Author Correction: The regulation, functions and clinical relevance of arginine methylation.

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The regulation, functions and clinical relevance of arginine methylation.

Methylation of arginine residues by protein arginine methyltransferases (PRMTs) is involved in the regulation of fundamental cellular processes, including transcription, RNA processing, signal transduction cascades, the DNA damage response and liquid-liquid phase separation. Recent studies have provided considerable advances in the development of experimental tools and the identification of clinically relevant PRMT inhibitors. In this review, we discuss the regulation of PRMTs, their various cellular roles and the clinical relevance of PRMT inhibitors for the therapy of neurodegenerative diseases and cancer.

Cellular pathways influenced by protein arginine methylation: Implications for cancer.

Arginine methylation is an influential post-translational modification occurring on histones, RNA binding proteins, and many other cellular proteins, affecting their function by altering their protein-protein and protein-nucleic acid interactions. Recently, a wealth of information has been gathered, implicating protein arginine methyltransferases (PRMTs), enzymes that deposit arginine methylation, in transcription, pre-mRNA splicing, DNA damage signaling, and immune signaling with major implications for cancer therapy, especially immunotherapy. This review summarizes this recent progress and the current state of PRMT inhibitors, some in clinical trials, as promising drug targets for cancer.

SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1.

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Arginine Methylation: The Coming of Age.

Arginine methylation is a common post-translational modification functioning as an epigenetic regulator of transcription and playing key roles in pre-mRNA splicing, DNA damage signaling, mRNA translation, cell signaling, and cell fate decision. Recently, a wealth of studies using transgenic mouse models and selective PRMT inhibitors helped define physiological roles for protein arginine methyltransferases (PRMTs) linking them to diseases such as cancer and metabolic, neurodegenerative, and muscular disorders. This review describes the recent molecular advances that have been uncovered in normal and diseased mammalian cells.

Update of the UMD-VHL database: classification of 164 challenging variants based on genotype-phenotype correlation among 605 entries.

BACKGROUND: The von Hippel-Lindau (VHL) disease is a hereditary tumour syndrome caused by germline mutations in VHL tumour suppressor gene. The identification of VHL variants requires accurate classification which has an impact on patient management and genetic counselling. METHODS: The TENGEN (French oncogenetics network of neuroendocrine tumors) and PREDIR (French National Cancer Institute network for Inherited predispositions to kidney cancer) networks have collected VHL genetic variants and clinical characteristics of all VHL-suspected patients analysed from 2003 to 2021 by one of the nine laboratories performing VHL genetic testing in France. Identified variants were registered in a locus-specific database, the Universal Mutation Database-VHL database (http://www.umd.be/VHL/). RESULTS: Here we report the expert classification of 164 variants, including all missense variants (n=124), all difficult interpretation variants (n=40) and their associated phenotypes. After initial American College of Medical Genetics classification, first-round classification was performed by the VHL expert group followed by a second round for discordant and ambiguous cases. Overall, the VHL experts modified the classification of 87 variants including 30 variants of uncertain significance that were as (likely)pathogenic variants for 19, and as likely benign for 11. CONCLUSION: Consequently, this work has allowed the diagnosis and influenced the genetic counselling of 45 VHL-suspected families and can benefit to the worldwide VHL community, through this review.

Limiting the DNA Double-Strand Break Resectosome for Genome Protection.

DNA double-strand break (DSB) resection, once thought to be a simple enzymatic process, is emerging as a highly complex series of coordinated activities required to maintain genome integrity. Progress in cell biology, biochemistry, and genetics has deciphered the precise resecting activities, the regulatory components, and their ability to properly channel the resected DNA to the appropriate DNA repair pathway. Herein, we review the mechanisms of regulation of DNA resection, with an emphasis on negative regulators that prevent single-strand (ss)DNA accumulation to maintain genome stability. Interest in targeting DNA resection inhibitors is emerging because their inactivation leads to poly(ADP-ribose) polymerase inhibitor (PARPi) resistance. We also present detailed regulation of DNA resection machineries, their analysis by functional assays, and their impact on disease and PARPi resistance.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA:DNA hybrids.

Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often causes catastrophic conflicts during replication, resulting in DNA double-strand breaks and genomic instability. Preventing such conflicts requires hybrid dissolution by helicases and/or RNase H. Little is known about how such helicases are regulated. Herein, we identify DDX5, an RGG/RG motif-containing DEAD-box family RNA helicase, as crucial player in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner, leading to R-loop degradation by the XRN2 exoribonuclease. DDX5-deficient cells accumulate R-loops at loci with propensity to form such structures based on RNA:DNA immunoprecipitation (DRIP)-qPCR, causing spontaneous DNA double-strand breaks and hypersensitivity to replication stress. DDX5 associates with XRN2 and resolves R-loops at transcriptional termination regions downstream of poly(A) sites, to facilitate RNA polymerase II release associated with transcriptional termination. Protein arginine methyltransferase 5 (PRMT5) binds and methylates DDX5 at its RGG/RG motif. This motif is required for DDX5 interaction with XRN2 and repression of cellular R-loops, but not essential for DDX5 helicase enzymatic activity. PRMT5-deficient cells accumulate R-loops, resulting in increased formation of γH2AX foci. Our findings exemplify a mechanism by which an RNA helicase is modulated by arginine methylation to resolve R-loops, and its potential role in regulating transcription.

[Communicating uncertainty: a necessary harm?] / Communiquer l'incertitude : un mal nécessaire ?

Uncertainty is omnipresent in medical practice. It occurs when the available data do not allow predicting with accuracy the patient's outcome. Paradoxically, despite the exponential progress of medical knowledge, uncertainty in medical practice seems to increase. Even though uncertainty is present at every stage of medical practice, this uncertainty is rarely discussed with the patient. However, there is an ethical and legal necessity to communicate it with the patient. The aim of this review is to describe the barriers and impacts of the communication of uncertainty with the patient and suggest strategies to enhance its communication.

L'incertitude est omniprésente dans la pratique médicale. Elle apparaît quand les données à disposition ne permettent pas de prédire avec exactitude le devenir du patient. Paradoxalement, malgré l'augmentation des connaissances médicales, elle semble s'accroître dans la pratique médicale. Bien que présente à chaque étape de la prise en charge du patient, cette incertitude est peu discutée avec lui. Cependant, il existe une nécessité éthique et légale à sa communication avec le patient. Cet article a pour but de décrire les barrières et les conséquences de la communication de l'incertitude avec le patient et de proposer des stratégies pour améliorer sa communication.

Novel germline MET pathogenic variants in French patients with papillary renal cell carcinomas type I.

Hereditary papillary renal cell carcinoma (HPRC) is a rare inherited renal cancer syndrome characterized by bilateral and multifocal papillary type 1 renal tumors (PRCC1). Activating germline pathogenic variants of the MET gene were identified in HPRC families. We reviewed the medical and molecular records of a large French series of 158 patients screened for MET oncogenic variants. MET pathogenic and likely pathogenic variants rate was 12.4% with 40.6% among patients with familial PRCC1 and 5% among patients with sporadic PRCC1. The phenotype in cases with MET pathogenic and likely pathogenic variants was characteristic: PRCC1 tumors were mainly bilateral (84.3%) and multifocal (87.5%). Histologically, six out of seven patients with MET pathogenic variant harbored biphasic squamoid alveolar PRCC. Genetic screening identified one novel pathogenic variant MET c.3389T>C, p.(Leu1130Ser) and three novel likely pathogenic variants: MET c.3257A>T, p.(His1086Leu); MET c.3305T>C, p.(Ile1102Thr) and MET c.3373T>G, p.(Cys1125Gly). Functional assay confirmed their oncogenic effect as they induced an abnormal focus formation. The genotype-phenotype correlation between MET pathogenic variants and PRCC1 presentation should encourage to widen the screening, especially toward nonfamilial PRCC1. This precise phenotype also constitutes a strong argument for the classification of novel missense variants within the tyrosine kinase domain when functional assays are not accessible.

Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication.

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein-mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.

Arsenic 3 methyltransferase (AS3MT) automethylates on cysteine residues in vitro.

Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.

Genetic evidence for partial redundancy between the arginine methyltransferases CARM1 and PRMT6.

