The m6A Reader IGF2BP2 Regulates Glutamine Metabolism and Represents a Therapeutic Target in Acute Myeloid Leukemia [RNA-seq]. The m6A Reader IGF2BP2 Regulates Glutamine Metabolism and Represents a Therapeutic Target in Acute Myeloid Leukemia [RNA-seq]

We report that m6A reader IGF2BP2 participates in the regulation of glutamine metabolism in AML. Targeting IGF2BP2 with our developed inhibitor CWI1-2 shows promising therapeutic efficacy in AML.To explore the mechanism of the inhibitor, we performed RNA-seq in AML cells upon treatment with CWI1-2 or DMSO. On the other hand, to distinguish the mechanism between IGF2BP2 and YTHDF2, we compare RNA-seq data of IGF2BP2 KD with YTHDF2 KD. Overall design: Total RNA from MonoMac6 cells with different treatments (i.e., CWI1-2 vs DMSO, YTHDF2 KD vs shNS) was isolated using the TRIzol reagent (Thermo fisher Scientific). Library construction of 1 µg RNA per sample was made using the NEBNext® Ultra RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. The library preparations were sequenced on an Illumina NovaSeq platform and 150 bp paired-end reads were generated.

本研究报道，m6A阅读蛋白（m6A reader）IGF2BP2参与调控急性髓系白血病（AML）细胞的谷氨酰胺代谢。我们开发的抑制剂CWI1-2靶向IGF2BP2，在AML模型中展现出颇具前景的治疗潜力。为探究该抑制剂的作用机制，我们对经CWI1-2或二甲基亚砜（DMSO）处理的AML细胞开展了RNA测序（RNA-seq）。另一方面，为区分IGF2BP2与YTHDF2的调控机制差异，我们对比了IGF2BP2敲低（KD）组与YTHDF2敲低组的RNA测序数据。
实验整体设计如下：使用TRIzol试剂（Thermo Fisher Scientific）从经不同处理的MonoMac6细胞中提取总RNA，处理组包括CWI1-2处理组与DMSO处理组、YTHDF2敲低组与阴性短发夹对照（shNS）组。每个样本取1μg总RNA，依照制造商操作指南，使用适配Illumina®测序平台的NEBNext® Ultra RNA文库制备试剂盒（NEB，美国）构建测序文库。PCR产物经AMPure XP磁珠系统纯化后，通过Agilent Bioanalyzer 2100生物分析仪评估文库质量。携带索引标签的样本聚类流程依照制造商说明，在cBot集群生成系统上使用TruSeq PE Cluster Kit v3-cBot-HS（Illumina）完成。最终将制备好的文库置于Illumina NovaSeq测序平台进行测序，生成150bp双端读段。