研究目的：

静息、自我更新潜能及化疗抵抗等，是白血病干细胞（LSC）的重要干性特征，与急性髓系白血病（AML）的复发和治疗预后不良密切相关。LSC主要定位于低氧骨髓龛中，其干性特征的维持依赖于与骨髓微环境的相互作用。DNA损伤诱导转录因子4（DDIT4）是一种在低氧条件下被诱导表达的分子，它通过抑制mTOR信号通路参与多种生物学过程，但在AML中的具体作用机制尚未被充分阐明。本研究通过对骨髓不同部位白血病细胞的转录组特征分析，发现DDIT4在骨髓龛中的白血病细胞高表达，拟通过探究骨髓龛对白血病细胞中DDIT4表达的调控作用，并进一步揭示DDIT4在AML1-ETO9a（AE9a）白血病细胞化疗抵抗、静息及自我更新中的功能，以及其在白血病发生中的作用，以最终阐明白血病细胞干性的重要外在和内在分子调节机制。

研究方法：

采用基因集富集分析（GSEA）对AE9a白血病小鼠骨髓龛和骨髓腔白血病细胞的转录组数据进行分析，并通过qRT-PCR和Western blot检测骨髓龛中白血病细胞DDIT4的表达水平。将AE9a细胞与骨髓龛基质细胞共培养后检测DDIT4的表达变化。分析TCGA和HOVON数据库中AML患者的转录组数据，分析DDIT4表达与预后的相关性。在体内药物化疗实验中，用阿糖胞苷（Ara-C）处理AE9a白血病小鼠，检测骨髓龛与骨髓腔白血病细胞中Ddit4表达。用AE9a病毒感染野生型（Ddit4^+/+）和Ddit4敲除（Ddit4^-/-）小鼠骨髓c-Kit⁺细胞（分别获得AE9a/Ddit4^+/+和AE9a/Ddit4^-/-细胞），与成骨细胞系共培养，流式细胞术分析细胞G0期比例，连续集落形成实验检测细胞集落形成能力。用AE9a病毒感染Ddit4^+/+和Ddit4^-/-小鼠c-Kit⁺细胞并移植到受体鼠中，构建AE9a-Ddit4^+/+和AE9a-Ddit4^-/-白血病小鼠模型，监测外周血GFP阳性率及观察生存期，Western blot检测发病小鼠骨髓细胞中DDIT4的表达。二代移植实验观察生存期，流式细胞术检测骨髓细胞免疫表型。极限稀释移植实验确定白血病干细胞频率。对发病小鼠白血病细胞进行转录组分析，qRT-PCR检测Hoxa家族分子mRNA水平。在Kasumi-1和KG-1a细胞系中过表达DDIT4，检测细胞G0期比例，用柔红霉素处理后检测细胞凋亡水平，qRT-PCR检测细胞系中HOXA家族mRNA表达。构建过表达Hoxa6的AE9a-Ddit4^-/-白血病细胞，移植至受体鼠，监测外周血RFP⁺GFP⁺细胞比例及观察生存期。

研究结果：

GSEA分析显示骨髓龛中的AE9a白血病细胞表现出低氧相关转录特征，且Ddit4表达升高。qRT-PCR和Western blot验证了骨髓龛白血病细胞中DDIT4的高表达，且AE9a白血病细胞与成骨细胞共培养后DDIT4表达显著上调。TCGA和HOVON数据库分析表明DDIT4高表达的AML患者预后较差。机制研究发现，化疗药物处理后，骨髓龛中高表达Ddit4的白血病细胞群被富集；在与成骨细胞系共培养条件下，与AE9a/Ddit4^+/+细胞相比，AE9a/Ddit4^-/-细胞的G0期比例降低，静息维持能力减弱，集落形成能力下降，自我更新能力受损，表明DDIT4缺失引起白血病细胞干性降低。成功构建AE9a-Ddit4^+/+和AE9a-Ddit4^-/-白血病小鼠模型，体内原代移植及二次移植实验显示AE9a-Ddit4^-/-移植小鼠发病时间及生存期显著延长，表明DDIT4敲除显著抑制白血病的发生。两组发病小鼠的骨髓细胞免疫表型相同，均为AML表型。极限稀释移植实验显示AE9a-Ddit4^-/-小鼠白血病干细胞频率显著降低。转录组分析AE9a-Ddit4^-/-白血病细胞中Hoxa家族分子mRNA水平降低，而过表达Ddit4后Hoxa家族分子表达上调，表明DDIT4可上调Hoxa家族分子表达。过表达DDIT4的Kasumi-1和KG-1a细胞G0期比例显著升高，用柔红霉素处理后，过表达DDIT4的细胞凋亡水平显著降低，对化疗药物的抵抗性增强，表明DDIT4可促进白血病细胞的静息及化疗抵抗。此外，过表达DDIT4的Kasumi-1和KG-1a细胞系中HOXA家族分子mRNA水平升高。进一步发现过表达Hoxa6的AE9a-Ddit4^-/-小鼠生存期显著缩短，部分挽救了由Ddit4缺失引起的白血病发生缺陷。

研究结论：

白血病细胞中DDIT4的表达可被低氧骨髓龛上调。DDIT4的高表达与AML病人预后不良相关。DDIT4高表达的白血病细胞更耐受化疗、静息细胞比例更高、自我更新能力增强。DDIT4可促进白血病的发生、发展，并维持白血病细胞的静息、自我更新及化疗抵抗等干性特征。HOXA家族分子介导了DDIT4对白血病发生的促进作用。

Objective:

