Data Sheet 1_Dual oncogenic role of RNF220 in AML: linking metabolic rewiring to cell proliferation and immune evasion.pdf

BackgroundAcute myeloid leukemia (AML) remains a clinical challenge with suboptimal long-term survival. While circular RNAs derived from the RNF220 host gene have been implicated in AML pathogenesis, the functional role and regulatory mechanisms of RNF220 itself in AML are poorly understood.
MethodsWe integrated bioinformatics analyses of public databases (TCGA-LAML, TARGET-LAML) and local cohort with in vitro functional assays. RNF220 was knocked down and overexpressed in AML cell lines using lentivirus. Transcriptomic profiling (RNA-seq), metabolic pathway enrichment (GSVA, GSEA), and immune microenvironment deconvolution (xCELL, CIBERSORT, MCP-counter) were performed. Transcription factor binding sites were predicted across five databases (JASPAR, ENCODE, GTRD, etc.). Validation of transcriptional regulation was performed using ChIP-PCR and luciferase reporter assays.
ResultsRNF220 overexpression correlated with poor prognosis in AML, drove an immunosuppressive microenvironment characterized by reduced CD8+ T cells, and inhibited NK activity and M2 polarization of macrophage. RNF220 promoted tumor proliferation by suppressing apoptosis and preventing G1 arrest. Knockdown of RNF220 dysregulated metabolic pathways, notably suppressing glycolysis and phenylalanine metabolism. Mechanistically, FOXA1 was identified as an upstream negative regulator of RNF220, where high FOXA1 predicted favorable survival and inversely correlated with RNF220-associated metabolic reprogramming.
ConclusionNF220 acts as an oncogenic ubiquitin ligase in AML by coordinating dual pro-leukemic mechanisms: cell-intrinsic metabolic rewiring (glycolysis/phenylalanine) and immune evasion via microenvironment suppression. Targeting the FOXA1–RNF220 axis may offer novel therapeutic strategies for high-risk AML.

背景
急性髓系白血病（Acute myeloid leukemia, AML）仍是临床亟待攻克的难题，长期生存率仍未达到理想水平。尽管源自RNF220宿主基因的环状RNA（circular RNAs）已被证实与AML的发病机制相关，但RNF220本身在AML中的功能角色及调控机制仍有待深入阐明。

方法
本研究整合了公共数据库（TCGA-LAML、TARGET-LAML）及本地队列的生物信息学分析，并结合体外功能实验。通过慢病毒（lentivirus）介导，在AML细胞系中敲低或过表达RNF220。随后开展了转录组测序（RNA-seq）、代谢通路富集分析（基因集变异分析GSVA、基因集富集分析GSEA）以及免疫微环境反卷积分析（xCELL、CIBERSORT、MCP-counter）。通过五大数据库（JASPAR、ENCODE、GTRD等）预测转录因子结合位点，并采用染色质免疫沉淀-聚合酶链式反应（ChIP-PCR）及荧光素酶报告基因实验（luciferase reporter assays）验证转录调控关系。

结果
RNF220过表达与AML患者不良预后显著相关，可驱动以CD8+ T细胞浸润减少为特征的免疫抑制微环境，抑制自然杀伤细胞（natural killer cell, NK）活性并促进巨噬细胞M2极化。RNF220通过抑制细胞凋亡、阻滞G1期细胞周期进程来促进肿瘤增殖。敲低RNF220会打乱代谢通路稳态，显著抑制糖酵解及苯丙氨酸代谢通路。机制层面，本研究鉴定出叉头框蛋白A1（FOXA1）是RNF220的上游负调控因子；高表达FOXA1预示患者预后良好，且与RNF220介导的代谢重编程呈负相关。

结论
RNF220作为一种致癌性泛素连接酶（ubiquitin ligase），在AML中通过协同两种促白血病机制发挥作用：细胞内在的代谢重编程（糖酵解/苯丙氨酸代谢）以及通过抑制肿瘤微环境实现的免疫逃逸。靶向FOXA1-RNF220轴可为高危AML患者提供全新的治疗策略。