (Supplementary Fig.

-KO BM细胞显示出约3 fmol每10⁷个细胞。相反，在WT BM中未检测到信号（补充图）。

). Loss of

). 损失

Dnmt3a

increased the self-renewal ability of Lineage

增加了谱系的自我更新能力

c-Kit

加号

Sca-1

加号

(LSK) cells and enhanced their repopulating capacity in the colony-forming unit (CFU) assay (Supplementary Fig.

(LSK)细胞，并在集落形成单位(CFU)测定中增强了它们的再填充能力(补充图)。

). Furthermore, inhibition of STING with the small molecule C-176 [

). 此外，用小分子C-176抑制STING [

] effectively reduced the colony-forming capability of

有效地降低了菌落形成能力

Dnmt3a

-KO cells at the third replating (Supplementary Fig.

第三次传代的KO细胞（补充图）。

). To genetically investigate the role of STING in hematopoietic disorders associated with

). 为了从基因层面研究STING在与造血障碍相关的疾病中的作用，

Dnmt3a

deficiency, we utilized

缺陷，我们利用了

Dnmt3a

f/f

，

Sting

刺痛

−/−

，

Mx1-Cre

mice (hereafter named

小鼠（以下简称

Dnmt3a; Sting

Dnmt3a；Sting

-DKO) to explore the impact of

-DKO）来探索影响

Sting

刺痛

loss on the hematopoietic phenotype mediated by

造血表型介导的损失

Dnmt3a

deficiency (Fig.

缺陷（图。

). Transcriptome analysis revealed that loss of

)。转录组分析显示，丢失了

Sting

刺痛

effectively reduced the expression of inflammatory genes in

有效降低了炎症基因的表达

Dnmt3a

-deficient c-Kit

缺陷型c-Kit

加号

cells (Supplementary Fig.

细胞（补充图）。

). Furthermore, the downstream pathways of STING, such as the type-I interferon response and cytokine-related signaling pathways, were also significantly diminished, as evidenced by GO analysis data between

此外，STING的下游通路，如I型干扰素反应和细胞因子相关信号通路，也显著减弱，这由基因本体（GO）分析数据证明。

Dnmt3a; Sting

Dnmt3a；Sting

-DKO and

-DKO 和

Dnmt3a

-KO samples (Supplementary Fig.

-KO 样本（补充图）。

二维

). Taken together, these data demonstrate that the cell-intrinsic inflammatory responses in

). 综上所述，这些数据表明细胞内在的炎症反应在

Dnmt3a

-KO HSPCs were dependent on the activation of the STING pathway.

-KO HSPCs 依赖于 STING 通路的激活。

Fig. 1: Loss of

图1：损失

Sting

刺痛

impairs the increased self-renewal of HSPCs mediated by

损害了由...介导的HSPCs自我更新能力的增强

Dnmt3a

deficiency.

不足。

Schematic of the strategy used to generate

生成策略的示意图

Dnmt3a; Sting

Dnmt3a； Sting

-DKO mice. The phenotypes of these mice were analyzed 16 weeks after poly(I:C) injection.

-DKO小鼠。这些小鼠的表型在聚(I:C)注射后16周进行了分析。

Deletion of

删除

Sting

刺痛

reduced the repopulating capacity of

减少了再生能力

Dnmt3a

-deficient LSK cells in CFU assays. LSK cells from different mice were sorted by FACS, and the number of CFUs was counted 7 days after plating. IFNβ was used at a concentration of 1 U/ml.

在CFU实验中，对LSK细胞进行缺陷分析。通过FACS分选不同小鼠的LSK细胞，并在接种后7天计数CFUs的数量。IFNβ的使用浓度为1 U/ml。

Loss of

丢失

Sting

刺痛

suppressed the increased self-renewal of

抑制了增强的自我更新能力

Dnmt3a

-deficient LSK cells in vivo (

-缺陷型LSK细胞在体内（

= 5).

= 5)。

The proportions of myeloid and lymphoid cells in peripheral blood were measured by FACS (

通过FACS测量外周血中髓系和淋巴系细胞的比例 (

= 5).

= 5)。

Deletion of

删除

Sting

刺痛

specifically reduced the proliferation of

特别减少了

Dnmt3a

-KO HSPCs. The HSPCs were stained with an antibody against Ki67 and analyzed by FACS (

-KO HSPCs。HSPCs用抗Ki67抗体染色，并通过FACS分析（

= 6). Data are presented as mean ± s.e.m., with *

= 6)。数据以均值 ± 标准误表示，*

< 0.05, **

< 0.01, ***

< 0.005, ****

< 0.0001, and “ns” not significant.

< 0.0001，且“ns”表示不显著。

Full size image

全尺寸图像

STING is involved in the

STING 参与了

Dnmt3a

deficiency-induced CH

缺陷诱导的CH

Similar to the treatment with the inhibitor, deletion of

类似于用抑制剂处理，删除

Sting

刺痛

significantly inhibited the colony-forming capability of

显著抑制了集落形成的能力

Dnmt3a

-deficient cells at the fourth replating in the CFU assay (Fig.

在CFU测定中第四次传代时的缺陷细胞（图。

). In contrast, IFNβ, a cytokine typically induced by the activated STING pathway, significantly enhanced the repopulating capacity of

). 相比之下，IFNβ是一种通常由激活的STING通路诱导的细胞因子，显著增强了

Dnmt3a; Sting

Dnmt3a；Sting

-DKO cells (Fig.

-DKO细胞（图。

). This suggests that the increased repopulating capacity of

）。这表明增加的再生能力

Dnmt3a

-deficient LSK cells requires the

缺陷型LSK细胞需要

Sting

刺痛

-dependent inflammatory response. We then evaluated the impact of deleting

依赖性炎症反应。然后我们评估了删除的影响

Sting

刺痛

on hematopoietic differentiation in

造血分化过程中

Dnmt3a

-KO mice. To avoid aging-induced inflammatory responses, such as tumor necrosis factor alpha (TNFα) signaling [

-KO小鼠。为避免衰老诱导的炎症反应，如肿瘤坏死因子α（TNFα）信号传导[

二十三

], and the increased risk of acquiring other genetic lesions mediated by

], 以及由以下因素引起的获得其他遗传损伤的风险增加：

Dnmt3a

deficiency, we conducted the analysis when the mice were 24 weeks old, with

缺陷时，我们对24周龄的小鼠进行了分析，

Dnmt3a

knockout occurring after 8 weeks. Loss of

在8周后发生击倒。失去

Dnmt3a

led to a 2-fold increase in the population of LSK cells compared to WT or

与野生型相比，导致LSK细胞群体增加了2倍

Sting

刺痛

-KO mice, while

-KO小鼠，而

Dnmt3a; Sting

Dnmt3a；Sting

-DKO mice showed a comparable population of LSK cells to that of WT mice (Fig.

-DKO小鼠显示出与WT小鼠相当的LSK细胞群体（图。

). The populations of downstream hematopoietic lineage cells showed no differences among the four genotypes (Fig.

）。下游造血谱系细胞的群体在四种基因型中没有显示出差异（图。

1天

), suggesting that the effect of

），这表明了

Dnmt3a

deficiency was limited to HSPCs at this time point. To verify this, we performed Ki67 staining to assess the proliferation index of HSPCs. The proportions of Ki67

缺陷仅限于此时点的造血干细胞和祖细胞（HSPCs）。为验证这一点，我们进行了Ki67染色以评估HSPCs的增殖指数。Ki67的比例

加号

cells in

细胞在

Dnmt3a

-KO HSPCs, including LSK cells, long-term HSCs (LT-HSCs), and short-term HSCs (ST-HSCs), were twice as high as those in

-KO HSPCs，包括LSK细胞、长期HSCs（LT-HSCs）和短期HSCs（ST-HSCs），是其两倍高

Dnmt3a; Sting

Dnmt3a；Sting

-DKO, WT, and

-DKO、WT 和

Sting

刺痛

-KO cells (Fig.

-KO细胞（图。

). This suggests that the increased proliferation of HSPCs due to

）。这表明HSPCs的增殖增加是由于

Dnmt3a

deficiency requires STING.

缺陷需要STING。

To assess the cell intrinsic effects of deleting STING in promoting

为了评估删除STING在促进中的细胞内在效应

Dnmt3a

deficiency-mediated CH, we conducted transplantation assays (Fig.

我们进行了移植实验（图。

). 20 weeks post-transplantation, recipients with

移植后 20 周，接受者

Dnmt3a

-KO BM cells exhibited a 2-fold increase in spleen weight compared to recipients transplanted with WT,

与移植了WT的受体相比，KO BM细胞的脾脏重量增加了2倍。

Sting

刺痛

-KO, or

-击倒，或者

Dnmt3a

;

；

Sting

刺痛

-DKO BM cells, with the latter three showing comparable spleen weights (Fig.

-DKO BM细胞，后三者显示出相似的脾脏重量（图。

). Additionally, the loss of

）。此外，失去

Dnmt3a

significantly increased the proportion of myeloid cells in the peripheral blood during the assay duration, while deletion of

在实验期间显著增加了外周血中髓系细胞的比例，而删除

Sting

刺痛

in the context of

在……的背景下

Dnmt3a

deficiency alleviated myeloid cell expansion in the recipients with comparable levels to those in WT and

缺陷减轻了受体中髓样细胞的扩增，其水平与野生型相当。

Sting

刺痛

-KO recipients (Fig.

