Overexpression Of Protein Arginine Methyltransferase 5 Research Articles

Protein arginine methyltransferase 6 mediates antiviral immunity in plants

Viral suppressor RNA silencing (VSR) is essential for successful infection. Nucleotide-binding and leucine-rich repeat (NLR)-based and autophagy-mediated immune responses have been reported to target VSR as counter-defense strategies. Here, we report a protein arginine methyltransferase 6 (PRMT6)-mediated defense mechanism targeting VSR. The knockout and overexpression of PRMT6 in tomato plants lead to enhanced and reduced disease symptoms, respectively, during tomato bush stunt virus (TBSV) infection. PRMT6 interacts with and inhibits the VSR function of TBSV P19 by methylating its key arginine residues R43 and R115, thereby reducing its dimerization and small RNA-binding activities. Analysis of the natural tomato population reveals that two major alleles associated with high and low levels of PRMT6 expression are significantly associated with high and low levels of viral resistance, respectively. Our study establishes PRMT6-mediated arginine methylation of VSR as a mechanism of plant immunity against viruses.

PRMT6 facilitates EZH2 protein stability by inhibiting TRAF6-mediated ubiquitination degradation to promote glioblastoma cell invasion and migration

Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.

Abstract 1725: Role of PRMT5 in neuroendocrine prostate cancer development

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer responsible for an estimated 20-30% of castration resistant prostate cancer (CRPC) deaths. While NEPC can arise de novo, the majority of these cases emerge as treatment-induced NEPC (tNEPC). Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Methods: Here we generated an in vitro cell culture model and mouse model to mimic the clinical conditions in androgen signaling inhibitor (ASI) or androgen deprivation treatment (ADT) We developed an in vitro cell culture and in vivo model to mimic the clinical conditions in enzalutamide-resistant prostate cancer. Regulation and role of protein arginine methyltransferase 5 (PRMT5) and its cofactor methylosome protein 50 (MEP50) were determined by treating cells and mice tumors with doxycycline inducible-shRNAs and catalytic inhibitors followed by analysis of cell viability, neurite growth, and effects on neuroendocrine related gene transcription. Pro-NEPC development effects were analyzed by immunofluorescence, immunohistochemical analysis, and western blot. PRMT5:MEP50-dependent methyltransferase activity was further validated by observing histone deposition levels via western blot and ChIP-qPCR. For clinical correlation, PRMT5 and MEP50 expression levels were comprehensively analyzed in both CRPC and NEPC patient prostate tissue samples. Results: Our clinical and computational analyses have identified increased expression of PRMT5:MEP50 in NEPC. Overexpression of PRMT5 and MEP50 in prostate cancer cells is sufficient to induce neuroendocrine differentiation (NED) in prostate cancer cells. Gene depletion of PRMT5 and MEP50 prevented the NE phenotype induced by androgen receptor inhibition and resensitized the prostate cancer cells to ASI/ADT. PRMT5 and MEP50 facilitate chromatin modulation through the dimethylation of H4R3, which is highly correlated with the NED phenotype. Moreover, overexpression of PRMT5 and MEP50 in mouse prostate induces NEPC development, and gene downregulation can prevent NED in a xenograft mouse model. Elevated PRMT5 and MEP50 correlated with increased recurrence in prostate cancer patients receiving ADT and their recurrence probability after the treatment. Conclusion: Our data establish PRMT5:MEP50 as a key epigenetic regulator in NEPC and provide a potential predictive biomarker for tNEPC. Citation Format: Hye Seung Nam, Xuehong Deng, Chang-Deng Hu. Role of PRMT5 in neuroendocrine prostate cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1725.

