Triomics Analysis of Imatinib-Treated Myeloma Cells Connects Kinase Inhibition to RNA Processing and Decreased Lipid Biosynthesis

The combination of metabolomics, lipidomics, and phosphoproteomics that incorporates triple stable isotope labeling by amino acids in cell culture (SILAC) protein labeling, as well as 13C in vivo metabolite labeling, was demonstrated on BCR–ABL-positive H929 multiple myeloma cells. From 11 880 phosphorylation sites, we confirm that H929 cells are primarily signaling through the BCR–ABL–ERK pathway, and we show that imatinib treatment not only downregulates phosphosites in this pathway but also upregulates phosphosites on proteins involved in RNA expression. Metabolomics analyses reveal that BCR–ABL–ERK signaling in H929 cells drives the pentose phosphate pathway (PPP) and RNA biosynthesis, where pathway inhibition via imatinib results in marked PPP impairment and an accumulation of RNA nucleotides and negative regulation of mRNA. Lipidomics data also show an overall reduction in lipid biosynthesis and fatty acid incorporation with a significant decrease in lysophospholipids. RNA immunoprecipitation studies confirm that RNA degradation is inhibited with short imatinib treatment and transcription is inhibited upon long imatinib treatment, validating the triomics results. These data show the utility of combining mass spectrometry-based “-omics” technologies and reveals that kinase inhibitors may not only downregulate phosphorylation of their targets but also induce metabolic events via increased phosphorylation of other cellular components.

本研究将代谢组学、脂质组学与磷酸化蛋白质组学技术联用，结合了细胞培养氨基酸稳定同位素标记（SILAC）的三重稳定同位素标记策略，并辅以体内13C同位素代谢物标记，以BCR-ABL阳性的H929多发性骨髓瘤细胞为实验对象开展研究。从11880个磷酸化位点的检测数据中，我们证实H929细胞主要通过BCR-ABL-ERK信号通路进行信号转导；同时发现伊马替尼处理不仅下调了该通路中的磷酸化位点，还上调了参与RNA表达过程的相关蛋白上的磷酸化位点。代谢组学分析显示，H929细胞内的BCR-ABL-ERK信号通路可激活磷酸戊糖途径（PPP）与RNA生物合成，而通过伊马替尼抑制该通路后，会导致磷酸戊糖途径显著受损、RNA核苷酸蓄积，并对mRNA产生负调控作用。脂质组学数据同样显示，整体脂质生物合成与脂肪酸掺入水平均出现下降，且溶血磷脂的含量显著降低。RNA免疫沉淀实验证实，短期伊马替尼处理会抑制RNA降解，而长期伊马替尼处理则会抑制转录过程，该结果验证了三组学联用的实验结论。本研究数据证明了基于质谱的“组学”技术联用的实用价值，并揭示了激酶抑制剂不仅可下调其靶标的磷酸化水平，还可通过上调其他细胞组分的磷酸化水平诱导代谢相关事件。