-KO受体（图。

C, D). We next examined the HSPC populations in the BM of the different recipient mice. Consistent with previous work, deletion of

C、D）。我们接下来检查了不同受体小鼠骨髓中的HSPC群体。与之前的研究一致，删除

Dnmt3a

resulted in an expansion of the stem cell pool [

导致干细胞池的扩增 [

], as evidenced by increased populations of LSK cells, ST-HSCs, and LT-HSCs when compared with WT recipients. However, these expansions were significantly mitigated when combined with the deletion of

], 与野生型受体相比，LSK细胞、ST-HSCs和LT-HSCs的数量增加证明了这一点。然而，当与删除结合时，这些扩增显著减轻。

Sting

刺痛

(Fig.

（图。

). The populations of LSK cells and LT-HSCs in recipients with

）。接受者体内LSK细胞和LT-HSCs的数量

Dnmt3a

;

；

Sting

刺痛

-DKO BM were comparable to those from WT recipients. Only a slight increase in ST-HSCs was observed in the

-DKO BM 与来自 WT 受体的相似。仅观察到 ST-HSCs 有轻微增加。

Dnmt3a

;

；

Sting

刺痛

-DKO recipients compared to WT recipients, whereas deletion of

与WT受体相比，DKO受体，而删除

Dnmt3a

alone resulted in a 3-fold increase in this cell population (Fig.

单独导致该细胞群体增加了3倍（图。

). We also examined the populations of lineage progenitor cells in these mice and found a significant increase in GMP cells in

）。我们还检查了这些小鼠中谱系祖细胞的群体，发现GMP细胞显著增加。

Dnmt3a

-KO recipients. Similarly, this effect was reversed by the deletion of

-KO受体。同样，这种效应通过删除得以逆转。

Sting

刺痛

(Fig.

（图。

, bottom panel).

，底部面板）。

Fig. 2: Deletion of

图2：删除

Sting

刺痛

inhibits the development of CH associated with

抑制了与CH相关的发展

Dnmt3a

deficiency in transplantation models.

移植模型中的缺陷。

Diagram of the whole bone marrow transplantation assay.

整个骨髓移植试验的示意图。

Loss of

丢失

Sting

刺痛

alleviated the splenomegaly in recipients of

缓解了接受者的脾肿大症状

Dnmt3a

-deficient donor bone marrow. The weight of spleens in recipient mice was measured 20 weeks after transplantation. Quantitative data are shown on the right (

-缺陷供体骨髓。移植后20周测量受体小鼠脾脏重量。定量数据如右图所示（

= 8).

The plot graph shows the frequency of myeloid cells in the peripheral blood of recipient mice over the course of the transplantation assay (

图表显示了在移植实验过程中受体小鼠外周血中髓系细胞的频率 (

= 11).

Deletion of

删除

Sting

刺痛

inhibited the myeloid differentiation bias mediated by

抑制了由...介导的髓系分化偏差

Dnmt3a

deficiency. The proportions of myeloid and lymphoid cells in peripheral blood were measured by FACS 20 weeks post-transplantation (

缺陷。移植后20周，通过FACS测量外周血中髓系和淋巴系细胞的比例（

= 8).

Deletion of

删除

Sting

刺痛

restrained the self-renewal ability of

限制了自我更新能力

Dnmt3a

-deficient HSPCs. The proportions of LSK cells, LT-HSCs, ST-HSCs, CMPs, GMPs, and MEPs were measured by FACS. Gating plots are shown on the left, and quantitative data are shown on the right (

缺陷型HSPCs。通过FACS测量LSK细胞、LT-HSCs、ST-HSCs、CMPs、GMPs和MEPs的比例。门控图显示在左侧，定量数据显示在右侧 (

= 8). Data are presented as mean ± s.e.m., with *

= 8）。数据以平均值 ± 标准误表示，*

< 0.05, **

< 0.01, ***

< 0.005, ****

< 0.0001, and “ns” not significant.

小于0.0001，且“ns”表示不显著。

Full size image

全尺寸图像

One hallmark of CH is the increased self-renewal activity of HSCs, which leads to an expansion of lineage cells in the hematopoietic system when competing with WT HSCs. To further validate the role of STING in

CH的一个标志是HSCs自我更新活性的增加，这会导致在与WT HSCs竞争时造血系统中谱系细胞的扩增。为进一步验证STING的作用，

Dnmt3a

-associated CH, we conducted a competitive transplantation assay (Fig.

相关CH，我们进行了一项竞争性移植试验（图。

). We generated

）。我们生成了

Vav-Cre

mediated

调解的

Dnmt3a

conditional knockout mice to avoid the acute inflammatory response associated with

条件性基因敲除小鼠以避免与之相关的急性炎症反应

Mx1-Cre

induction in this assay (hereafter named

在该试验中的诱导（以下称为

Dnmt3a

-KO

-故障

Vav

). To explore the therapeutic potential of targeting STING in vivo, we employed the STING inhibitor C-176. Consistent with data from the whole BM transplantation assay,

为了探索在体内靶向STING的治疗潜力，我们采用了STING抑制剂C-176。与全骨髓移植实验的数据一致，

Dnmt3a

-KO

-击倒

Vav

cells in recipients treated with DMSO displayed a significant competitive advantage in the PB, and recipients had enlarged spleens compared to WT recipients. In contrast, inhibition of STING with C-176 reduced the CD45.2 population to 25% and halved the spleen weight compared to

用DMSO处理的受体细胞在PB中显示出显著的竞争优势，且受体的脾脏比野生型受体更大。相反，使用C-176抑制STING后，CD45.2群体降至25%，脾脏重量比对照组减少了一半。

Dnmt3a

-KO

-故障输出

Vav

recipients treated with DMSO (Fig.

用DMSO处理的受体（图。

3B, C

3B，C

).Twelve weeks post-transplantation, the myeloid cell population in the

移植后十二周，髓系细胞群体在

Dnmt3a

-KO

Vav

recipients in the DMSO group was 3-fold higher than those in the WT or the

DMSO组的受体是WT组或

Dnmt3a

-KO

-故障

Vav

recipients treated with C-176, and C-176 injection showed no effects on T/B cell differentiation in either the WT or

接受C-176治疗的受体，以及C-176注射对WT或T/B细胞分化均无影响。

Dnmt3a

-KO

-击倒

Vav

groups (Fig.

组（图。

). The increased populations of LSK cells and LT-HSCs in

）。LSK细胞和LT-HSCs的数量增加在

Dnmt3a

-KO

-故障

Vav

cell recipients also returned to levels comparable to WT recipients upon inhibition of STING (Fig.

细胞接受者在抑制STING后也恢复到与WT接受者相当的水平（图。

3E, F

3E，F

). Moreover, the loss of

). 此外，损失了

Dnmt3a

specifically resulted in the expansion of multipotent progenitor cell 2 (MPP2) and CMP cells, while inhibition of STING with C-176 mitigated this effect. Recipients with

特别导致了多能祖细胞2（MPP2）和CMP细胞的扩增，而使用C-176抑制STING则减轻了这种效果。接受者 với

Dnmt3a

-KO

-击倒

Vav

cells exhibited similar populations of MPP3, MPP4, and GMP cells compared to WT controls and showed an increase in MEP cells, which was not significantly reduced by the inhibition of STING, potentially due to the time constraints of this assay (Fig.

细胞显示出与野生型对照相似的MPP3、MPP4和GMP细胞群体，并且MEP细胞增加，这种增加并未因STING的抑制而显著减少，可能是由于该实验的时间限制所致（图。

3楼

). Taken together, these data suggest that the activation of STING plays a crucial role in mediating the development of

). 综合来看，这些数据表明STING的激活在介导发展过程中起着至关重要的作用。

Dnmt3a

-associated CH. Furthermore, targeting STING is an effective way to prevent a series of hematopoietic disorders induced by

相关的CH。此外，针对STING是预防由其引起的一系列造血障碍的有效方法

Dnmt3a

deficiency in vivo.

体内缺乏。

Fig. 3: Inhibition of

图3：抑制

Sting

刺痛

reduces the self-renewal ability of

减少了自我更新能力

Dnmt3a

-deficient HSPCs.

-缺陷的HSPCs。

Diagram of competitive transplantation assay.

竞争性移植试验的示意图。

Injection of the STING inhibitor C-176 impaired the competitive advantage of

注射STING抑制剂C-176削弱了竞争优势

Dnmt3a

-deficient donors over the course of the competitive assay (

- 在竞争性实验过程中，缺乏供体 (

= 8).

Inhibition of STING alleviated the splenomegaly mediated by

抑制STING减轻了由其介导的脾肿大

Dnmt3a

deficiency (

不足（

= 5).

= 5)。

The bar graph shows the proportions of myeloid and lymphoid cells in the recipients (

条形图显示了受试者体内髓系细胞和淋巴系细胞的比例（

= 5).