Abstract 4663: Establishing the role of PRMT5 in lung cancer tumorigenicity and modulation of expression in smokers

Abstract Studies show that PRMT5 (protein arginine methyl transferase 5) is responsible for increased tumorigenicity by activation of the PI3K/Akt signaling pathway promoting increased cancer cell proliferation. However, the role of PRMT5 in modulating EGFR TKI resistance is minimally studied. We hypothesize that over-expression of PRMT5 could promote tumorigenesis and TKI resistance by activating PI3K-Akt-mTOR signaling pathways. Hence, modulating this key biomarker may help reduce tumorigenicity and overcome TKI resistance. We performed qPCR to study the expression of PRMT5 in normal lung cells and NSCLC cell lines. We also compared the expression of these biomarkers in erlotinib resistant (ER) and Osimertinib resistant (OR) cells using Western blotting. PRMT5 was inhibited using GSK591 (PRMT5i) to evaluate its sensitivity to EGFR TKIs (ER and OR). PRMT5 expression in normal lung cells and NSCLC cells treated with Cigarette smoke extract (CSE) were also studied and correlated with immunostaining results from smoker and non-smoker lung tumor tissue samples. Changes in PRMT5 expression were analyzed in WT-EGFR lung cancer cell lines (H358, H2170), mutant EGFR lung cancer cell lines (H3255, H1975, PC9) and normal lung fibroblast cells (HLF). PRMT5 expression was upregulated by 1.6-2.1 fold in HLF, H2170 and H358 cells after 1 week of treatment with CSE. CSE treatment also upregulated expression of inflammatory cytokines (IL-1β, IL-8 and TNF-α) in H358 cells by 1.6-7 fold as analyzed by qPCR. PRMT5 expression was upregulated in EGFR-TKI resistant cell lines, by 1.5-1.9 fold in PC9ER, PC9OR and H2170ER cells, compared to parental cells. Similar results were found using qPCR, with 1.6-2.3 fold increase in PRMT5 mRNA expression in H2170OR, PC9ER/OR and H3255OR cells compared to parental cells. An MTT assay was done to assess percentage viability of cells after treatment of PRMT5i. While 2μM PRMT5i treatment of PC9OR cells decreased cell viability by 42%, an inhibitory effect that was more than additive was seen with a combination of 2μM of PRMT5i and 21nM of OR. Similar results were also observed in H2170ER cells. Migratory properties of OR resistant cells, were studied by wound healing assays. Percentage wound area in PRMT5i treated PC9OR cells was 29.3% as compared to 6.1% and 10.5% in diluent and OR treated cells respectively after 48 hrs. Combination treatment of PRMT5i and OR further inhibited cell migration with higher wound area of 41.7%. Immunostaining of lung tumor sections from smokers and non-smokers showed high expression of PRMT5 localized in the cytoplasm and nucleus. Microscopic analysis of 20 tumor sections from smokers showed higher expression of PRMT5 (p<0.05) compared to non-smokers. We can conclude that PRMT5 is upregulated in TKI resistant cells causing tumorigenesis. Modulation of PRMT5 can prove to be groundbreaking and revolutionary, as it may increase the efficacy of EGFR TKIs. Citation Format: Syed N. Ali, Meet Patel, Bamby Dieng, Cody Lund, Neelu Puri. Establishing the role of PRMT5 in lung cancer tumorigenicity and modulation of expression in smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4663.

PRMT6-FOXO3A ATTENUATES APOPTOSIS BY UPREGULATING PARKIN EXPRESSION IN INTESTINAL ISCHEMIA-REPERFUSION INJURY.

