Quantitative Global Proteomics of Formalin-Fixed Paraffin-Embedded Tumors Identifies Dimethylated G3BP1 at Arginine 460 as a Robust Pharmacodynamic Biomarker of Methylthioadenosine Phosphorylase-Selective Protein Arginine Methyltransferase 5 Inhibition

MTA-cooperative inhibition of protein arginine methyltransferase 5 (PRMT5) is synthetic lethal with methylthioadenosine phosphorylase (MTAP)-deficient cancers. PRMT5's enzymatic activity can be assessed by measuring symmetric dimethylarginine (SDMA) modification levels of protein substrates. However, conventional assays that attempt to measure total SDMA levels lack the specificity to measure individual PRMT5 substrates, and thus potentially reduce selectivity and sensitivity. This study aims to identify and characterize specific DMA peptides that can serve as clinical pharmacodynamic (PD) biomarkers for PRMT5 inhibition in formalin-fixed paraffin-embedded solid tumors, leveraging data-independent acquisition-mass spectrometry (DIA-MS)-based global proteomics without additional DMA enrichment. We evaluated 145 DMA peptides in xenograft models treated by a novel MTAP-selective PRMT5 inhibitor, AZD3470. Arginine dimethylation of G3BP1 at residue R460 (G3BP1(R460)) was identified as the primary PD biomarker for PRMT5 inhibition due to its high abundance and significant post-treatment reduction that correlated with dose. The global proteomics assay with a limit of detection/quantification characterized the quantitative performance and allowed for confident detection of G3BP1(R460). Using this quantitative assay, more than 90% reduction in G3BP1(R460) modification at 100 mg/kg was reproducibly detected in MTAP-null xenograft models. Our findings suggested that the DIA-MS proteomics assay can provide high specificity and sensitivity in the detection of PRMT5 inhibition.