伴DNMT3A突变急性髓细胞白血病代谢重构和免疫逃逸机制研究

Patients with acute myeloid leukemia (AML) who had a DNMT3A mutation had poorer prognosis. The mechanism is not clear, which seriously restricts the effective targeted therapies. Previous studies showed that DNMT3A mutations caused a metabolic remodeling responsible for resisting daunorubicin-induced apoptosis. Recent studies found that the metabolic remodeling in AML cells lead to a suppressive effect on immune microenvironment. Thus, we speculate that the metabolic remodeling in AML cells is a key links of the effect mechanism of DNMT3A mutation, and it also indirectly mediates immunosuppressive effects of leukemia cells. In this study, several mutant AML cell lines with DNMT3A R882H are established. Differentially expressed genes are screened among mutant and wild clones. By targeted regulation of possible candidate genes, it will be confirmed that remodeling the amino acid metabolic passways is helpful for anti-apoptosis and immunosuppressive effects of AML cells with DNMT3A mutation. The mouse models carrying DNMT3A mutant clones are constructed and the reversal effect is observed by using the tryptophan pathway IDO1/TDO inhibitor 735D. The above phenomenon was further confirmed by using samples from patients with newly diagnosed AML. If successfully completed the research, it is very possible to uncover the effect mechanism of DNMT3A mutations in AML cells and find new potential therapeutic targets from the remodeling metabolic passways, which will provide theoretical basis and new strategies for breaking the treatment bottlenecks of AML with DNMT3A mutation.

急性髓细胞白血病（AML）伴DNMT3A突变预后差，效应机制未明，缺乏高效靶向治疗方法。前期研究发现DNMT3A突变AML细胞存在重构的氨基酸代谢介导的凋亡抵抗现象。新近发现，重构的代谢对免疫微环境存在抑制效应。据此我们推测AML细胞重构代谢是DNMT3A突变效应机制的关键环节，且间接介导了白血病细胞的免疫抑制能力。本课题拟借助于构建的DNMT3A R882H突变AML克隆和野生克隆，筛选差异表达基因并靶向调控，证实DNMT3A R882H突变造成的重构氨基酸代谢有利于AML细胞抗凋亡和产生免疫抑制效应。构建携带突变克隆的小鼠模型，借助于色氨酸通路限速酶IDO1/TDO抑制剂735D的免疫干预，观察潜在的逆转效应；利用初诊AML病人标本证实上述现象。本课题的完成，将进一步阐明DNMT3A突变效应机制，从重构的代谢通路发现新的作用靶点，为突破DNMT3A突变治疗瓶颈提供新的策略和理论依据。

急性髓细胞白血病伴DNMT3A突变预后较差，效应机制未明。本研究旨在探讨DNMT3A突变AML对免疫微环境的作用及调控机制，探寻潜在治疗靶点。我们构建了DNMT3A敲除克隆和DNMT3A R882H突变克隆的细胞株。通过RNA-seq检测发现DNMT3A突变体中差异基因显著富集在炎症免疫相关通路，并且转录因子和巨噬细胞炎症蛋白均显著下调。我们将AML细胞与巨噬细胞共培养后发现DNMT3A突变的AML细胞在体内外均能减弱M1型巨噬细胞的极化并抑制其杀伤作用。在共培养上清中，发现实验组促炎细胞因子（MIP-1α、MIP-1β和IL-1β）的表达明显低于对照组，而免疫抑制因子（IL-10和TGF-β）的表达明显高于对照组。共培养后，突变体细胞促炎细胞因子表达量的增加明显低于对照组，而免疫抑制因子的表达量无明显差异。在小鼠荷瘤模型中，实验组肿瘤体积明显大于对照组，M2型巨噬细胞比例也明显高于对照组。研究表明抵抗素可以显著促进AML细胞炎症相关蛋白（MIP-1α、MIP-1β和IL-1β）的表达。并可以削弱DNMT3A突变的抑制作用，促进实验组共培养巨噬细胞表型恢复，通过调节免疫微环境，进而增强对化疗药物敏感性。我们的研究一方面初步阐释了DNMT3A突变AML对免疫微环境可能的调控机制。另一方面，研究意还外发现，构建的DNMT3A突变的细胞株中较野生型异常高表达CD44v6，并且CD44v6 CAR-T 对DNMT3A突变 AML 具有明显的靶向杀伤效应，提示CD44v6可能是细胞免疫治疗靶向DNMT3A突变AML新的潜在靶点，后续研究将进一步证实其有效性和安全性。本课题的进一步深入研究，将论证细胞免疫治疗策略克服伴分子遗传学异常AML不良预后这一新理念；研究成果即可丰富DNMT3A突变AML的治疗选择，也具有重要临床转化价值。