Intestinal ischemia-reperfusion injury (IIRI) is a serious disease with high morbidity and mortality. This study aims to investigate the potential regulatory mechanisms involving protein arginine methyltransferase 6 (PRMT6), Forkhead box O3a (FoxO3a), and Parkin in IIRI and elucidate their roles in mediating cell apoptosis. The IIRI animal model was established and confirmed using hematoxylin and eosin staining. Oxygen-glucose deprivation and reperfusion (OGD/R) cell model was established to mimic ischemic injury in vitro . Transient transfection was used to overexpress or knock down genes. Cell death or apoptosis was assessed by propidium iodide staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and flow cytometry. The expression of proteins was detected by western blot. The histopathology observed by hematoxylin and eosin staining suggested that the IIRI animal model was successfully established. Our findings revealed that IIRI resulted in increased Bax and decreased Bcl-2 levels. In vitro experiments showed that overexpression of Parkin decreased OGD/R injury and suppressed elevation of Bax/Bcl-2. PRMT6 regulated the methylation level of FoxO3a. Moreover, FoxO3a directly binds to Parkin, and FoxO3a overexpression reduced OGD/R-induced cell death and regulation of Parkin. Overexpression of PRMT6 can attenuate the downregulation of Parkin and elevation of Bax/Bcl-2 caused by OGD/R. Knockdown of PRMT6 promoted apoptosis in intestinal epithelial cells of OGD/R group, while PRMT6 overexpression exhibited the opposite effect. Notably, the levels of PRMT6, FoxO3a, and Parkin were decreased in IIRI mouse intestinal tissue. Knocking out PRMT6 causes a significant decrease in the lifespan of mice. Altogether, our results demonstrated that PRMT6 upregulated the expression of Parkin by regulating FoxO3a methylation level, attenuating the apoptosis induced by IIRI.

PRMT1 accelerates cell proliferation, migration, and tumor growth by upregulating ZEB1/H4R3me2as in thyroid carcinoma.

Thyroid carcinoma (TC) represents the most prevalent malignancy of the endocrine system. Protein arginine methyltransferase 1 (PRMT1) is a critical member of the protein arginine methyltransferase family in mammals and is involved in multiple biological processes. This study aimed to investigate the function of PRMT1 in TC. In the present study, human TC cell lines (8505C, CAL62, and BCPAP) and a normal human thyroid cell line Nthy‑ori 3‑1 were employed. Small interfering RNA targeting PRMT1 was used to knock down PRMT1 expression in 8505C cells, and PRMT1 overexpression plasmids were transfected into BCPAP cells. Cell viability was assessed using a CCK‑8 and colony formation assays. Apoptosis was measured using flow cytometry and TUNEL assays. Cell migration was assessed using wound healing and Transwell assays. Reverse transcription‑quantitative PCR was used to determine the mRNA expression levels of PRMT1. Western blotting was used to detect the protein expression levels of PRMT1, E‑cadherin, vimentin, H4R3me2as, and zinc‑finger E homeobox‑binding 1 (ZEB1). Notably, PRMT1 expression was elevated in TC (P<0.01). PRMT1 knockdown inhibited TC cell proliferation and migration and concurrently enhanced migration. Furthermore, PRMT1 knockdown suppressed tumor growth and metastasis in a mouse model of TC. PRMT1 downregulation increased E‑cadherin expression and decreased the expression of vimentin, H4R3me2as, and ZEB1 in the TC cells and the mouse model of TC. Conversely, PRMT1 overexpression had the opposite effect on TC malignant characteristics (P<0.05). These findings suggest that PRMT1 knockdown inhibited TC malignancy by downregulating H4R3me2as/ZEB1, thereby highlighting novel therapeutic targets and diagnostic markers for the management of TC.

Protein arginine methyltransferase 5 mediates arginine symmetric dimethylation of influenza A virus PB2 and supports viral replication.

Influenza A virus (IAV) relies on intricate and highly coordinated associations with host factors for efficient replication and transmission. Characterization of such factors holds great significance for development of anti-IAV drugs. Our study identified protein arginine methyltransferase 5 (PRMT5) as a novel host factor indispensable for IAV replication. Silencing PRMT5 resulted in drastic repression of IAV replication. Our findings revealed that PRMT5 interacts with each protein component of viral ribonucleoproteins (vRNPs) and promotes arginine symmetric dimethylation of polymerase basic 2 (PB2). Overexpression of PRMT5 enhanced viral polymerase activity in a dose-dependent manner, emphasizing its role in genome transcription and replication of IAV. Moreover, analysis of PB2 protein sequences across various subtypes of IAVs demonstrated the high conservation of potential RG motifs recognized by PRMT5. Overall, our study suggests that PRMT5 supports IAV replication by facilitating viral polymerase activity by interacting with PB2 and promoting its arginine symmetric dimethylation. This study deepens our understanding of how IAV manipulates host factors to facilitate its replication and highlights the great potential of PRMT5 to serve as an anti-IAV therapeutic target.