= 5)。

C-176 treatment reduced the proportion of

C-176处理减少了比例

Dnmt3a

-deficient LSK cells in the recipients (

-受体中缺乏LSK细胞 (

= 5).

The bar graph shows the frequencies of LT-HSCs, ST-HSCs, CMPs, GMPs, MEPs and MPP2/3/4 across different groups of recipient mice (

条形图显示了LT-HSC、ST-HSC、CMP、GMP、MEP和MPP2/3/4在不同组受体小鼠中的频率（

= 5). Data are presented as mean ± s.e.m., with *

= 5)。数据以平均值 ± 标准误表示，*

< 0.05, **

< 0.01, ***

< 0.005 and “ns” not significant.

小于0.005，且“ns”表示不显著。

Full size image

全尺寸图像

Hypomethylation of ERVs in the genome leads to the activation of STING in

基因组中ERVs的低甲基化导致STING的激活

Dnmt3a

-KO HSPCs

-KO 造血干细胞

We next investigated how the STING pathway was activated in

我们接下来研究了STING通路是如何被激活的

Dnmt3a

-KO

-故障输出

Vav

HSPCs. One possible mechanism is DNA damage, which could cause increase micronuclei (MN) formation and the releasement of genomic DNA into the cytoplasm. To test this hypothesis, we assessed the DNA damage level in WT and

造血干细胞。一个可能的机制是DNA损伤，这可能导致微核（MN）形成增加，并将基因组DNA释放到细胞质中。为了验证这一假设，我们评估了WT和

Dnmt3a

-KO

-故障

Vav

BM cells. There were no detectable γH2AX signals in either

BM细胞。在两者中均未检测到γH2AX信号

Dnmt3a

-deficient c-Kit

缺乏的c-Kit

加号

or c-Kit

或 c-Kit

cells (Supplementary Fig.

细胞（补充图）。

). Additionally,

). 此外，

Dnmt3a

-KO

-击倒

Vav

Lineage

血统

c-Kit

加号

(LK) cells showed no differences from WT LK cells in the comet assay (Supplementary Fig.

(LK) 细胞在彗星实验中与 WT LK 细胞没有差异（补充图）。

), which is commonly used to measure overall DNA damage levels. We also counted the number of MN in both WT and

)，常用于衡量整体DNA损伤水平。我们还计数了WT和

Dnmt3a

-KO

-击倒

Vav

LSK cells and observed no significant changes between the two cell populations (Supplementary Fig.

LSK细胞，并观察到两个细胞群体之间没有显著变化（补充图）。

). These findings suggest that loss of

）。这些发现表明丧失了

Dnmt3a

does not trigger an autonomous DNA damage response in HSPCs. Recently, a study reported that mutations in

不会在造血干细胞中触发自主的DNA损伤反应。最近，一项研究报道了

DNMT3A

result in mitochondrial DNA (mtDNA) release and activation of the STING pathway in macrophages [

导致线粒体DNA（mtDNA）释放并激活巨噬细胞中的STING通路[

]. We speculated that this mechanism might also operate in HSPCs. Using the same approach, we evaluated mtDNA release in

]. 我们推测这一机制可能也在HSPCs中起作用。使用相同的方法，我们评估了线粒体DNA的释放。

Dnmt3a

-KO

-故障

Vav

c-Kit

加号

cells. However, we observed no increase in mtDNA levels in

细胞。然而，我们没有观察到mtDNA水平的增加在

Dnmt3a

-KO

-击倒

Vav

c-Kit

加号

cells compared to WT cells (Supplementary Fig.

与WT细胞相比（补充图）。

）。

The reverse-transcribed cDNA of ERVs acts as another stimulus of the cGAS-STING pathway [

ERVs的反转录cDNA充当cGAS-STING通路的另一个刺激物[

]. To explore this possibility, we first examined the expression of several transposable elements in

]. 为了探讨这种可能性，我们首先检查了几个转座元件的表达情况。

Dnmt3a

-KO and WT HSPC lineages. Specifically, we observed that the expression of ERVs, which belong to the group of long terminal repeat (LTR) retrotransposons [

-KO和WT HSPC谱系。具体来说，我们观察到属于长末端重复（LTR）逆转录转座子组的ERVs的表达[

], was upregulated in

], 在...中上调

Dnmt3a

-KO HSPCs (Fig.

-KO HSPCs（图。

). Typically, the expression of ERVs is silenced by host surveillance mechanisms, such as DNA methylation [

）。通常，内源性逆转录病毒（ERVs）的表达会被宿主监控机制（如DNA甲基化）所沉默。

]. We quantified the level of methylated cytosine in

]. 我们量化了甲基化胞嘧啶的水平在

Dnmt3a-

KO c-Kit

敲除c-Kit

加号

cells using liquid chromatography–mass spectrometry (LC-MS). Indeed, the total methylated cytosine level, including hydroxy-methyl cytosine levels in the genome of

细胞使用液相色谱-质谱联用技术（LC-MS）。事实上，包括基因组中羟甲基胞嘧啶水平在内的总甲基化胞嘧啶水平，

Dnmt3a

-KO

Vav

c-Kit

加号

cells was significantly reduced compared to WT cells (Supplementary Fig.

细胞显著减少，与WT细胞相比（补充图）。

). We then analyzed the whole-genome bisulfite sequencing data of HSCs from previously published data [

）。然后，我们从之前发表的数据中分析了HSCs的全基因组亚硫酸氢盐测序数据 [

，

], finding that the average methylation level of ERV genic regions was significantly reduced in

]，发现ERV基因区域的平均甲基化水平显著降低在

Dnmt3a-

KO HSCs (Fig.

敲除HSCs（图。

）。

Fig. 4:

图4：

Dnmt3a

deficiency induces the expression of ERVs in HSPCs.

缺陷诱导HSPCs中ERVs的表达。

Pie charts display the percentages of upregulated repetitive elements in each class (top), and a heatmap shows the relative expression levels of the top 7 upregulated repetitive elements (bottom) across different

饼图显示了每个类别中上调的重复元件的百分比（顶部），而热图则显示了前7个上调的重复元件在不同条件下的相对表达水平（底部）。

Dnmt3a

knockout HSPC lineages.

敲除HSPC谱系。

Deletion of

删除

Dnmt3a

leads to hypomethylation of ERVs. The profile of average CpG methylation levels is displayed across regions including ERVs and their 2-kb flanking areas. The methylation levels of various ERV subfamilies are illustrated at the bottom.

导致ERVs的低甲基化。平均CpG甲基化水平的分布图展示了包括ERVs及其2kb侧翼区域在内的区域。底部说明了各种ERV亚家族的甲基化水平。

The upregulation of ERVs in

内源性逆转录病毒（ERVs）的上调

Dnmt3a

-deficient c-Kit

缺乏的c-Kit

加号

cells was measured using bulk RNA-seq (left) and the specific transcripts (right, top panel) and cDNA (right, bottom panel) of ERVs in c-Kit

使用批量RNA-seq测量细胞（左图），以及c-Kit中ERVs的特定转录本（右图，上方面板）和cDNA（右图，下方面板）。

加号

cells were quantified using q-PCR. A scatter plot illustrates the differentially expressed genes (circles) and transposable elements (triangles) between

使用q-PCR对细胞进行了量化。散点图展示了差异表达基因（圆圈）和转座元件（三角形）之间的关系。

Dnmt3a

-KO

-故障

Vav

and WT c-Kit

和野生型c-Kit

加号

cells, with log

细胞，带有日志

(fold change) >1 and

（倍数变化）>1 且

< 0.05 (

= 3).

Dnmt3a

deficiency results in the hypomethylation of genic ERV loci in c-Kit

缺陷导致c-Kit中基因ERV位点的低甲基化

加号

cells.

细胞。

FACS analysis showing the populations of specific cell types in the bone marrow of RTi-treated recipient mice (

FACS分析显示了RTi处理的受体小鼠骨髓中特定细胞类型的人群 (

= 5). Data are presented as mean ± s.e.m., with **

= 5）。数据以平均值 ± 标准误表示，**

< 0.01, ***

< 0.005 and “ns” not significant.

< 0.005，且“ns”表示不显著。

Full size image

全尺寸图像

To explore the overall expression profiles of ERVs and genes, we analyzed our RNA-seq data performed with c-Kit

为了探索 ERVs 和基因的整体表达谱，我们分析了使用 c-Kit 进行的 RNA-seq 数据。

加号

cells from

细胞来自

Dnmt3a

-KO

-击倒

Vav

and WT mice. We identified several subsets of ERVs, such ERV1 and ERVK, that were upregulated in

和WT小鼠。我们鉴定出几个ERVs亚群，如ERV1和ERVK，在

Dnmt3a

-KO

-击倒

Vav

c-Kit

加号

cells and confirmed the upregulation of various ERVs in c-Kit

细胞并证实了 c-Kit 中多种 ERVs 的上调表达

加号

cells, both in the mRNA level and in the cDNA level by q-PCR (Fig.

细胞，在mRNA水平和cDNA水平通过q-PCR（图）。

). And this upregulation of ERVs‘ transcribes was also detected in the

）。并且在

Dnmt3a

-KO

-故障输出

Vav

LSK cells (Supplementary Fig.

