The role of protein arginine methylation 5 in DNA damage repair and cancer therapy

Protein arginine methylation, a post-translational modification (PTM), is fundamental in regulating protein function and stability. Among the nine protein methyl transferases (PRMT), PRMT5 plays a critical role in promoting oncogenic processes including tumor proliferation, invasiveness, immune escape and DNA damage repair through different signaling pathways. It is also a target in cancer therapy, with numerous inhibitors in clinical trial. In this review, we focus on the biological functions of PRMT5 in DNA damage repair and maintenance of genome stability in cancer, and summarize the development advance of PRMT5 inhibitors in cancer therapy.


Introduction
Post-translational modification (PTM) is a fundamental protein regulation that realizes or abolishes the function of a protein.Methylation modification on arginine is a common PTM in mammals, catalyzed by the protein arginine methyltransferases (PRMTs) family, which includes nine members from PRMT1 to PRMT9 (Hwang et al., 2021).The methyl groups provided by S-adenosylmethionine (SAM) are transferred by PRMTs to the guanidinium nitrogen atoms of the arginine residue, forming various types of methylated arginine in the substrate protein (Wang et al., 2018).PRMTs are classified into three types depending on the type of methylation modification they catalyze (Fig. 1).PRMT5, a member of the type II PRMT, which consists of 637 amino acids, is gaining increasing attention due to its critical role in cancer and other diseases (Kim & Ronai, 2020;Motolani et al., 2021).PRMT5 catalyzes mono-and symmetric dimethylation modifications on arginine.Its substrate specificity is highly dependent on RGG/RG sequences (PRMT5 target spots), especially the arginine residue, both sides of which are linked to glycine (Gly-Arg-Gly sequence) (Musiani et al., 2019) (Fig. 2).PRMT5 substrates include both histone and non-histone proteins.For histone substrates, particularly the N-terminal histone tails such as H2AR3, H3R2, H3R8, and H4R3, symmetric dimethylation of the aforementioned PRMT5-modified arginines recruits different readers to activate or suppress gene transcription (Kim & Ronai, 2020); for non-histone substrate proteins, methylation of the arginines induce functional alteration in the substrates involving different signaling pathways.
PRMT5 has complicated biological function in cancer cells and plays a critical role in many important cellular processes such as transcription, splicing, translation, metabolism, signal transduction and DNA damage repair (DDR) (Yuan et al., 2021).PRMT5 is not only a star molecule in oncogenesis research, but also a valuable target for cancer therapy with a series of inhibitors in clinical trials or approved by the FDA (Wu et al., 2021).Here, we focused on the role of PRMT5 in responding to DNA damage and maintaining genome stability to better understand its potential in cancer research and therapy.

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The role of PRMT5 in DNA damage repair and genome stability maintenance PRMT5 plays a fundamental role in DDR by multiple pathways.By epigenetic regulation, PRMT5 promotes the expression of DDR genes through histone arginine methylation.In prostate cancer, PRMT5 cooperates with partner proteins such as plCln and WDR77 to form an epigenetic activator that upregulates the expression of DDR genes involved in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, such as KU70/80, RAD51 and BRCA1/2 (Owens et al., 2020).In malignant glioblastoma, PRMT5 promotes the transcription of RNF168, an E3 ligase that activates H2AX and prevents its degradation to enhance the DNA damage response and induce tumor chemoradiation resistance (Du et al., 2019).In addition to double-strand break (DSB) repair, PRMT5  Translation initiation factor PRMT5 participates in the interaction between eIF4E and 5′-cap structure of c-Myc or cyclin D1 mRNA DDR and cell cycle is regulated also facilitates inter-cross-link damage (ICL) repair through epigenetic regulation (Du et al., 2021).For example, PRMT5 upregulates H3R2me1 in the promoter regions of FA genes, promoting their expression in cancer cells, resulting in cellular resistance to ICL reagents (Du et al., 2021).Beside histone arginine methylation, PRMT5 also regulates the expression of DDR proteins by targeting RNA, including splicing and RNA m6A modification.PRMT5 has been shown to play an essential role in maintaining splicing fidelity and genome-level stability by methylating Sm proteins (Sachamitr et al., 2021).In PRMT5-deficient hematopoietic stem cells, gene expression is impaired and DDR-related gene splicing is abnormal, exon skipping and intron retention events increase significantly (Tan et al., 2019).For example, loss of PRMT5 leads to abnormal splicing of TIP60 and SUV4-20H2, both of which play critical roles in chromosome remodeling and DDR protein recruitment during DSB repair (Hamard et al., 2018).PRMT5 also promotes BRCA1 expression after doxorubicin treatment in breast cancer, where PRMT5 attenuates m6A methylation of BRCA1 mRNA, thereby enhancing its stability through methylation of RNA demethylase AlkB homolog 5 (ALKBH5) resulting from translocation of ALKBH5 from the nucleus to the cytoplasm for demethylation of its mRNA substrates including BRCA1 (Wu et al., 2022).
Another regulatory pathway of PRMT5 for genome stability is PTM.Several DDR proteins have been identified as substrates for PRMT5, and their symmetrically dimethylated arginine residues regulate their functions, stability, DNAbinding ability, and interaction with other proteins.A typical example is p53, a central protein in the DDR that maintains genome stability, whose R335 and R337 have been dimethylated by PRMT5 (Hwang et al., 2021).Methylated p53 not only enhances its function in DNA damage response and cell cycle arrest, but also promotes the expression of other DDR genes such as the FA family (Du et al., 2016).Similarly, p53-binding protein 1 (53BP1) is also a substrate of PRMT5, whose R1355 has been methylated by PRMT5 (Hwang et al., 2020).53BP1 functions as a scaffold protein for the recruitment of DDR proteins to damaged chromatin and promotes NHEJ signaling pathways by limiting end resection after a double-strand break (Clarke et al., 2017).Methylated 53BP1 enhances its proteostasis to facilitate DNA repair through the NHEJ pathway, which could be blocked by Src, a kinase that catalyzes inhibitory phosphorylation of PRMT5 at Y324 (Hwang et al., 2020).In addition to DSB repair, PRMT5 also promotes DNA single-stranded damage (SSB) repair ability by methylating critical members involved in base excision repair (BER) and nucleotide excision repair (NER).PRMT5 enhances the efficiency of BER, by mediating symmetric dimethylation of Flap endonuclease 1 (FEN1) at its R192 (Guo et al., 2010), thereby improving cellular resistance to oxidation-induced DNA damage.For the NER process, PRMT5 dimethylates TDP1 at its R361 and R586, increasing its binding affinity to topoisomerase 1 (Top1) to form a complex that repairs damaged single-stranded DNA (Rehman et al., 2018).Arginine methylation of TDP1 also increases the association of XRCC1 with TDP1 in response to camptothecin, a Top1 inhibitor that causes DNA damage (Rehman et al., 2018).
Above all, PRMT5 modulates DDR and maintains genome stability by multiple pathways.We summarized the targets and regulation mechanism of PRMT5 in DDR process, see Table 1 and Fig. 3.

