MYCN amplification is an independent poor prognostic factor in patients with high-risk neuroblastoma (NB). Further exploring the molecular regulatory mechanisms in MYCN-amplified NB will help to develop novel therapy targets. In this study, methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) was identified as the differentially expressed gene (DEG) highly expressed in MYCN-amplified NB, and it showed a positive correlation with MYCN and was associated with a poor prognosis of NB patients. Knockdown of MTHFD1 inhibited proliferation and migration, and induced apoptosis of NB cells in vitro. Mouse model experiments validated the tumorigenic effect of MTHFD1 in NB in vivo. In terms of the mechanism, ChIP-qPCR and dual-luciferase reporter assays demonstrated that MTHFD1 was directly activated by MYCN at the transcriptional level. As an important enzyme in the folic acid metabolism pathway, MTHFD1 maintained the NADPH redox homeostasis in MYCN-amplified NB. Knockdown of MTHFD1 reduced cellular NADPH/NADP⁺ and GSH/GSSG ratios, increased cellular reactive oxygen species (ROS) and triggered the apoptosis of NB cells. Moreover, genetic knockdown of MTHFD1 or application of the anti-folic acid metabolism drug methotrexate (MTX) potentiated the anti-tumor effect of JQ1 both in vitro and in vivo. Taken together, MTHFD1 as an oncogene is a potential therapeutic target for MYCN-amplified NB. The combination of MTX with JQ1 is of important clinical translational significance for the treatment of patients with MYCN-amplified NB.

MYCN 扩增是高危神经母细胞瘤 (NB) 患者的独立不良预后因素。进一步探索 MYCN 扩增 NB 的分子调控机制将有助于开发新的治疗靶点。本研究中，亚甲基四氢叶酸脱氢酶1（MTHFD1）被确定为MYCN扩增的NB中高表达的差异表达基因（DEG），其与MYCN呈正相关，并与NB患者的不良预后相关。敲除 MTHFD1 可抑制体外 NB 细胞的增殖和迁移，并诱导细胞凋亡。小鼠模型实验验证了MTHFD1在NB体内的致瘤作用。在机制方面，ChIP-qPCR和双荧光素酶报告基因检测表明MTHFD1在转录水平上被MYCN直接激活。作为叶酸代谢途径中的重要酶，MTHFD1 维持 MYCN 扩增的 NB 中的 NADPH 氧化还原稳态。 MTHFD1 的敲低降低了细胞 NADPH/NADP ⁺和 GSH/GSSG 比率，增加了细胞活性氧 (ROS) 并引发了 NB 细胞的凋亡。此外，MTHFD1的基因敲除或抗叶酸代谢药物甲氨蝶呤（MTX）的应用增强了JQ1在体外和体内的抗肿瘤作用。综上所述，MTHFD1 作为癌基因是 MYCN 扩增 NB 的潜在治疗靶点。 MTX联合JQ1对于治疗MYCN扩增的NB患者具有重要的临床转化意义。