CARM1 is a protein arginine methyltransferase (PRMT) that acts as a coactivator in a number of transcriptional programs. CARM1 orchestrates this coactivator activity in part by depositing the H3R17me2a histone mark in the vicinity of gene promoters that it regulates. However, the gross levels of H3R17me2a in CARM1 KO mice did not significantly decrease, indicating that other PRMT(s) may compensate for this loss. We thus performed a screen of type I PRMTs, which revealed that PRMT6 can also deposit the H3R17me2a mark in vitro CARM1 knockout mice are perinatally lethal and display a reduced fetal size, whereas PRMT6 null mice are viable, which permits the generation of double knockouts. Embryos that are null for both CARM1 and PRMT6 are noticeably smaller than CARM1 null embryos, providing in vivo evidence of redundancy. Mouse embryonic fibroblasts (MEFs) from the double knockout embryos display an absence of the H3R17me2a mark during mitosis and increased signs of DNA damage. Moreover, using the combination of CARM1 and PRMT6 inhibitors suppresses the cell proliferation of WT MEFs, suggesting a synergistic effect between CARM1 and PRMT6 inhibitions. These studies provide direct evidence that PRMT6 also deposits the H3R17me2a mark and acts redundantly with CARM1.

MET alterations in biphasic squamoid alveolar papillary renal cell carcinomas and clinicopathological features.

Biphasic squamoid alveolar papillary renal cell carcinoma (BSA-PRCC) is a recently studied lesion considered a morphologic variant of papillary renal cell carcinoma (RCC), more closely related to type 1. Considering the role of proto-oncogene MET in both sporadic type 1 papillary RCC and hereditary papillary RCC, we aimed to explore the role of MET activation in the oncogenesis of BSA-PRCC. We identified 17 patients with either unique (n = 14) or multiple (n = 3) BSA-PRCC, all localized, and performed an integrative analysis of MET status in 18 formalin-fixed paraffin-embedded tumors combining next-generation sequencing analysis, fluorescent in situ hybridization and immunohistochemistry. Trisomy 7 was found in 86% of tumors (14/16) without MET amplification at 7q31 (15/15). A pathogenic MET genetic variant was identified in 60% (9/15) of cases, at the germline level in 57% (4/7) of tested patients or at the somatic level (5/11). MET expression was observed in all tumors with a higher value of combined score in large cells (mean 97%, range 80-100%) than in small cells (mean 74%, range 10-100%) and was lower in two cases without MET copy number gain. In conclusion, our study provides additional evidence to consider biphasic squamoid alveolar papillary RCC as a morphological variant of type 1 papillary renal RCC. Our data strongly suggest that MET represents a major oncogenic driver gene in BSA-PRCC, harboring a higher frequency of MET mutation that encourages to further explore the benefice of anti-MET targeted therapies for aggressive BSA-PRCC.

CRAF Methylation by PRMT6 Regulates Aerobic Glycolysis-Driven Hepatocarcinogenesis via ERK-Dependent PKM2 Nuclear Relocalization and Activation.

BACKGROUND AND AIMS: Most tumor cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and promote tumorigenicity and drug resistance. Intriguingly, the molecular mechanisms underlying this phenomenon are not well understood. In this work, using gain-of-function and loss-of-function in vitro studies in patient-derived organoid and cell cultures as well as in vivo positron emission tomography-magnetic resonance imaging animal models, we showed that protein arginine N-methyltransferase 6 (PRMT6) regulates aerobic glycolysis in human hepatocellular carcinoma (HCC) through nuclear relocalization of pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. APPROACH AND RESULTS: We found PRMT6 to methylate CRAF at arginine 100, interfering with its RAS/RAF binding potential, and therefore altering extracellular signal-regulated kinase (ERK)-mediated PKM2 translocation into the nucleus. This altered PRMT6-ERK-PKM2 signaling axis was further confirmed in both a HCC mouse model with endogenous knockout of PRMT6 as well as in HCC clinical samples. We also identified PRMT6 as a target of hypoxia through the transcriptional repressor element 1-silencing transcription factor, linking PRMT6 with hypoxia in driving glycolytic events. Finally, we showed as a proof of concept the therapeutic potential of using 2-deoxyglucose, a glycolysis inhibitor, to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC. CONCLUSIONS: Our findings indicate that the PRMT6-ERK-PKM2 regulatory axis is an important determinant of the Warburg effect in tumor cells, and provide a mechanistic link among tumorigenicity, sorafenib resistance, and glucose metabolism.

Defining the RGG/RG motif.