Targeting PRMT5 in cancer therapy
Various PRMT5 inhibitors have been developed and translated into clinical trials (Table 2).Currently, the most commonly used pharmacological approaches for PRMT5 inhibitors include SAM competitive inhibitors, SAM noncompetitive inhibitors, and substrate-competitive inhibitors (Fu et al., 2022).In the clinic, PRMT5 inhibitors are classified into two generations according to their target binding site: First-generation inhibitors target the PRMT5 protein directly (e.x.PF0693999) and second-generation inhibitors target the PRMT5-MTA complex (e.x.MRTX1719).Based on the reports from ongoing or finished clinical trials, the therapeutic outcome of PRMT5 inhibitors are encouraging.For example, in a clinical trial organized by MD Anderson Cancer Center, PF-06939999 was proved to dose-dependent and manageable toxicities and 6 mg QD was identified as the recommended monotherapy dose for expansion (NCT03854227).In another multi-center clinical trial, the PRMT5 inhibitor PRT543 showed an excellent anti-inflammation effect and symptom remission rate in selected cancer patients; moreover, a prolonged progression free survival was observed in the patients with refractory myelofibrosis (NCT03886831).With regards to safety, the first-generation inhibitors have been reported to have a high incidence of severe adverse events in clinical trials, including thrombocytopenia, anemia, and gastrointestinal reactions, putting their clinical use at risk (Feustel & Falchook, 2022).In contrast, the second-generation PRMT5 inhibitors were clinically well tolerated and showed acceptable toxicity.The development of second-generation PRMT5 inhibitors is based on the understanding of the enzyme MTAP, which catalyzes the cleavage of methylthioadenosine (MTA) into adenine and 5-methylthioribose-1-phosphate (Bertino et al., 2011).Deletion of MTAP is observed in many cancers, resulting in accumulation of MTA that binds to PRMT5 and attenuates its enzyme activity, leading to lethal synthesis of PRMT5 and MTAP (Kryukov et al., 2016).The second-generation PRMT5 inhibitors mimic MTA to inhibit PRMT5 activity, with optimal clinical effect and acceptable toxicity (Smith et al., 2022).In addition, some marketed drugs and plantderived natural reagents also have inhibitory effects on PRMT5 (Liu et al., 2022;Prabhu et al., 2023), highlighting their epigenetic and DDR regulatory effects and therapeutic potential in cancer treatment.In summary, given the importance of PRMT5 for DDR and maintenance of genome stability, deciphering mechanisms underlying PRMT5 expression, activity, and subcellular localization will advance the development of PRMT5related therapies in cancer.Although many questions remain about PRMT5, such as the upstream activation pathway of PRMT5 and its shuttle mechanism between the cytoplasm and nucleus, we strongly believe that PRMT5 can be profoundly understood with the help of comprehensive research and will transform cancer therapy in the future with increasingly encouraging clinical trial results.

Declarations
Conflict of interest There is no conflict of interests among the authors.
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Fig. 3
Fig. 3 Regulatory mechanism of PRMT5 to DNA damage repair

Table 2
The clinical trial of PRMT5 inhibitors