LSK细胞（补充图。

). Additionally, the genic methylation levels of the upregulated ERVs, such as MMVL30, MMLV, and MuSD, were also reduced in

）。此外，上调的ERVs（如MMVL30、MMLV和MuSD）的基因甲基化水平也降低了。

Dnmt3a

-KO

-故障

Vav

c-Kit

加号

cells (Fig.

细胞（图。

). To further validate the link between genome hypomethylation, ERV expression, and inflammation induction, we treated WT c-Kit

为了进一步验证基因组低甲基化、ERV表达和炎症诱导之间的联系，我们处理了WT c-Kit

加号

cells with decitabine, a hypomethylation agent. As expected, decitabine treatment induced the upregulation of similar subsets of ERVs as observed in

用低甲基化剂地西他滨处理细胞。正如预期的那样，地西他滨处理诱导了与观察到的相似的内源性逆转录病毒（ERVs）亚群的上调。

Dnmt3a-

韩国语

Vav

cells and significantly induced inflammatory cytokines (Supplementary Fig.

细胞并显著诱导炎症细胞因子（补充图）。

). The hypomethylation of the genome and ERV loci in decitabine-treated c-Kit

`). 基因组和ERV位点在地西他滨处理的c-Kit中的低甲基化`

加号

cells was confirmed through LC-MS and bisulfite sequencing (Supplementary Fig.

通过LC-MS和亚硫酸盐测序确认了细胞（补充图）。

). Additionally, as a biomarker of STING pathway activation, the phosphorylation of TBK1 and STING was observed in the decitabine-treated cells (Supplementary Fig.

此外，作为STING通路激活的生物标志物，在地西他滨处理的细胞中观察到TBK1和STING的磷酸化（补充图）。

). Collectively, these data suggesting that ERV activation was due to the hypomethylation.

这些数据共同表明，ERV的激活是由于低甲基化引起的。

The accumulation of ERV transcripts in the cytoplasm can directly activate the viral RNA-sensing pathway and trigger downstream inflammatory responses [

ERV转录物在细胞质中的积累可以直接激活病毒RNA感知通路并触发下游炎症反应[

]. To address this, we silenced the expression of

]. 为了解决这个问题，我们抑制了

Mavs

小牛队

[

], a key adaptor mediating the RNA innate immunity response, in

]，一种介导RNA先天免疫反应的关键适配器，在

Dnmt3a

-KO

-故障

Vav

c-Kit

加号

cells and conducted a serial CFU assay to test the repopulating capacity of these cells. The knockdown of

细胞并进行了连续的CFU测定，以测试这些细胞的再填充能力。削减

Mavs

小牛队

showed no inhibitory effects on the repopulating capacity of

对再增殖能力没有显示出抑制作用

Dnmt3a

-KO

-击倒

Vav

cells (Supplementary Fig.

细胞（补充图）。

), suggesting that the viral RNA-sensing pathway is not responsible for the phenotypes associated with

），这表明病毒RNA感知通路不是与相关表型的原因。

Dnmt3a

deficiency. However, when we used a reverse transcriptase inhibitor to prevent the synthesis of ERV cDNA in

不足。然而，当我们使用逆转录酶抑制剂来阻止ERV cDNA的合成时，

Dnmt3a

-KO

-故障输出

Vav

cells, the repopulating capacity of these cells was significantly impaired (Supplementary Fig.

细胞，这些细胞的再增殖能力显著受损（补充图）。

). This finding is consistent with the previous reports that reverse-transcribed ERV cDNA acts as an inducer of the STING pathway [

). 这一发现与之前的报告一致，即逆转录的ERV cDNA充当STING通路的诱导剂 [

，

]. We next evaluated the effects of RTi treatment in vivo. Recipient mice transplanted with WT or

]. 随后，我们评估了RTi治疗在体内的效果。接受WT或移植的小鼠受体

Dnmt3a

-KO

-故障输出

Vav

BM cells were administered RTi by oral gavage every other day for one month. This treatment resulted in a significant increase in the LT-HSC population in

BM细胞每隔一天通过口服灌胃给予RTi，持续一个月。这种处理导致LT-HSC群体显著增加。

Dnmt3a

-KO

-击倒

Vav

recipients compared to WT recipients. However, the ST-HSC population in DMSO-treated

与WT受体相比，受体。然而，DMSO处理的ST-HSC群体

Dnmt3a

-KO

-故障

Vav

recipients was four times higher than that in both WT recipients and RTi-treated

受体数量是野生型受体和RTi处理组的四倍。

Dnmt3a

-KO

-故障输出

Vav

recipients (Fig.

收件人（图。

). For progenitor cells, RTi treatment reduced the expansion of CMP and CLP populations in

）。对于祖细胞，RTi处理减少了CMP和CLP群体的扩增。

Dnmt3a

-KO

-击倒

Vav

recipients, bringing them to levels comparable to those observed in WT recipients. Additionally, a slight increase in the proportion of MEPs was noted in RTi-treated

受体，使它们达到与WT受体中观察到的水平相当的水平。此外，在RTi处理的受体中注意到MEPs比例略有增加。

Dnmt3a

-KO

-故障输出

Vav

recipients compared to the DMSO-treated group (Fig.

与DMSO处理组相比的受体（图。

). Collectively, these results indicate that

). 总体而言，这些结果表明

Dnmt3a

deficiency results in the hypomethylation of ERV genic loci, and the reverse-transcribed cDNA of ERVs triggers the activation of the cGAS-STING pathway in

缺乏会导致ERV基因位点的低甲基化，并且ERV的反转录cDNA会触发cGAS-STING通路的激活。

Dnmt3a

-KO HSPCs, ultimately driving lineage differentiation bias in mice.

-KO HSPCs，最终导致小鼠的谱系分化偏差。

Inhibiting STING delays the progression of leukemia associated with DNMT3A mutations

抑制STING可延缓与DNMT3A突变相关的白血病进展

To assess whether STING can be targeted to prevent the evolution of leukemia, we generated a transgenic mouse model,

为了评估STING是否可以被靶向以防止白血病的演变，我们生成了一个转基因小鼠模型，

Dnmt3a

f/+

, FLT3

，FLT3

ITD/+

，

Mx1-Cre

, to replicate a common mutation combination seen in AML patients. To validate the effects of targeting STING in this model, we used AC220, an FDA-approved drug currently used to treat leukemia with FLT3-ITD rearrangement, as a benchmark for evaluation (Fig.

，以复制在AML患者中常见的突变组合。为了验证在此模型中靶向STING的效果，我们使用了AC220（一种FDA批准的药物，目前用于治疗具有FLT3-ITD重排的白血病）作为评估基准（图。

). In the transplantation models, recipients treated with AC220, C-176, or a combination of both exhibited significant reductions in the populations of CD45.2

）。在移植模型中，接受AC220、C-176或两者联合治疗的受体表现出CD45.2群体的显著减少。

加号

cells in the PB during the assay period (Fig.

在测定期间PB中的细胞（图。

). Inhibition of STING decreased the proportion of c-Kit

）。抑制STING降低了c-Kit的比例

加号

cells in the PB, whereas AC220 alone had no effect on this parameter. Interestingly, the pattern of inhibition was reversed for the proportions of myeloid cells, with AC220 showing significant inhibitory effects, whereas C-176 had no impact on these cells. Moreover, the combination of these two agents demonstrated a synergistic effect, reducing the expansion of both c-Kit.

PB中的细胞，而单独使用AC220对此参数没有影响。有趣的是，对于髓样细胞的比例，抑制模式发生了逆转，AC220显示出显著的抑制作用，而C-176对这些细胞没有影响。此外，这两种药物的组合表现出协同效应，减少了c-Kit的扩增。

加号

and myeloid cells in the PB by about 50% and 10%, respectively, compared to DMSO (Fig.

与DMSO相比，PB中的髓系细胞分别减少了约50%和10%（图。

). In the BM, C-176 and AC220 showed comparable reductions in the proportions of c-Kit

). 在BM中，C-176和AC220显示出相当的c-Kit比例减少。

加号

cells, LSK cells, and LT-HSCs, and neither had any effect on the population of ST-HSCs. The combination of these two drugs exhibited a synergistic effect on these cell populations, especially on ST-HSCs, showing a nearly 60% decrease compared to DMSO and a 40% decrease compared to either C-176 or AC220 alone (Fig. .

细胞、LSK细胞和LT-HSCs，且对ST-HSCs群体没有任何影响。这两种药物的组合对这些细胞群体表现出协同效应，尤其是对ST-HSCs，与DMSO相比减少了近60%，与单独使用C-176或AC220相比减少了40%（图。

). Neither drug had an effect on the populations of MPP2 and MPP3, yet they both demonstrated a 50% inhibition efficiency on the proportion of MPP4 (Fig.

). 两种药物均未对MPP2和MPP3的群体产生影响，但它们都对MPP4的比例表现出50%的抑制效率（图。

). For progenitor cells, targeting STING yielded better inhibitory effects compared to AC220; the proportion of CMP cells was reduced by about 30% with STING targeting, but increased when treated with AC220. Both drugs reduced the population of GMP cells, and a cooperative effect was observed when they were used in combination (Fig. .

