Objectives: To examine the regulatory role of PCNA in MM, we have targeted PCNA with the experimental drug ATX-101 in three commercial cell lines (JJN3, RPMI 1660, AMO) and seven in-house patient-derived cell lines with a more primary cell-like phenotype (TK9, 10, 12, 13, 14, 16 and 18) and measured the systemic molecular effects.Methods: We have used a multi-omics untargeted approach, measuring the gene expression (transcriptomics), a subproteomics approach measuring mainly signalling proteins and proteins in complex with these (signallomics) and quantitative metabolomics. These results are supplemented with traditional analysis, e.g., viability, Western and ELISA analysis.Results: The sensitivity of the cell lines to ATX-101 varied, including between three cell lines derived from the same patient at different times of disease. A trend towards increased sensitivity to ATX-101 during disease progression was detected. Although with different sensitivities, ATX-101 treatment resulted in numerous changes in signalling and metabolite pools in all cell lines. Transcriptomics and signallomics analysis of the TK cell lines revealed that elevated endogenous expression of ribosomal genes, elevated proteasomal and endoplasmic reticulum (ER) stress and low endogenous levels of NAD+ and NADH were associated with ATX-101 hypersensitivity. ATX-101 treatment further enhanced the ER stress, reduced primary metabolism and reduced the levels of the redox pair GSH/GSSG in sensitive cells. Signallome analysis suggested that eleven proteins (TPD52, TNFRS17/BCMA, LILRB4/ILT3, TSG101, ZNRF2, UPF3B, FADS2, C11orf38/SMAP, CGREF1, GAA, COG4) were activated only in the sensitive MM cell lines (TK13, 14 and 16 and JJN3), and not in nine other cancer cell lines or in primary monocytes. These proteins may therefore be biomarkers of cells with activated proteasomal and ER stress even though the gene expression levels of these proteins were not elevated. Interestingly, carfilzomib-resistant cells were at least as sensitive to ATX-101 as the wild-type cells, suggesting both low cross-resistance between ATX-101 and proteasome inhibitors and elevated proteasomal stress in carfilzomib-resistant cells.Conclusions: Our multi-omics approach revealed a vital role of PCNA in regulation of proteasomal and ER stress in MM.
目的：为探究PCNA在多发性骨髓瘤（MM）中的调控作用，本研究采用实验药物ATX-101靶向PCNA，对三种商业化细胞系（JJN3、RPMI 1660、AMO）及七株具有更接近原代细胞表型的内部患者来源细胞系（TK9、10、12、13、14、16和18）进行处理，并系统评估其分子效应。方法：采用多组学非靶向研究策略，包括基因表达谱分析（转录组学）、以信号通路蛋白及其复合物为主的亚蛋白质组分析（信号组学）以及定量代谢组学检测。上述结果辅以传统分析方法验证，如细胞活力检测、Western印迹及ELISA分析。结果：各细胞系对ATX-101的敏感性存在差异，其中同一患者不同疾病阶段来源的三株细胞系间亦呈现敏感性分化，且发现疾病进展过程中对ATX-101敏感性增强的趋势。尽管敏感性不同，ATX-101处理均引起所有细胞系信号通路及代谢物池的广泛改变。对TK细胞系的转录组学与信号组学分析显示，核糖体基因内源性高表达、蛋白酶体与内质网（ER）应激水平升高以及NAD+与NADH内源性水平降低与ATX-101超敏反应相关。ATX-101处理进一步加剧敏感细胞的内质网应激，抑制基础代谢并降低氧化还原对GSH/GSSG水平。信号组分析提示，11种蛋白（TPD52、TNFRS17/BCMA、LILRB4/ILT3、TSG101、ZNRF2、UPF3B、FADS2、C11orf38/SMAP、CGREF1、GAA、COG4）仅在敏感MM细胞系（TK13、14、16及JJN3）中被激活，而在其他九种癌细胞系及原代单核细胞中未见激活。因此，这些蛋白可能成为蛋白酶体与内质网应激激活细胞的生物标志物，尽管其基因表达水平并未升高。值得注意的是，卡非佐米耐药细胞对ATX-101的敏感性至少与野生型细胞相当，这提示ATX-101与蛋白酶体抑制剂间交叉耐药性较低，且卡非佐米耐药细胞中蛋白酶体应激水平可能升高。结论：本研究通过多组学策略揭示了PCNA在调控多发性骨髓瘤蛋白酶体及内质网应激中的关键作用。
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