PO.MCB09.03 · 分子与细胞生物学
一种多重LC-MS/MS代谢物检测法，用于实现MTA协同型PRMT5抑制剂的患者分层和药效学监测
A multiplexed LC-MS/MS metabolite assay to enable patient stratification and pharmacodynamic monitoring for MTA-cooperative PRMT5 inhibitors
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摘要 Abstract
中文摘要
蛋白质精氨酸甲基转移酶5（PRMT5）抑制剂的开发，尤其是选择性靶向MTAP缺失癌症的MTA协同型药物，迫切需要可靠的生物标志物来识别目标患者群体并确认药效学（PD）靶点结合。本研究的目的是开发并应用一种高灵敏度、多重的液相色谱-质谱（LC-MS/MS）检测法，以定量关键的PRMT5相关代谢物，如甲硫基腺苷（MTA）、对称二甲基精氨酸（SDMA）、S-腺苷甲硫氨酸（SAM）和非对称二甲基精氨酸（ADMA），以及叶酸循环、甲硫氨酸循环和多胺代谢中的相互作用代谢物。所开发的多重LC-MS/MS方法能够从细胞裂解物、肿瘤组织和生物体液中同时定量MTA、SDMA、SAM、SAH及其他12种相关代谢物。该检测法利用同位素标记的内标和碎片离子监测，实现对关键PRMT生物标志物ADMA（I型PRMT，如PRMT1）和SDMA（PRMT5）的可靠定量与分辨。随后，我们将该检测法应用于来自n=6例人类患者的配对腺癌肿瘤组织和癌旁正常组织（NAT）标本。结果显示，肿瘤中SAM和MTA以及S-腺苷同型半胱氨酸（SAH）水平出现具有统计学意义（p<0.05）的升高。升高的MTA水平由一部分患者所驱动，提示这些个体的肿瘤存在MTAP缺失。SAM和SAH分别是PRMT5和PRMT1等I型PRMT的反应底物和产物。肿瘤中SAM和SAH的升高提示PRMT活性增强。在检查SDMA和ADMA水平时，我们发现肿瘤中ADMA（PRMT1生物标志物）升高了40%，而SDMA（PRMT5生物标志物）的升高不到20%。总体而言，这些数据证明了所开发的代谢物检测法在PRMT5疗法开发中的实用价值。肿瘤与NAT代谢物谱之间的差异与肿瘤中PRMT1（或其他I型PRMT）上调相一致，从而导致ADMA、SAM和SAH升高。另一方面，PRMT5在MTAP缺失的肿瘤中可能因MTA水平升高而受到抑制，尽管肿瘤中SDMA水平未显著增加，但这使其对MTA协同型PRMT5抑制剂更加敏感。
查看英文原文 English abstract
The development of Protein Arginine Methyltransferase 5 (PRMT5) inhibitors, particularly MTA-cooperative agents that selectively target MTAP -deleted cancers, has created an urgent need for robust biomarkers to identify target patient populations and confirm pharmacodynamic (PD) target engagement. The purpose of this study was to develop and apply a highly sensitive, multiplexed liquid chromatography-mass spectrometry (LC-MS/MS) assay to quantify key PRMT5-related metabolites such as methylthioadenosine (MTA), symmetric dimethylarginine (SDMA), S-adenosylmethionine (SAM), and asymmetric dimethylarginine (ADMA), as well as interacting metabolites in the folate cycle, methionine cycle, and polyamine metabolism. The developed multiplexed LC-MS/MS method enables simultaneous quantification of MTA, SDMA, SAM, and SAH, and 12 other related metabolites from cell lysates, tumor tissue, and biofluids. The assay leverages isotopically labeled internal standards and fragment ion monitoring for robust quantitation and resolution of key PRMT biomarkers ADMA (Type I PRMTs, e.g., PRMT1) and SDMA (PRMT5). We then applied this assay to matched adenocarcinoma tumor and normal adjacent tissue (NAT) specimens from n=6 human patients. The results showed statistically significant (p<0.05) increases in SAM and MTA as well as S-adenosylhomocysteine (SAH) levels in the tumor. The elevated MTA levels was driven by a subset of patients, suggesting these individuals have MTAP- deleted tumors. SAM and SAH are reactants and products, respectively, of PRMT5 and Type I PRMTs like PRMT1. The elevation of SAM and SAH in the tumor suggests elevated PRMT activity. In examining SDMA and ADMA levels, we found that ADMA (PRMT1 biomarker) was elevated by 40% in the tumor, while SDMA (PRMT5 biomarker) showed less than 20% elevation. In total, these data demonstrate the utility of the developed metabolite assay for the development of PRMT5 therapies. The differences between tumor and NAT metabolite profiles are consistent with upregulated PRMT1 (or another Type I PRMT) in the tumor, leading to elevated ADMA, SAM, and SAH. PRMT5, on the other hand, is likely inhibited by elevated MTA levels in the MTAP -deleted tumors, making them more susceptible to MTA-cooperative PRMT5 inhibitors, despite not significantly increasing SDMA levels in the tumor.
利益披露 Disclosure
E. Stancliffe, None..
A. Mehta, None..
D. Guzior, None..
A. Richardson, None..
T. Cohen, None..
K. Cho, None.