). 对于祖细胞，靶向STING相比AC220产生了更好的抑制效果；通过靶向STING，CMP细胞的比例减少了约30%，但在使用AC220处理时反而增加。两种药物均减少了GMP细胞的数量，并且在联合使用时观察到了协同效应（图.

五楼

). Collectively, these data indicate that the primary effect of targeting STING appears to be on the expansion of HSPCs rather than on the proliferation of leukemia blasts.

). 总体而言，这些数据表明靶向STING的主要作用似乎在于HSPCs的扩增，而非白血病母细胞的增殖。

Fig. 5: Inhibition of

图5：抑制作用

STING

刺痛

restrains the leukemogenesis of mouse leukemia cells associated with

抑制与小鼠白血病细胞相关的白血病发生

Dnmt3a

insufficiency.

不足。

Diagram of comparing the biological activity of C-176 and FLT3 inhibitor AC220.

比较C-176和FLT3抑制剂AC220生物活性的图表。

Injection of either C-176 or AC220 inhibited the myeloid differentiation bias of

注射C-176或AC220抑制了髓系分化偏向

Dnmt3a

-deficient leukemia cells over the course of the transplantation assay (

- 缺陷型白血病细胞在移植实验过程中 (

= 9).

Inhibition of

抑制

STING

刺痛

restrained the expansion of naïve hematopoietic cells in the peripheral blood of recipient mice (

抑制了受体小鼠外周血中未成熟造血细胞的扩增 (

= 8).

C-176 and AC220 showed a combined effect in inhibiting the expansion of

C-176 和 AC220 在抑制扩张方面显示出联合效应

Dnmt3a

+/−

;

；

Flt3

ITD/+

leukemia stem cells (

白血病干细胞 (

= 8).

C-176 and AC220 specifically inhibited the expansion of MPP4 cells (

C-176 和 AC220 特异性地抑制了 MPP4 细胞的扩增 (

= 8).

= 8）。

C-176 and AC220 treatment reduced the proportions of CMP and GMP cells in

C-176 和 AC220 治疗减少了 CMP 和 GMP 细胞的比例。

Dnmt3a

+/−

;

；

Flt3

ITD/+

mice (

鼠标（

= 8). Data are presented as mean ± s.e.m., with *

= 8）。数据以平均值 ± 标准误表示，*

< 0.05, **

< 0.01, ***

< 0.005 and “ns” not significant.

< 0.005，且“ns”表示不显著。

Full size image

全尺寸图像

We further assessed the effects of targeting STING on a patient sample with DNMT3A R882H and FLT3-ITD mutations. Inhibition of STING effectively restricted the proliferation of these leukemia cells in culture, showing a comparable inhibitory effect to AC220 treatment, while both chemicals showing no effects on the proliferation cord blood CD34.

我们进一步评估了靶向STING在携带DNMT3A R882H和FLT3-ITD突变的患者样本中的效果。抑制STING有效地限制了这些白血病细胞在培养中的增殖，显示出与AC220处理相当的抑制效果，同时这两种化合物对脐带血CD34的增殖均无影响。

cells (Fig.

细胞（图。

). We then used this patient sample to establish a patient-derived xenograft (PDX) model in NOD/SCID/γ-chain knockout (NSG) mice, to evaluate the in vivo effects of STING inhibition. Treatment with H-151, a STING inhibitor which targets human STING, led to a threefold reduction in the expansion of human leukemia cells with .

）。然后，我们使用该患者样本在NOD/SCID/γ链基因敲除（NSG）小鼠中建立了一个患者来源的异种移植（PDX）模型，以评估STING抑制的体内效果。使用针对人类STING的STING抑制剂H-151进行治疗，使得人类白血病细胞的扩增减少了三倍，且。

DNMT3A

mutation in the PB during the transplantation assays (Fig.

在移植试验中PB的突变（图。

). Six weeks after initiating H-151 treatment, the proportion of leukemia cells in the PB decreased by fourfold compared to the DMSO group, while no significant changes were observed in the proportion of immature myeloid cells between the H-151 and DMSO groups (Fig.

). 在开始H-151治疗六周后，外周血（PB）中白血病细胞的比例比DMSO组减少了四倍，而H-151组和DMSO组之间未成熟髓样细胞的比例没有显著变化（图。

). In the spleen and bone marrow, H-151 treatment reduced the proportion of leukemia stem cells threefold compared to the DMSO group, with no effect on the population of blast cells (Fig.

）。在脾脏和骨髓中，与DMSO组相比，H-151处理使白血病干细胞的比例减少了三倍，而对原始细胞群体没有影响（图。

6D, E

6D，E

). These results further underscore that inhibiting STING primarily delays leukemia development by restraining the expansion of stem cells. Furthermore, H-151 treatment significantly extended the survival of mice harboring DNMT3A-mutated leukemia cells. In contrast, no increase in lifespan was observed in mice with DNMT3A wild-type leukemia cells treated with H-151.

这些结果进一步强调了抑制STING主要通过限制干细胞的扩增来延缓白血病的发展。此外，H-151治疗显著延长了携带DNMT3A突变白血病细胞的小鼠的生存期。相反，在接受H-151治疗的DNMT3A野生型白血病细胞小鼠中，未观察到寿命的延长。

(Fig. .

（图。

6楼

). Collectively, these findings suggest that STING is a viable therapeutic target in DNMT3A-mutated hematopoietic disorders.

这些发现共同表明，STING 是 DNMT3A 突变型造血障碍中一个可行的治疗靶点。

Fig. 6: Targeting STING inhibits leukemia development in the PDX model.

图6：在PDX模型中，靶向STING可抑制白血病的发展。

The effects of H-151 and AC220 treatments on patient-derived leukemia cells (left) and cord blood CD34

H-151 和 AC220 治疗对患者来源的白血病细胞（左）和脐带血 CD34 的影响

加号

cells (right) were evaluated in vitro using the CCK8 assay.

细胞（右）使用CCK8测定法在体外进行评估。

Inhibition of STING restrains the expansion of DNMT3A-mutated leukemia cells in NSG mice (

抑制STING可限制DNMT3A突变的白血病细胞在NSG小鼠中的扩增 (

= 3 biological replicates). The DNMT3A-mutated patient sample harbors DNMT3A R882H and FLT3-ITD mutations, whereas the DNMT3A-WT sample carries CEBPA c.295_300del and TP53 P72R homozygous mutations.

= 3个生物学重复）。DNMT3A突变的患者样本携带DNMT3A R882H和FLT3-ITD突变，而DNMT3A-WT样本携带CEBPA c.295_300del和TP53 P72R纯合突变。

Bar graphs display the proportions of human leukemia cells (hCD45

条形图显示了人类白血病细胞的比例 (hCD45

加号

) and immature myeloid cells (hCD45

）和未成熟的髓样细胞（hCD45

加号

hCD34

人CD34

hCD33

人源CD33

加号

) (

= 3 biological replicates). Targeting STING diminishes the proportions of leukemia stem cells in the spleen (

= 3个生物学重复）。靶向STING可减少脾脏中白血病干细胞的比例（

) and bone marrow (

）和骨髓（

) of recipient mice. The proportions of human leukemia cells, blast cells (hCD45

)受体小鼠。人类白血病细胞、原始细胞（hCD45

加号

hCD34

人CD34

加号

hCD38

人CD38

加号

), and leukemia stem cells (hCD45

），以及白血病干细胞（hCD45

加号

hCD34

人CD34

加号

hCD38

人源CD38

) were analyzed via FACS (

通过FACS分析了（

= 3 biological replicates).

= 3个生物学重复）。

Survival analysis of recipient mice treated with DMSO or H-151, presented as a Kaplan–Meier curve (

接受DMSO或H-151治疗的受体小鼠的生存分析，以Kaplan-Meier曲线表示（

= 3 with DMSO, 6 with H-151). Statistical significance was assessed by t-test (

= 3使用DMSO，6使用H-151）。通过t检验评估统计显著性（

–

). Data are mean ± s.e.m., *

）。数据为平均值±标准误，*

< 0.05; **

< 0.05；**

< 0.01.

小于0.01。

Full size image

全尺寸图像

Discussion

讨论

As a major driver mutation in CH and hematopoietic malignancies,

作为 CH 和造血系统恶性肿瘤中的主要驱动突变，

DNMT3A

mutations contribute to nearly 40% of genetic events in these diseases. Therefore, identifying the factors and pathways involved in the development of hematopoietic disorders associated with

突变在这些疾病中贡献了近40%的遗传事件。因此，确定与造血功能障碍发展相关的因素和通路

DNMT3A

mutations is crucial for developing clinical strategies to improve patient outcomes. In this study, we demonstrated that

突变对于制定改善患者预后的临床策略至关重要。在这项研究中，我们证明了

Dnmt3a

-deficient HSPCs exhibit autonomously activated STING pathways and chronic inflammation. This inflammatory state promotes the expansion and skewed differentiation of HSPC populations associated with

-缺乏的HSPCs表现出自主激活的STING通路和慢性炎症。这种炎症状态促进了与之相关的HSPC群体的扩增和分化偏移。

Dnmt3a

deficiency. Mechanistically, the STING pathway in

不足。从机制上讲，STING通路在

Dnmt3a

-deficient HSPCs is activated by the induction of ERVs, which become hypomethylated in the genome following

-缺陷的HSPCs通过ERVs的诱导而被激活，这些ERVs在基因组中随后发生低甲基化

Dnmt3a

deletion. Both genetic and pharmacological inhibition of STING effectively slowed down the progression of hematopoietic disorders mediated by

删除。STING的遗传和药理学抑制均有效减缓了由其介导的造血障碍的进展。

Dnmt3a

deficiency, both in

不足，无论是在

vitro

体外

and in vivo (Fig.

