Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues

Paul B Noto,Craig D Wagner,Helai P Mohammad,Rocio Montes De Oca,Francesca Zappacosta,Yan Liu,Roland S Annan,Charles F Mchugh,Matthew E Szapacs,Steven P Piccoli,Timothy W Sikorski,Caretha L Creasy

doi:10.1038/s41598-020-78800-6

Paul B Noto, Craig D Wagner + Show 10 more

Open Access

https://doi.org/10.1038/s41598-020-78800-6

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Abstract

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Highlights

Substrates are typically methylated on arginine residues within glycine-arginine-rich (GAR) motifs, and RBPs, such as small and heterogenous nuclear ribonucleoproteins, have been extensively characterized accounting for over 60% of asymmetric dimethyl arginine (ADMA) levels in the nucleus (10)
While a decrease in ADMA and an increase in MMA was observed in non-stimulated PBMCs, upon T Cell Receptor (TCR)-activation, treatment with GSK3368712 resulted in a robust decrease in ADMA and an increase in both MMA and symmetric dimethyl arginine (SDMA)
Type I Protein arginine methyltransferases (PRMTs) catalyze the formation of asymmetric dimethyl arginine (ADMA) on a wide range of cellular substrates spanning histones, signaling molecules and RNA splicing factors

Summary

Introduction

Substrates are typically methylated on arginine residues within glycine-arginine-rich (GAR) motifs, and RBPs, such as small and heterogenous nuclear ribonucleoproteins (sn/hnRNPs), have been extensively characterized accounting for over 60% of ADMA levels in the nucleus (10). While the pharmacodynamic effects of GSK3368715 on global arginine methylation are broad and consistent across cell types, the dynamic range and magnitude of these changes are limited, posing a challenge for use in the clinic. We describe the identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition, and the development of novel methodologies to accurately and precisely quantitate changes in the levels of ADMA on hnRNP-A1 in both blood and tumor compartments. These methodologies can be readily implemented in the clinical setting to evaluate GSK3368715 target engagement

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