Protein arginine methyltransferase 5-mediated arginine methylation stabilizes Kruppel-like factor 4 to accelerate neointimal formation.

Accumulating evidence supports the indispensable role of protein arginine methyltransferase 5 (PRMT5) in the pathological progression of several human cancers. As an important enzyme-regulating protein methylation, how PRMT5 participates in vascular remodelling remains unknown. The aim of this study was to investigate the role and underlying mechanism of PRMT5 in neointimal formation and to evaluate its potential as an effective therapeutic target for the condition. Aberrant PRMT5 overexpression was positively correlated with clinical carotid arterial stenosis. Vascular smooth muscle cell (SMC)-specific PRMT5 knockout inhibited intimal hyperplasia with an enhanced expression of contractile markers in mice. Conversely, PRMT5 overexpression inhibited SMC contractile markers and promoted intimal hyperplasia. Furthermore, we showed that PRMT5 promoted SMC phenotypic switching by stabilizing Kruppel-like factor 4 (KLF4). Mechanistically, PRMT5-mediated KLF4 methylation inhibited ubiquitin-dependent proteolysis of KLF4, leading to a disruption of myocardin (MYOCD)-serum response factor (SRF) interaction and MYOCD-SRF-mediated the transcription of SMC contractile markers. Our data demonstrated that PRMT5 critically mediated vascular remodelling by promoting KLF4-mediated SMC phenotypic conversion and consequently the progression of intimal hyperplasia. Therefore, PRMT5 may represent a potential therapeutic target for intimal hyperplasia-associated vascular diseases.

Constitutive PRMT5 Expression Drives Spontaneous Lymphoblastic Lymphoma In Vivo

Protein arginine methyltransferase 5 (PRMT5) is a highly conserved enzyme that catalyzes monomethylation and symmetric dimethylation of arginine residues on many target proteins, including histones and non-histone proteins (P53, P65/NFκB, E2F1, AKT and spliceosome proteins). (Shailesh, 2018) PRMT5 is overexpressed in numerous solid and hematologic malignancies. (Yang, 2013) It is associated with several oncogenic mechanisms, from driving the malignant phenotype to promoting tumor cell survival and invasiveness. While preliminary data has shown PRMT5 to be upregulated in T-ALL/LBL, the mechanism by which PRMT5 promotes T-ALL/LBL has yet to be established. (Mei, 2019) Preclinical transgenic models of leukemia and lymphoma driven by a variety of oncogenes (MYC, NOTCH) have identified PRMT5 activity as a critical component for the driver phenotype. (Li, 2015) To test the hypothesis that overexpression of this enzyme is capable of malignant driver activity, we developed a transgenic (Tg) mouse model designed to over-express PRMT5 in the lymphoid compartment. (Figure 1) Tg mice were created by injecting a linearized pBH-Eμ-PRMT5 vector into FVB/N pronuclei and implanted into pseudo-pregnant FVB/N mice. We obtained five founder lines crossed with wild-type FVB/N mice to obtain the F1 generation. RT-PCR for PRMT5 showed preferential overexpression in Tg B lymphocytes compared to control FVB/N mice (p<0.01) but also overexpression in T and NK cell compartments due to leaky Eμ phenotype. We observed a high percentage of F1 progeny from all five founder lines to develop spontaneous lymphoproliferative disease compared to control mice (p<0.001). Immunophenotypic and pathologic evaluation was performed by flow cytometry, immunohistochemistry, and clonality by IgH and TCR Vβ PCR analysis. Pathologic features showed a range of lymphoid neoplasms, with pre-T lymphoblastic immunophenotype being the most common. Tumor-bearing animals demonstrated diffuse involvement of all organs, peripheral blood, and bone marrow. Pre-T lymphoblastic lymphomas (LBL) showed expression of cytoplasmic CD3, surface CD4 (dim), TdT, and variable Ki67 with constitutive overexpression of PRMT5, MYC, CYCLIN D1, Ki67, and CD99. We developed a T-LBL cell line (Tg813) derived from a thymic tumor that displayed clonal TCR Vβ.17 gene rearrangement, intracellular TdT and CD3 expression with dim surface CD4 expression, immunophenotypic characteristics consistent with T-ALL/LBL. This cell line was maintained in culture and successfully passaged in immune-competent FVB mice, which led to disseminated disease, suggesting that PRMT5 drives an immune escape program in vivo. Preliminary data demonstrate that tumor-bearing animals display a prominent population of normal and neoplastic T-cell subsets with strong PD1 expression. PD1 expression in T-ALL/LBL has been linked with oncogenic function (Yang, 2019) and, in normal T-cell subsets, is a marker for exhaustion. (Lee, 2015) In addition, treatment of our Tg813 in vitro model with PRMT5 knockdown (CRISPR CAS9) and selective inhibition with a SAM-competitive inhibitor (C220 and PRT382, Prelude Therapeutics) showed a reduction of global symmetric dimethyl arginine, loss of the PRMT5 epigenetic mark H4R3(Sme2) and decreased proliferation with cell cycle arrest by day 6, findings consistent with PRMT5-dependent driver activity. Engraftment of Tg813 into SCID and immunocompetent FVB/N mice led to disseminated lymphomas (mean: 21 days). Tg813 engrafted animals treated with C220 (10mg/kg) showed improved survival compared to vehicle-treated mice (p<0.001). This preclinical model supports the hypothesis that PRMT5 over-expression provides cancer driver activity and a unique opportunity to study the role of this oncogene in an immune-competent, in vivo model system. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oxidative stress state inhibits exosome secretion of hPDLCs through a specific mechanism mediated by PRMT1.