Motifs rich in arginines and glycines were recognized several decades ago to play functional roles and were termed glycine-arginine-rich (GAR) domains and/or RGG boxes. We review here the evolving functions of the RGG box along with several sequence variations that we collectively term the RGG/RG motif. Greater than 1,000 human proteins harbor the RGG/RG motif, and these proteins influence numerous physiological processes such as transcription, pre-mRNA splicing, DNA damage signaling, mRNA translation, and the regulation of apoptosis. In particular, we discuss the role of the RGG/RG motif in mediating nucleic acid and protein interactions, a function that is often regulated by arginine methylation and partner-binding proteins. The physiological relevance of the RGG/RG motif is highlighted by its association with several diseases including neurological and neuromuscular diseases and cancer. Herein, we discuss the evidence for the emerging diverse functionality of this important motif.

Germline mutations in the new E1' cryptic exon of the VHL gene in patients with tumours of von Hippel-Lindau disease spectrum or with paraganglioma.

BACKGROUNDS: The incidence of germline mutations in the newly discovered cryptic exon (E1') of VHL gene in patients with von Hippel-Lindau (VHL) disease and in patients with paraganglioma or pheochromocytoma (PPGL) is not currently known. METHODS: We studied a large international multicentre cohort of 1167 patients with a previous negative genetic testing. Germline DNA from 75 patients with a single tumour of the VHL spectrum ('Single VHL tumour' cohort), 70 patients with multiple tumours of the VHL spectrum ('Multiple VHL tumours' cohort), 76 patients with a VHL disease as described in the literature ('VHL-like' cohort) and 946 patients with a PPGL were screened for E1' genetic variants. RESULTS: Six different genetic variants in E1' were detected in 12 patients. Two were classified as pathogenic, 3 as variants of unknown significance and 1 as benign. The rs139622356 was found in seven unrelated patients but described in only 16 patients out of the 31 390 of the Genome Aggregation Database (p<0.0001) suggesting that this variant might be either a recurrent mutation or a modifier mutation conferring a risk for the development of tumours and cancers of the VHL spectrum. CONCLUSIONS: VHL E1' cryptic exon mutations contribute to 1.32% (1/76) of 'VHL-like' cohort and to 0.11% (1/946) of PPGL cohort and should be screened in patients with clinical suspicion of VHL, and added to panels for Next Generation Sequencing (NGS) diagnostic testing of hereditary PPGL. Our data highlight the importance of studying variants identified in deep intronic sequences, which would have been missed by examining only coding sequences of genes/exomes. These variants will likely be more frequently detected and studied with the upcoming implementation of whole-genome sequencing into clinical practice.

Machine learning model to predict oncologic outcomes for drugs in randomized clinical trials.

Predicting oncologic outcome is challenging due to the diversity of cancer histologies and the complex network of underlying biological factors. In this study, we determine whether machine learning (ML) can extract meaningful associations between oncologic outcome and clinical trial, drug-related biomarker and molecular profile information. We analyzed therapeutic clinical trials corresponding to 1102 oncologic outcomes from 104 758 cancer patients with advanced colorectal adenocarcinoma, pancreatic adenocarcinoma, melanoma and nonsmall-cell lung cancer. For each intervention arm, a dataset with the following attributes was curated: line of treatment, the number of cytotoxic chemotherapies, small-molecule inhibitors, or monoclonal antibody agents, drug class, molecular alteration status of the clinical arm's population, cancer type, probability of drug sensitivity (PDS) (integrating the status of genomic, transcriptomic and proteomic biomarkers in the population of interest) and outcome. A total of 467 progression-free survival (PFS) and 369 overall survival (OS) data points were used as training sets to build our ML (random forest) model. Cross-validation sets were used for PFS and OS, obtaining correlation coefficients (r) of 0.82 and 0.70, respectively (outcome vs model's parameters). A total of 156 PFS and 110 OS data points were used as test sets. The Spearman correlation (rs ) between predicted and actual outcomes was statistically significant (PFS: rs = 0.879, OS: rs = 0.878, P < .0001). The better outcome arm was predicted in 81% (PFS: N = 59/73, z = 5.24, P < .0001) and 71% (OS: N = 37/52, z = 2.91, P = .004) of randomized trials. The success of our algorithm to predict clinical outcome may be exploitable as a model to optimize clinical trial design with pharmaceutical agents.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.