以及在体内（图。

). When evaluating the potential of targeting STING to inhibit the proliferation of AML cells, the STING inhibitor exhibited effects comparable to an FDA-approved targeted drug for FLT3-ITD. This was observed in both the

）。在评估靶向STING抑制AML细胞增殖的潜力时，STING抑制剂表现出与FDA批准的FLT3-ITD靶向药物相当的效果。这在两者中均被观察到。

Dnmt3a

-KO; FLT3-ITD mouse model and a human leukemia sample with the similar mutation background in a PDX model. Furthermore, the combined use of these two agents showed a cooperative effect, suggesting that targeting STING could be a strategy to enhance the clinical management of leukemia.

-KO；FLT3-ITD小鼠模型和具有相似突变背景的人类白血病样本在PDX模型中。此外，这两种药物的联合使用显示出协同效应，表明靶向STING可能是一种增强白血病临床管理的策略。

Fig. 7: Activation of the STING pathway by ERVs promotes increased self-renewal and skewed lineage populations in

图7：ERVs通过激活STING通路促进自我更新增加和谱系群体偏斜

DNMT3A

-mutated HSCs.

突变的造血干细胞。

The mutated

变异的

DNMT3A

in HSCs leads to genome-wide hypomethylation. Beyond impacting gene regulation, this also results in the upregulation of previously silenced ERVs due to the absence of cytosine methylation-associated transcriptional inhibition. The increased ERV RNA can be reverse-transcribed into cDNA, which in turn activates the STING pathway and induces chronic inflammation in .

在HSCs中导致全基因组低甲基化。除了影响基因调控外，这还导致先前被沉默的内源性逆转录病毒（ERVs）因缺乏与胞嘧啶甲基化相关的转录抑制而上调。增加的ERV RNA可以逆转录为cDNA，从而激活STING通路并诱导慢性炎症。

DNMT3A

-mutated HSCs. This STING-dependent autonomous inflammation leads to increased self-renewal and biased lineage differentiation in

-突变的HSCs。这种STING依赖性的自主炎症导致自我更新增加，并在

DNMT3A

-mutated HSCs. Inhibition of STING mitigates these phenotypes and prevents the development of leukemia associated with

-突变的HSCs。抑制STING可以减轻这些表型，并防止与之相关的白血病发展。

DNMT3A

mutations in mouse models.

小鼠模型中的突变。

Full size image

全尺寸图像

Inflammation plays a crucial role in promoting the development of CH and the initiation of hematopoietic malignancies. In a mouse CH model with mutated

炎症在促进CH的发展和造血恶性肿瘤的启动中起着至关重要的作用。在一个具有突变的CH小鼠模型中，

Dnmt3a

, microbial infection-induced IFNγ expression drove the expansion of

，微生物感染诱导的IFNγ表达驱动了

Dnmt3a

mutant CH [

突变体 CH [

]. Additionally, similar phenotypes were observed in cases of CH involving mutated

]. 此外，在涉及突变的CH病例中观察到相似的表型，

TET2

. Microbial signals can trigger the release of cytokines such as TNF-α and IL-6. These elevated environmental cytokines result in inflammation-induced toxicity to WT HSCs [

微生物信号可以触发细胞因子的释放，如TNF-α和IL-6。这些升高的环境细胞因子导致对WT HSCs的炎症诱导毒性 [

，

], while

]，而

Tet2

-deficient clones exhibit enhanced resistance to the inflammatory environment, gaining a competitive advantage over WT clones. Despite short-term exposure to infection-induced acute inflammatory responses, the mutated HSCs largely remain in a state of homeostasis throughout the majority of their lifespan.

缺陷克隆对炎症环境表现出增强的抵抗力，从而获得了相对于野生型克隆的竞争优势。尽管在短期内暴露于感染诱导的急性炎症反应，突变的造血干细胞在其生命周期的大部分时间里仍基本保持稳态。

This means that mutated clones are not consistently exposed to environments with high levels of inflammatory factors. Therefore, it raises an intriguing question: What is the driving force that promotes the development of CH in the absence of infections? One of the significant stressors associated with CH is aging [.

这意味着突变克隆并不会持续暴露于具有高水平炎症因子的环境中。因此，这引发了一个有趣的问题：在没有感染的情况下，是什么驱动力促进了CH的发展？与CH相关的重要应激源之一是衰老[。

]. One mechanism driving aging-related progression of CH is thought to be “inflammaging”, a term that describes a baseline increase in the production of pro-inflammatory factors such as IFN-γ, TNF-α, and IL-6 over time across various tissues and organs, including the hematopoietic system [

]. 一种驱动与衰老相关的CH进展的机制被认为是“炎症老化”，这个术语描述了各种组织和器官（包括造血系统）中促炎因子（如IFN-γ、TNF-α和IL-6）的基线水平随着时间的推移而增加的现象 [

，

]. Additionally, metabolism also contributes to the homeostasis of the hematopoietic hierarchy. Increased blood glucose levels have been shown to drive leukemogenesis in mice with

]. 此外，代谢也有助于造血层次的稳态。较高的血糖水平已被证明会促使患有疾病的鼷鼠产生白血病。

TET2

mutations, partly due to the expression of the antiapoptotic, long noncoding RNA

突变，部分原因是抗凋亡的长链非编码RNA的表达

Morrbid

莫比德

[

]. Furthermore, cohort analyses have revealed that individuals with unhealthy diets—characterized by high obesity rates, high cholesterol, and low vitamin intake—show a higher prevalence of CH compared to those with a balanced diet [

]. 此外，队列分析显示，饮食不健康（肥胖率高、胆固醇高、维生素摄入低）的个体比饮食均衡的人群显示出更高的CH患病率 [

，

]. One plausible explanation for these findings is the dietary intake of vitamin C, as various studies have demonstrated that vitamin C is crucial for maintaining HSC homeostasis and balanced lineage differentiation by regulating the stability of the epigenetic landscape [

]. 这些发现的一个合理解释是维生素C的摄入，因为多项研究表明，维生素C通过调节表观遗传景观的稳定性，对维持造血干细胞稳态和平衡谱系分化至关重要 [

，

The cGAS-STING pathway plays a crucial role in various physiological and pathological processes, including immune defense, tumorigenesis, DNA damage response, cellular senescence and so on. Recently, a study reported that ERVs can activate the STING pathway to enhance erythropoiesis under conditions such as pregnancy and serial bleeding [.

cGAS-STING 通路在多种生理和病理过程中起着至关重要的作用，包括免疫防御、肿瘤发生、DNA 损伤反应、细胞衰老等。最近，一项研究报道，在怀孕和连续出血等条件下，ERVs 可以激活 STING 通路以增强红细胞生成。

]. This work provides compelling evidence supporting the role of ERV-induced STING activation in unique hematopoietic processes. However, the study did not address why ERVs are upregulated in the hematopoietic cells of pregnant or bleeding mice. Based on our data,

]. 这项工作提供了令人信服的证据，支持ERV诱导的STING激活在独特的造血过程中的作用。然而，该研究并未解释为什么在怀孕或出血小鼠的造血细胞中ERVs会上调。根据我们的数据，

Dnmt3a

-deficient hematopoietic cells or treatment with hypomethylating agents can induce ERV expression. We hypothesize that when the hematopoietic system is under pressure to rapidly produce cells, the acceleration of proliferation leads to incomplete genomic methylation, which subsequently upregulates ERV expression and activates the STING pathway.

缺陷型造血细胞或用低甲基化剂治疗可以诱导ERV表达。我们假设，当造血系统承受快速产生细胞的压力时，增殖的加速会导致基因组甲基化不完全，随后上调ERV表达并激活STING通路。

This low-grade inflammatory response may create a positive feedback loop that further promotes hematopoiesis in these unique contexts. Further studies are required to gain a deeper understanding of the role of STING in various hematopoietic contexts and to validate the underlying mechanisms..

这种低级别的炎症反应可能会创建一个正反馈循环，进一步促进这些独特环境中的造血作用。需要进一步的研究来更深入地了解STING在各种造血环境中的作用，并验证其潜在机制。

Mutations in

突变

DNMT3A

and

和

TET2

account for nearly 70% of all genetic mutation events in CH [

占CH中所有基因突变事件的近70% [

，

]. As DNA cytosine-modifying enzymes, mutations in these two proteins exhibit highly similar phenotypes in HSC maintenance and skewed lineage differentiation. Therefore, researchers have spent over a decade identifying the shared mechanisms in hematopoietic disorders mediated by these two mutated enzymes.

