A multiplexed LC-MS/MS metabolite assay to enable patient stratification and pharmacodynamic monitoring for MTA-cooperative PRMT5 inhibitors

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.