Periodontitis, the most common chronic inflammation characterized by persistent alveolar bone resorption in the periodontitis, affects almost half of the adult population worldwide. Oxidative stress is one of the pathophysiological mechanisms underlying periodontitis, which affects the occurrence and development of periodontitis. Exosomes are increasingly recognized as vehicles of intercellular communication and are closely related to periodontitis. However, the effects of oxidative stress on exosome secretion and the specific mechanisms remain elusive in human periodontal ligament cells (hPDLCs). The relationship between exosome secretion and the osteogenic differentiation of hPDLCs also needs to be investigated. Isolated PDLSCs were identified using flow cytometry. Osteogenesis was measured using alizarin red staining and ALP staining. Expression of exosomal markers and PRMT1 was analyzed using western blot. Immunofluorescence was used to measure exosome uptake and the expression of EEA1. The secretion capacity of exosomes was markedly suppressed under oxidative stress. Protein arginine methyltransferase 1 (PRMT1) has been strongly associated with both oxidative stress and inflammation, and PRMT1 was significantly upregulated under oxidative stress conditions. Lentivirus-mediated overexpression of PRMT1 caused a significant reduction in the secretion of exosomes, but multivesicular bodies (MVBs) containing a large number of intraluminal vesicles (ILVs) were increased. Rab11a and Rab27a expression, which mediate MVBs fusion with cell membranes, decreased, although this phenomenon was restored after knocking down PRMT1 expression under oxidative stress. These results indicated that PRMT1 mediated a decrease in exosome secretion of hPDLCs. The decrease in Rab11a and Rab27a leads to a large accumulation of MVBs in cells and is one of the main reasons for impaired exosome secretion. The decrease in osteogenic differentiation of hPDLCs caused by H2 O2 may originate in part from the inhibition of exosome secretion.

Adriamycin induces cardiac fibrosis in mice via PRMT5-mediated cardiac fibroblast activation.