]. 作为DNA胞嘧啶修饰酶，这两种蛋白质的突变在HSC维持和偏倚谱系分化中表现出高度相似的表型。因此，研究人员花了十多年的时间来识别由这两种突变酶介导的造血障碍中的共同机制。

For example, one work reported that DNMT3A and TET2 both contributed to 5hmC maintenance [.

例如，一项工作报道说 DNMT3A 和 TET2 都有助于 5hmC 的维持 [。

]. According to this study, DNMT3A and TET2 collaborate to suppress the expression of Klf1, a transcription factor specific to the erythropoietic lineage, in HSCs. Sequencing analysis revealed that

]. 根据这项研究，DNMT3A 和 TET2 协同作用抑制 HSCs 中红细胞生成谱系特异性转录因子 Klf1 的表达。测序分析显示，

Klf1

gains 5hmC in DNMT3A-deficient HSCs and loses 5hmC at the transcription start site (TSS) when TET2 is absent. Another cohort study found that individuals with mutations in

在DNMT3A缺陷的HSCs中获得5hmC，并在TET2缺失时在转录起始位点（TSS）失去5hmC。另一项队列研究发现，携带突变的个体中，

DNMT3A

或

TET2

acquired additional mutations at a higher rate, with odds ratios of 1.404 and 1.0937, respectively [

以较高的速率获得了额外的突变，其优势比分别为1.404和1.0937 [

]. This suggests that loss of function mutations in these two genes increase the chance that HSCs acquire additional genetic lesions. Another study reported that deficiencies in DNMT3A or TET2 in the macrophages isolated from human atherosclerotic plaques led to activation of the cGAS-STING pathway, triggered by the release of mtDNA [.

].这表明这两个基因的功能缺失突变增加了HSCs获得额外遗传损伤的机会。另一项研究报告称，从人类动脉粥样硬化斑块中分离的巨噬细胞中DNMT3A或TET2的缺陷导致了cGAS-STING通路的激活，这是由mtDNA的释放触发的[。

]. This finding suggested that the STING pathway might be a commonly affected pathway in hematopoietic cells with mutations of

]. 这一发现表明，STING 通路可能是具有以下突变的造血细胞中普遍受影响的通路：

DNMT3A

或者

TET2

. Recently, our group identified that CH harboring

最近，我们小组发现CH携带

TET2

mutations have a DNA damage-activated cGAS-STING pathway, which in turn stimulates a chronic inflammatory response and promotes the pathogenesis of CH in a non-infectious context [

突变激活了DNA损伤相关的cGAS-STING通路，这反过来刺激了慢性炎症反应，并在非感染性背景下促进CH的发病机制。

]. Including data from our previous research, we demonstrated here that hematopoietic disorders associated with mutations in

]. 包括我们之前研究的数据，我们在此证明了与突变相关的造血障碍在

DNMT3A

或

TET2

both rely on the activated STING pathway to create a chronic inflammatory environment, which promotes the clonal expansion of mutated HSPCs. Our findings suggest that targeting STING could be a highly effective strategy to prevent or delay the progression to AML of CH-associated with mutations in DNA-modifying enzymes.

两者都依赖于激活的STING通路来创造一个慢性炎症环境，这促进了突变HSPC的克隆扩增。我们的研究结果表明，针对STING可能是一种非常有效的策略，可以预防或延缓与DNA修饰酶突变相关的CH进展为AML。

More broadly, the concept of dampening inflammatory processes in the bone marrow might also be applicable to delay the progression of other preleukemic states like MDS and MPN..

更广泛地说，抑制骨髓中炎症过程的概念也可能适用于延缓其他癌前状态（如MDS和MPN）的进展。

Methods

方法

Mouse models

小鼠模型

Dnmt3a

f/f

mice were generated as previously described [

小鼠按之前描述的方法生成 [

]. The source of

]. 来源是

Sting

刺痛

-KO,

-KO，

Mx1-Cre

and

和

Vav-Cre

mice were described in ref. [

小鼠在参考文献[中被描述。

Dnmt3a

f/f

，

Mx1-

Cre and

Cre 和

Sting

刺痛

-KO mice were crossed to produce

-KO小鼠被杂交以产生

Dnmt3a

f/f

Mx1-

Cre and

Cre 和

Dnmt3a

f/f

Mx1-

Cre

创建

Sting-

刺痛-

KO mice. To induce

敲除小鼠。诱导

Dnmt3a

conditional knock-out, the

条件性基因敲除，

Mx1-

Cre transgene was induced in 4-week-old mice using poly(I:C) at 250 μg/body administered intraperitoneally every other day for 8 times. All mice were bred on a C57BL/6 genetic background. CD45.1 recipient mice (B6.SJL) were kindly provided by Prof. Xiaolong Liu. Immuno-deficient (M-NSG) mice were obtained from Shanghai Model Organisms Center (NM-NSG-001) and used for establishing AML PDX models..

在4周龄的小鼠中使用250 μg/体重的poly(I:C)每隔一天腹腔注射一次，共诱导Cre转基因8次。所有小鼠均在C57BL/6遗传背景下繁殖。CD45.1受体小鼠（B6.SJL）由刘小龙教授惠赠。免疫缺陷（M-NSG）小鼠购自上海模型生物中心（NM-NSG-001），并用于建立AML PDX模型。

Bisulfite PCR sequencing

双硫酸盐PCR测序

Genomic DNA (500 ng) was treated with EZ DNA methylation kit (D5001, Zymo). Bisulfite-treated DNA was amplified with gene-specific primers using Taq polymerse (T201, EnzyArtisan). PCR primers were listed in the Supplementary Table

基因组DNA（500 ng）用EZ DNA甲基化试剂盒（D5001，Zymo）处理。经亚硫酸盐处理的DNA使用Taq聚合酶（T201，EnzyArtisan）和基因特异性引物进行扩增。PCR引物列于补充表中。

. PCR products were cloned into T vector (C601, Vazyme), transformed into DH5α and sequenced.

PCR产物克隆到T载体（C601，Vazyme），转入DH5α并进行测序。

RNA-seq library preparation

RNA-seq文库制备

About 300 hematopoietic cells per group were sorted directly into Smart-seq2 lysis buffer by FACS. Sorted cells were lysed and the reverse-transcribed RNA was amplified to obtain enough cDNA by a modified SMART-Seq2 protocol. cDNA was quantified by Qubit 3 (Q33327, Invitrogen), and 5 ng cDNA was used for cDNA library construction with TruePrep DNA Library Prep Kit V2 for Illumina (TD502, Vazyme)..

每组约300个造血细胞通过荧光激活细胞排序（FACS）直接分选到Smart-seq2裂解缓冲液中。分选后的细胞被裂解，逆转录的RNA通过改良的SMART-Seq2协议进行扩增以获得足够的cDNA。使用Qubit 3（Q33327，Invitrogen）对cDNA进行定量，并使用TruePrep DNA建库试剂盒V2 for Illumina（TD502，Vazyme）以5 ng cDNA进行cDNA文库构建。

RNA data processing

RNA数据处理

Raw data were trimmed by Trim Galore (v0.6.7) with default setting. The clean data were mapped to mm10 reference genome using Hisat2 (v7.5.0) in paired-end mode with default parameters. Gene count matrices were calculated by featureCounts (v2.0.1) with parameters -M -O. DEG analysis was performed by using DESeq2 (v1.34.0) package.

原始数据使用Trim Galore（v0.6.7）以默认设置进行修剪。清洁数据使用Hisat2（v7.5.0）以成对模式和默认参数映射到mm10参考基因组。基因计数矩阵通过featureCounts（v2.0.1）使用参数-M -O计算。差异表达基因分析使用DESeq2（v1.34.0）包进行。

Only genes with at least a twofold change and an adjusted .

仅有至少两倍变化且经过调整的基因。

value less than 0.05 were considered to be differentially expressed. GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses were performed by using clusterProfiler (v4.10.0) package. To assess the transcription level of TE, TE transcripts (v.2.2.3) was applied to count TE and gene with the parameter mode multi.

小于0.05的值被认为差异表达。使用clusterProfiler（v4.10.0）包进行GO（基因本体）和KEGG（京都基因与基因组百科全书）通路富集分析。为了评估TE的转录水平，应用TE转录本（v.2.2.3）通过参数模式multi对TE和基因进行计数。

Differentially expressed TE analysis was performed by using DESeq2 package. Only TE with at least a 2-fold change and an .

使用DESeq2包进行差异表达的TE分析。只有变化至少2倍且...的TE被考虑。

value less than 0.05 were considered to be differentially expressed.

小于0.05的值被认为是有差异表达。

WGBS data processing

WGBS数据处理

Raw data were trimmed by Trim Galore (v0.6.7) with default setting. The clean data were mapped to mm10 reference genome using bismark (v0.23.1) in paired-end mode. PCR deduplicates were removed using deduplicate function of bismark. All replicates were merged for further analysis. Then, the methylation levels of CpG sites were quantified using extractor function of bismark.