Quiescence, self-renewal potential, and chemoresistance are critical stemness features of leukemia stem cells (LSCs), which are closely associated with relapse and poor prognosis in acute myeloid leukemia (AML). LSCs primarily reside in hypoxic bone marrow niches, where their stemness is maintained through interactions with the bone marrow microenvironment. DNA damage-inducible transcript 4 (DDIT4), a molecule induced under hypoxic conditions, regulates various biological processes by inhibiting the mTOR signaling pathway. However, its role in AML is poorly understood. This study aims to investigate the regulatory role of the bone marrow niche in DDIT4 expression in leukemia cells and to elucidate the function of DDIT4 in chemotherapy resistance, quiescence, and self-renewal of AML1-ETO9a (AE9a) leukemia cells, as well as its role in leukemogenesis, ultimately revealing the intrinsic and extrinsic molecular mechanisms underlying leukemia cell stemness.

Methods:

Gene Set Enrichment Analysis (GSEA) was performed on transcriptomic data from AE9a leukemia cells in the bone marrow niche and central bone marrow of mouse models. DDIT4 expression in leukemia cells within the bone marrow niche was validated by qRT-PCR and Western blot. AE9a cells were co-cultured with bone marrow (BM) niche stromal cells to assess the promotion effect of BM niche on DDIT4 expression. The correlation between DDIT4 expression and prognosis in AML patients was analyzed using transcriptomic data from the TCGA and HOVON databases. In vivo chemotherapy experiments were conducted using cytarabine (Ara-C) to treat AE9a leukemia mice, and DDIT4 expression was measured in leukemia cells. C-Kit⁺ bone marrow cells from wild-type (Ddit4^+/+) and Ddit4 knockout (Ddit4^-/-) mice were transfected with AE9a retrovirus, and the proportion of cells in the G0 phase and colony formation ability were determined. AE9a-Ddit4^+/+ and AE9a-Ddit4^-/- leukemia mouse models were established, and survival, peripheral blood GFP⁺ cell rates, and DDIT4 expression were monitored. Secondary transplantation and limiting dilution assays were performed to assess leukemia stem cell frequency. Flow cytometry was used to analyze immunophenotypes of bone marrow cells. Transcriptomic analysis was conducted and Hoxa family gene expression was evaluated by qRT-PCR. DDIT4 was overexpressed in Kasumi-1 and KG-1a cell lines, followed by assessment of the proportion of cells in the G0 phase and HOXA family mRNA expression. AE9a-Ddit4^-/- leukemia cells were transfected with Hoxa6 retrovirus and then transplanted into recipient mice to monitor survival and RFP⁺GFP⁺ cell rates.

Results:

GSEA revealed that AE9a leukemia cells in the bone marrow niche exhibited hypoxia-related transcriptomic signatures and elevated DDIT4 expression. High DDIT4 expression in leukemia cells within the bone marrow niche was confirmed by qRT-PCR and Western blot, in addition, DDIT4 expression was significantly upregulated upon co-culture with osteoblasts. TCGA and HOVON database analyses indicated that high DDIT4 expression was associated with poor prognosis in AML patients. After chemotherapy treatment, leukemia cell populations with high DDIT4 expression were enriched in the bone marrow niche. AE9a/Ddit4^-/- cells showed reduced G0 phase proportion, impaired quiescence maintenance, and diminished colony formation capacity compared to AE9a/Ddit4^+/+ cells under co-culture with osteoblasts. DDIT4-mediated quiescence required direct interactions between leukemia cells and osteoblasts. AE9a-Ddit4^+/+ and AE9a-Ddit4^-/- leukemia mouse models were successfully established. Primary transplantation and secondary transplantation experiments showed that AE9a-Ddit4^-/- mice exhibited prolonged survival, indicating that knockout of DDIT4 significantly inhibits the leukemia initiation and progression. Both AE9a-Ddit4^+/+ and AE9a-Ddit4^-/- mice displayed AML immunophenotypes. Limiting dilution transplantation assay indicated that AE9a-Ddit4^-/- cells exhibited a reduced leukemia stem cell frequency, further confirming the critical role of DDIT4 in leukemia cell self-renewal and leukemia initiation. Transcriptomic analysis revealed decreased Hoxa family gene expression in AE9a-Ddit4^-/- cells, which was restored by DDIT4 overexpression. In Kasumi-1 and KG-1a cell lines, overexpression of DDIT4 led to an increased proportion of cells in the G0 phase. After daunorubicin treatment, apoptosis levels were significantly lower in DDIT4-overexpressing cells, indicating that DDIT4 overexpression enhances chemotherapy resistance in leukemia cells. Additionally, HOXA family mRNA levels were elevated in DDIT4-overexpressing Kasumi-1 and KG-1a cells. When Hoxa6 was re-expressed in AE9a-Ddit4^-/- cells, AE9a-Ddit4^-/--Hoxa6 cell-transplanted mice developed leukemia with a significantly shorter survival time compared with AE9a-Ddit4^-/- cell transplanted mice. Overexpression of Hoxa6 in AE9a-Ddit4^-/- cells partially rescued the leukemogenesis defect in AE9a-Ddit4^-/- mice.

Conclusions:

The expression of DDIT4 in leukemia cells can be upregulated by the hypoxic bone marrow niche. High DDIT4 expression is associated with poor prognosis in AML patients. DDIT4 is critical for chemoresistance, quiescence, and self-renewal in leukemia cells. DDIT4 promotes leukemogenesis and maintains leukemia cell stemness. The HOXA family molecules mediate the promoting effect of DDIT4 on leukemia development.