Project description:Glutamyl-prolyl-tRNA synthetase (EPRS) is a unique bifunctional aminoacyl-tRNA synthetase which catalyzes the ATP-dependent aminoacylation of glutamyl and prolyl residues to cognate tRNAs, constituting fundamental building blocks in protein synthesis. Genomic data suggest that EPRS promotes disease progression and is associated with poor prognosis in multiple myeloma (MM), while downregulation of EPRS triggers MM cell apoptosis implying an amino acid starvation response as a central mechanism. We developed a novel ATP competitive inhibitor, NCP26, targeting the prolyl-tRNA synthetase (PRS) component. The inhibitor demonstrates significant anti-tumor activity against human MM cell lines, regardless of their sensitivity to other therapeutic agents, against patient MM cells and is active in vivo using a MM xenograft mouse model. In contrast to the proline competitive PRS inhibitor halofuginone, NCP26 effects are not altered by exogenous proline levels. NCP26 treatment induces G0/G1 cell cycle arrest followed by apoptotic MM cell death, evidenced by depletion of mitochondrial membrane potential as well as caspase and PARP cleavage. Using combined transcriptomic and proteomic approaches we demonstrate a complex phenotypic response involving multiple pathways in protein quality control which center around the ribosome as integrating hub. Furthermore, we identified multiple proline rich motif containing protein targets of NCP26 as downstream effectors. These include myeloma survival factors such as syndecan 1 (SDC1, CD138), or basic helix-loop-helix transcription factors such as MYC, and transcription factor 3 (TCF3) suggesting that acute blockade of prolyl- aminoacylation evokes a complex pro-apoptotic response beyond the canonical amino acid starvation and integrated stress response. Taken together, our pre-clinical studies validate EPRS as a possible therapeutic target and provide the framework for evaluation of PRS inhibitors in a clinical setting.

Project description:Purpose: In mammals, glutamyl-prolyl-tRNA synthetase (EPRS) catalyzes the attachment of two amino acids, glutamic acid (E) and proline (P), to their cognate tRNAs for protein synthesis. We aim to study the mechanism of EPRS in translational regulation of cardiac fibrosis and establish EPRS or its downstream target genes as potential anti-fibrosis therapeutic targets. The goals of this study are to use next generation sequencing (NGS)-derived fibroblast transcriptome profiling (RNA-seq) and translatome profiling (polysome-seq) followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods to identify the downstream effector genes that are preferentially regulated by EPRS at the transcriptional and translational levels. Results: Using a well established data analysis workflow from the Genomic Research Center from University of Rochester Medical Center, we analyzed RNA-seq and polysome-seq data. We have identified novel Pro-rich (PRR) gene pathways that are preferentially regulated by EPRS via enhanced translation elongation at Pro-rich codons and antagonized by Halofuginone. Considering the translation active state in both light and heavy polysome fractions, we redefined the 4 groups by overlapping genes from heavy and light polysome associated transcripts compared to their total RNA expression change (p value <0.05 for differential expressed genes). 1371 genes were downregulated at translation (indicated by polysome-seq) and steady-state mRNA (indicated by RNA-seq) levels, and 775 genes were upregulated at both levels. There are a small number of genes whose gene regulation was changed in the opposite direction at the mRNA and translation levels. These observations indicate a coordinated regulatory effect between steady-state mRNA level and translation efficiency under the EPRS inhibitory condition. KEGG analysis of the genes that are reduced at RNA and translation levels revealed multiple pro-fibrotic pathways, including ECM-receptor interaction and proteoglycans. In contrast, the genes that are induced at RNA and translation levels are enriched in general translation factors, including ribosome and aminoacyl-tRNA biosynthesis, which suggests a compensatory amino acid starvation response upon EPRS inhibition. Altered expression of typical Pro-rich collagen and other genes was confirmed with qRT-PCR. Conclusions: Our study represents the first detailed analysis of fibroblast translatome in comparison with transcriptome under EPRS inhibitory condition (halofuginone treatment), with biological triplicates, generated by RNA-seq and polysome-seq technology. Our results show that the next generation sequencing offers a comprehensive qualitative and quantitative evaluation of mRNA content and its translation state within cells treated with translation inhibitory drug halofuginone (EPRS inhibitor). Based on our results, we conclude that Pro-rich motif bearing genes are preferential targets of EPRS, and mild inhibition of EPRS leads to inefficient translation of these PRR genes.

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Inhibitor of prolyl-tRNA synthetase (PRS) to reduce tubulointerstitial nephritis

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