原始数据使用Trim Galore（v0.6.7）进行默认设置的修剪。清洁数据使用bismark（v0.23.1）以双端模式比对到mm10参考基因组。使用bismark的deduplicate功能去除PCR重复。所有重复样本被合并用于进一步分析。然后，使用bismark的extractor功能量化CpG位点的甲基化水平。

Bigwig files were generated using bedGraphToBigWig (v.4) and visualized in Integrative Genomics Viewer (IGV). The annotation files of genes and repetitive elements (RepeatMasker) were downloaded from the UCSC Genome Browser. The methylation levels of different genomic elements were calculated with bedtools (v2.30.0).

Bigwig 文件使用 bedGraphToBigWig (v.4) 生成，并在 Integrative Genomics Viewer (IGV) 中可视化。基因和重复元件（RepeatMasker）的注释文件从 UCSC 基因组浏览器下载。不同基因组元件的甲基化水平使用 bedtools (v2.30.0) 计算。

Metaplots for 5mC and 5hmC levels were generated using deepTools (v3.5.5)..

使用deepTools（v3.5.5）生成了5mC和5hmC水平的元图。

Additional methods including bone marrow transplantation, FACS analysis, LC-MS etc. are presented in Supplementary Data.

包括骨髓移植、FACS分析、LC-MS等其他方法在补充数据中提供。

Data availability

数据可用性

All relevant data are within the manuscript and its additional files. The raw data of the RNA-seq analysis were uploaded to GEO database (GSE278208).

所有相关数据均在手稿及其附加文件中。RNA-seq分析的原始数据已上传至GEO数据库（GSE278208）。

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Acknowledgements

致谢

We acknowledge S.Chen for critical reading of the manuscript; Z. Lu for providing

我们感谢S. Chen对稿件的仔细阅读；Z. Lu提供

Sting

刺痛

-/-

mice; X. Liu for providing B6.SJL mice. This work was supported by the National Key R&D Program of China (Grant No. 2021YFA1102200 and No. 2022YFA1303000 to YS, No. 2023YFA1800400 to Guo-Liang Xu), the National Natural Science Foundation of China (32370625 to DZ; 31901011 and 82270163 to YS; 82400135 to JX), the China Postdoctoral Science Foundation (No.

小鼠；刘X提供了B6.SJL小鼠。这项工作得到了中国国家重点研发计划（资助号：2021YFA1102200和2022YFA1303000给YS，2023YFA1800400给许国亮），中国国家自然科学基金（32370625给DZ；31901011和82270163给YS；82400135给JX），中国博士后科学基金（编号：

2024M752000 to JX), the Medical Science Data Center in Shanghai Medical College of Fudan University and the Guangdong Major Project of Basic and Applied Basic Research (2023B0303000005)..

2024M752000 至 JX），复旦大学上海医学院医学科学数据中心，广东省基础与应用基础研究重大项目（2023B0303000005）。

Author contricutions

作者贡献

YHS, GX, and DZ conceived the original idea and, YHS, JH, JX, HG and DZ designed the experiments. Experiments were performed by JH and JX with the help of MS for the experiments associated with FACS; YXW for mouse modeling; YS and PC for mouse breeding and phenotyping; YW for Smart-seq2 library preparation and epigenetic data analysis; PZ for the leukemia samples collecting; SR and DY for LC-MS analysis, GX, YHS, DZ, SKB and HG wrote and revised the manuscript, with contributions from all other authors..

YHS、GX 和 DZ 提出了原始构思，YHS、JH、JX、HG 和 DZ 设计了实验。实验由 JH 和 JX 执行，在 FACS 相关实验中得到了 MS 的帮助；在小鼠建模方面得到了 YXW 的帮助；在小鼠繁殖和表型分析方面得到了 YS 和 PC 的帮助；在 Smart-seq2 文库制备和表观遗传数据分析方面得到了 YW 的帮助；在白血病样本收集方面得到了 PZ 的帮助；在 LC-MS 分析方面得到了 SR 和 DY 的帮助。GX、YHS、DZ、SKB 和 HG 撰写并修订了手稿，其他所有作者也做出了贡献。

Author information

作者信息

Author notes

作者笔记

These authors contributed equally: Jingru Huang, Jiaying Xie, Yin Wang.

这些作者贡献相同：黄静茹、谢佳颖、王寅。

Authors and Affiliations

作者与所属机构

Center of Precision Medicine for Blood Diseases, Huashan Hospital, Zhongshan-Xuhui Hospital, Chinese Academy of Medical Sciences (RU069), Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Medical College of Fudan University, Shanghai, 200032, China

中国医学科学院华山医院、中山徐汇医院血液疾病精准医疗中心，上海医学表观遗传学重点实验室，复旦大学医学院生物医学研究院，上海，200032，中国

Jingru Huang, Yin Wang, Mengyao Sheng, Yue Sun, Pingyue Chen, Shaoqin Rong, Dongrui Yin, Yuanxian Wang, Guo-Liang Xu, Hai Gao & Yuheng Shi

黄静茹，王寅，盛梦瑶，孙悦，陈平越，荣绍琴，尹冬瑞，王远贤，徐国良，高海，史宇恒

State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute; Shanghai Key Laboratory for Cancer Systems Regulation and Clinical Translation; Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

中国上海市，上海交通大学医学院附属仁济医院，癌症系统医学国家重点实验室，上海市癌症系统调控与临床转化重点实验室，上海癌症研究所

Jiaying Xie

谢佳颖

Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, Guangdong, 510100, China

广东省心血管病研究所，广东省人民医院，广东省医学科学院，南方医科大学，广州，广东，510100，中国

Ping Zhu

朱平

Guangdong Provincial Key Laboratory of Pathogenesis, Targeted Prevention and Treatment of Heart Disease, Guangzhou Key Laboratory of Cardiac Pathogenesis and Prevention, Guangzhou, China

广东省心脏病发病机制、靶向预防与治疗重点实验室，广州市心脏发病机制与预防重点实验室，中国广州

Ping Zhu

朱平

Leukaemia & Blood Cancer Research Unit, Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand

白血病与血液癌症研究室，分子医学与病理学系，奥克兰大学，奥克兰，新西兰

Stefan K. Bohlander

斯特凡·K·博兰德

CAS Key Laboratory of Epigenetic Regulation and Intervention, Shanghai Key Laboratory of Molecular Andrology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China

中国科学院分子细胞科学卓越创新中心，上海，200031，中国科学院表观遗传调控与干预重点实验室，上海市分子男科学重点实验室

Guo-Liang Xu

郭亮徐

Center for Medical Research and Innovation, Shanghai Pudong Hospital, Institutes of Biomedical Sciences, Medical College of Fudan University, Shanghai, 201399, China

复旦大学医学院生物医学研究院上海浦东医院医学研究中心，上海，201399，中国

Dan Zhou

丹州

Huadong hospital, Fudan university, Shanghai, 200040, China

中国上海复旦大学华东医院，邮编200040

Yuheng Shi

石宇恒

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Jingru Huang

黄静如

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Jiaying Xie

谢佳英

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Yin Wang

王银

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Mengyao Sheng

盛梦瑶

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Yue Sun

孙悦

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Pingyue Chen

陈平越

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Shaoqin Rong

荣少琴

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Dongrui Yin

尹东瑞

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Yuanxian Wang

王元宪

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Ping Zhu

朱平

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Stefan K. Bohlander

斯特凡·K·博赫兰德

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Guo-Liang Xu

郭亮徐

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Hai Gao

高海

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Dan Zhou

儋州

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Yuheng Shi

石宇恒

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Corresponding authors

通讯作者

Correspondence to

致信给

Hai Gao

高海

，

Dan Zhou

丹州

或

Yuheng Shi

石宇恒

。

Ethics declarations

伦理声明

Competing interests

竞争利益

The authors declare no competing interests.

作者声明不存在竞争性利益。

Ethics approval

伦理批准

All animal experiments described in this study were conducted in accordance with the ethical guidelines of the Department of Laboratory Animal Science Fudan University, No. 202307021S. The primary leukemia cells were collected from patient bone marrow cells and followed the ethical guidelines of Huashan Hospital, No.

本研究中描述的所有动物实验均按照复旦大学实验动物科学部的伦理指南进行，编号为202307021S。原代白血病细胞从患者骨髓细胞中收集，并遵循华山医院的伦理指南，编号为。

KY2015-269..

Additional information

更多信息

Publisher’s note

出版社说明

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature 对已发布地图中的管辖权声明和机构隶属关系保持中立。

Supplementary information

补充信息

Supplementary Figure and Method

补充图和方法

Primer list

引物列表

c-Kit RNAseq raw counts of gene

c-Kit RNAseq 基因的原始计数

c-Kit RNAseq raw counts of TE

c-Kit RNAseq 原始计数的 TE

HSPC SMARTseq raw counts of gene

HSPC SMARTseq 基因原始计数

HSPC SMARTseq raw counts of TE

HSPC SMARTseq TE原始计数

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根据本许可，您无权分享从本文或其部分内容衍生的改编材料。本文中的图像或其他第三方材料包含在文章的Creative Commons许可中，除非材料的署名行另有说明。如果材料未包含在文章的Creative Commons许可中，并且您的预期用途不被法律法规允许或超出了允许的使用范围，您需要直接从版权持有人处获得许可。

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引用本文

Huang, J., Xie, J., Wang, Y.

黄，J.，谢，J.，王，Y.