Long-term treatment with adriamycin (ADR) is associated with higher incidences of cumulative cardiotoxicity manifest as heart failure. ADR-induced cardiomyopathy is characterized by extensive fibrosis that is caused by cardiac fibroblast activation. To date, however, no specific treatment is available to alleviate ADR-induced cardiotoxicity. Protein arginine methyltransferase 5 (PRMT5), a major enzyme responsible for methylation of arginine, regulates numerous cellular processes such as cell differentiation. In the present study we investigated the role of PRMT5 in cardiac fibrosis. Mice were administered ADR (3 mg/kg, i.p., every 2 days) for 2 weeks. We showed that aberrant PRMT5 expression was largely co-localized with α-SMA-positive activated cardiac fibroblasts in ADR-injected mice and in ADR-treated cardiac fibroblasts in vitro. PRMT5-overexpression exacerbated, whereas PRMT5 knockdown alleviated ADR-induced cardiac fibrosis in vivo and TGF-β1-induced cardiac fibroblast activation in vitro. We demonstrated that PRMT5-overexpression enhanced methylated-Smad3 levels in vivo and in vitro. Pretreatment with a specific PRMT5 inhibitor EPZ015666 (5 nM) or overexpression of a catalytically inactive mutant of PRMT5, PRMT5(E444Q), reduced PRMT5-induced methylation of Smad3, thus suppressing PRMT5-mediated cardiac fibroblast activation in vitro. Furthermore, ADR activated cardiac fibroblasts was depending on autocrine TGF-β1. Taken together, our results demonstrate that PRMT5 promotes ADR-induced cardiac fibrosis via activating cardiac fibroblasts, suggesting that it may be a potential therapeutic target of ADR-caused cardiotoxicity.

Inhibition of protein arginine methyltransferase 1 alleviates liver fibrosis by attenuating the activation of hepatic stellate cells in mice.

Protein arginine methyltransferase 1 (PRMT1) has been reported to be involved in various diseases. The expression of PRMT1 was increased in cirrhotic livers from human patients. However, the role of PRMT1 in hepatic fibrogenesis remains largely unexplored. In this study, we investigated the effect of PRMT1 on hepatic fibrogenesis and its underlying mechanism. We found that PRMT1 expression was significantly higher in fibrotic livers of the mice treated with thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Immunofluorescence staining revealed that PRMT1 expression was augmented in both hepatocytes and hepatic stellate cells (HSCs) in the fibrotic livers. Applying a selective inhibitor of PRMT1, PT1001B, significantly suppressed PRMT1 activity and mitigated liver fibrosis in mice. Hepatocyte-specific Prmt1 knockout did not affect liver fibrosis in mice. PRMT1 overexpression promoted the expression of fibrotic genes in the LX-2 cells, whereas knockdown of PRMT1 or treatment with PT1001B exhibited reversal effects, suggesting that PRMT1 plays an important role in HSC activation. Additionally, HSC-specific Prmt1 knockout attenuated HSC activation and liver fibrosis in TAA-induced fibrotic model. RNA-seq analysis revealed that Prmt1 knockout in HSCs significantly suppressed pro-inflammatory NF-κB and pro-fibrotic TGF-β signals, and also downregulated the expression of pro-fibrotic mediators in mouse livers. Moreover, treatment with PT1001B consistently inhibited hepatic inflammatory response in fibrotic model. In conclusion, PRMT1 plays a vital role in HSC activation. Inhibition of PRMT1 mitigates hepatic fibrosis by attenuating HSC activation in mice. Therefore, targeting PRMT1 could be a feasible therapeutic strategy for liver fibrosis.

Inhibition of PRMT5 attenuates cerebral ischemia/reperfusion–Induced inflammation and pyroptosis through suppression of NF–κB/NLRP3 axis

