NSUN2 promotes leukemogenesis by FTH1-mediated anti-ferroptosis and FBL-mediated ribosome biogenesis in B-ALL

NOP2/Sun RNA methyltransferase family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modifications in tRNA, rRNA, and mRNA, playing critical roles in various malignancies. However, its expression and functional relevance in B-acute lymphoblastic leukemia (B-ALL) remain largely undefined. Lineage-negative progenitor cells from Nsun2 knockout (?/?) and wild-type (fl/fl) mice were used to generate BCR-ABL (P190)-driven murine B-ALL model. Furthermore, RNA sequencing (RNA-seq) was performed to identify NSUN2 targets. Here, we found that NSUN2 expression was significantly higher in bone marrow (BM) cells from B-ALL patients than normal controls (NCs). Knockdown of NSUN2 reduced m5C amounts, induced apoptosis and ferroptosis in B-ALL cells. Moreover, Nsun2 knockout significantly suppressed the leukemic burden and extended the OS in murine B-ALL model in vivo. RNA-seq and subsequent studies identified ferritin heavy chain 1 (FTH1) as a downstream target of NSUN2. Mechanistically, NSUN2 knockdown reduced m5C modifications on 3'UTR of FTH1 mRNA, impairing its stability and reducing FTH1 expression. Overexpression of wild-type (WT) NSUN2, but not its mutants, blocked NSUN2 knockdown-induced cytotoxic effects, indicating that NSUN2 enhances leukemogenesis in an m5C-dependent manner. In addition, NSUN2 facilitated global protein synthesis and ribosome biogenesis by enhancing fibrillarin (FBL) translation efficiency. In contrast, Nsun2 depletion was dispensable for hematopoiesis and exerted minimal impact on ferroptosis and protein synthesis in murine pre-B (B220+) cells, suggesting selective leukemic dependency. Our results demonstrate that NSUN2 facilitates leukemogenesis by increasing FTH1 expression and enhancing FBL-mediated ribosome biogenesis in an m5C-dependent manner. Targeting NSUN2 might provide a promising therapeutic strategy for B-ALL. Overall design: we established Mus musculus stem cell in which each target gene has been knocked down by shRNA.We then performed gene expression profiling analysis using data obtained from RNA-seq .Comparative gene expression profiling analysis of RNA-seq data for NELM-6 cells and its KD derivatives (shNUSN2).

NOP2/Sun RNA甲基转移酶家族成员2（NSUN2）可催化tRNA、rRNA及mRNA中的5-甲基胞嘧啶（5-methylcytosine，m5C）修饰，在多种恶性肿瘤中发挥关键作用。然而，其在B细胞急性淋巴细胞白血病（B-acute lymphoblastic leukemia，B-ALL）中的表达及功能相关性仍未完全阐明。本研究使用Nsun2敲除（(?/?)）与野生型（fl/fl）小鼠的谱系阴性祖细胞，构建了BCR-ABL（P190）驱动的小鼠B-ALL模型；同时通过RNA测序（RNA sequencing，RNA-seq）鉴定NSUN2的靶标基因。
本研究发现，B-ALL患者骨髓（bone marrow，BM）细胞中NSUN2的表达水平显著高于正常对照（normal controls，NCs）。敲低NSUN2可降低B-ALL细胞内的m5C水平，诱导细胞凋亡与铁死亡（ferroptosis）。体内实验显示，Nsun2敲除可显著抑制小鼠B-ALL模型的白血病负荷，并延长其总生存期（overall survival，OS）。通过RNA-seq及后续实验，本研究鉴定出铁蛋白重链1（ferritin heavy chain 1，FTH1）为NSUN2的下游靶标。机制层面，敲低NSUN2可降低FTH1 mRNA 3'非翻译区（3'UTR）的m5C修饰，削弱其mRNA稳定性并下调FTH1的表达。过表达野生型（wild-type，WT）NSUN2（而非其突变体）可阻断敲低NSUN2诱导的细胞毒性效应，表明NSUN2以m5C依赖的方式促进白血病发生。此外，NSUN2通过提高核仁蛋白（fibrillarin，FBL）的翻译效率，促进全局蛋白质合成与核糖体生物发生。与之相反，在小鼠前B（B220+）细胞中，敲除Nsun2对造血过程并非必需，且对铁死亡与蛋白质合成的影响极小，提示白血病细胞存在选择性的NSUN2依赖性。本研究结果证实，NSUN2可通过以m5C依赖的方式上调FTH1表达并增强FBL介导的核糖体生物发生，从而促进白血病发生。靶向NSUN2或可为B-ALL提供极具前景的治疗策略。
整体实验设计：本研究构建了通过短发夹RNA（shRNA）敲低各靶基因的小鼠干细胞模型。随后利用RNA-seq获得的数据开展基因表达谱分析，并对NELM-6细胞及其NSUN2敲低衍生物（shNUSN2）的RNA-seq数据进行比较基因表达谱分析。