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Chromatin accessibility in MC38/gp100 cells after type I PRMT inhibitor treatment

ABSTRACT: Immunotherapies have produced dramatic responses in reducing tumor growth and prolonging overall survival to unprecedented rates in melanoma, lung cancer, renal cancer and mismatch repair deficient colon cancer patients. Despite approvals for immune checkpoint inhibitors in a wide range of cancers, the majority of patients’ relapse following initial response to immunotherapy or fail to respond altogether. These failures can be attributed to an insufficient immune response against tumors and/or mechanisms co-opted by tumor cells to produce a suppressive tumor microenvironment. Therefore, identification of combination strategies to enhance the response rates of immunotherapies to a broader patient population are the current subject of intensive basic and clinical research. Type I PRMTs have been described as regulators of immune response pathways in several cell types by direct arginine methylation on specific substrates, as well as through indirect mechanisms. Recent work has suggested that arginine methylation may have a role in tumor immunity, yet these roles remain poorly understood. Using the skin cutaneous melanoma (SKCM) TCGA dataset, we show that increased expression of Type I PRMTs is associated with poor clinical response and decreased immune infiltration in melanoma patients. Through a series of preclinical tumor models, we demonstrate that inhibition of Type I PRMTs can increase hallmarks of an inflamed tumor microenvironment and sensitivity to inhibition of the PD-(L)1 signaling axis. Accordingly, Type I PRMT inhibition synergizes with immune checkpoint blockade to induce durable and effective antitumor responses in an array of immunocompetent tumor models. These data provide a rationale to combine Type I PRMT inhibitors with checkpoint blockade to maximize clinical benefit in cancer patients.

ORGANISM(S): Mus musculus

PROVIDER: GSE161909 | GEO | 2022/11/01

REPOSITORIES: GEO

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Project description:Protein arginine methylation, catalyzed by the protein arginine methyltransferase (PRMT) family, is recognized as a widespread post-translational modification (PTM) with implications in a plethora of biological processes in eukaryotes. PRMT proteins were classified into three types, type I, II and III, according to the final methyl-arginine products generated. Despite that thousands of substrates have been identified for Type I and II PRMTs, a full scope of arginine methylation catalyzed by the only type III PRMT, PRMT7, as well as its connection with that of type I and II PRMTs remain unknown, limiting our understanding of the network of arginine methylation and its functions in cells. In this study, global profiling of PRMT4 (type I), PRMT5 (type II) and PRMT7 (type III) substrates (also referred as methylome) revealed that PRMT7 methylated a GAR (glycine and arginine) motif similar as PRMT5, while PRMT4 uniquely methylated a motif in which proline was highly enriched. PRMT4, 5 and 7-methylome were all enriched with proteins functioning in mRNA splicing, and knockdown of PRMT4, 5 or 7 led to a global change of alternative splicing events, among which a cohort with implications in cancer development was co-regulated by all three PRMTs. PRMT4, 5 and 7-mediated arginine methylation on splicing factors such as hnRNPA1, though at different status, were shown to enhance RNA binding in general and participate in the regulation of PRMT4, 5 and 7 co-regulated alternative splicing events.The clinical significance of PRMT4, 5 and 7-mediated arginine methylation was underscored by the fact that these three PRMTs as well as hnRNPA1 arginine methylation were over-represented in multiple types of cancers, which were associated with aberrant gene alternative splicing. Consistently, silencing or pharmacological inhibition of PRMT4, 5 and 7 altered gene alternative splicing and suppressed cancer cell growth, and co-treatment exhibited synergistic effects. Taken together, our study provided an integrative view of substrates for the three types of PRMTs, revealing the important function of arginine methylation in RNA splicing and cancer.

Project description:Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (~0.1% of total arginine), Rme2a was highly abundant (~1.1%), and Rme2s was intermediate (~0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

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Chromatin accessibility in MC38/gp100 cells after type I PRMT inhibitor treatment

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