Protein methylation is a prevalent post-translational modification after cerebral ischemia. Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse the formation of methylated residues on histones and non-histone proteins. Accumulating evidence suggested that PRMT5 might play a carcinogenic role in various cancers. However, the role of PRMT5 in cerebral ischaemia/reperfusion (I/R) injury remains unclear. In this project, middle cerebral artery occlusion/reperfusion (MCAO/R) model in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model in human neuroblastoma SH-SY5Y cells were utilized to mimic disease state of cerebral I/R. We found that expression of inflammatory-related factors [Interleukin (IL)-1β and IL-6)] and pyroptotic-related factor [N-term cleaved Gasdermin-D (GSDMD-N)] were up-regulated in both MCAO/R mice and OGD/R SH-SY5Y cells. In addition, both in vivo and in vitro, PRMT5 was aberrantly upregulated during cerebral I/R. However, these alterations induced by I/R were blocked by PRMT5 inhibitor LLY-283, and enhanced by overexpression of PRMT5. Furthermore, rescue experiment proved that PRMT5 plays a pro-inflammatory and pro-pyroptotic role by activating nuclear factor kappa B (NF-κB)/nucleotide-binding oligomerization domainlike receptor pyrin domain containing 3 (NLRP3) axis. Finally, we observed that treatment of LLY-283 alleviated neurological deficits and reduced infarct volume in the MCAO/R mice. Taken together, PRMT5 may be a potential therapeutic target for cerebral I/R injury.

PRMT1 Confers Resistance to Olaparib via Modulating MYC Signaling in Triple-Negative Breast Cancer.

Treatment of triple-negative breast cancer (TNBC) remains an unmet clinical need owing to its lack of an efficient therapeutic target. The targeting of DNA repair by poly(ADP-ribose) polymerase (PARP) inhibitors has shown benefit for patients with the BRCA variation. However, sensitivities to the PARP inhibitors were reported regardless of BRCA status. Thus, exploring the underlying mechanisms is imperative. Herein, we identified that breast cancer cells with an elevated expression of protein arginine methyl transferase 1 (PRMT1) was associated with therapeutic sensitivity to the PARP inhibitor olaparib. The results of cell viability and colony formation assays indicated that the suppression of PRMT1 by small hairpin RNA or by the chemical inhibitor increased sensitivity to olaparib in human TNBC MDA-MB-231 and BT549 cells. Bioinformatic analysis revealed that PRMT1 expression was significantly associated with the MYC signature, and TNBC cells with higher PRMT1 and the MYC signature were associated with therapeutic sensitivity to olaparib. Mechanistic studies further demonstrated that knockdown of PRMT1 reduced the c-Myc protein level and downregulated the expression of MYC downstream targets, whereas overexpression of PRMT1 enhanced c-Myc protein expression. Moreover, the overexpression of PRMT1 promoted c-Myc protein stability, and the inhibition of PRMT1 downregulated c-Myc protein stability. Accordingly, the knockdown of PRMT1 inhibited homologous recombination gene expression. These data indicate that PRMT1 is instrumental in regulating DNA repair, at least in part, by modulating c-Myc signaling. Our data highlighted the PRMT1/c-Myc network as a potential therapeutic target in patients with TNBC.

PRMT5 Initiates an Epigenetic Program That Promotes Progression of Chronic Lymphocytic Leukemia

Background: Epigenetic alterations that occur as a result of arginine methyltransferase activity play a critical role in normal biological functions, and increasingly their importance has been shown in cancer initiation and progression. In particular, protein arginine methyltransferase 5 (PRMT5) is an important epigenetic regulator that influences differentiation, cell cycle progression, and many other processes. Aberrant PRMT5 activity has been implicated in tumorigenesis, however its role in the biology of chronic lymphocytic leukemia (CLL) has not been established. Despite recent improvements in therapy, CLL remains an incurable disease. While newer therapies targeting the B cell receptor pathway have shown remarkable efficacy, complete responses are not frequent. In particular, transformation of CLL to aggressive lymphoma (Richter syndrome; RS) occurs in up to 15% of patients and confers a poor prognosis; however, the mechanisms by which RS occurs are poorly understood. A better understanding of CLL progression may facilitate the development of new targeted therapies. We hypothesized that PRMT5 dysregulation initiates an epigenetic program in CLL that contributes to disease progression and potentially transformation. Moreover, we provide evidence that inhibition of PRMT5 is a promising strategy in CLL.Methods: Eμ-PRMT5 transgenic mice were generated on the C57BL/6 background via pronuclear injection of human PRMT5 cDNA under the heavy chain promoter/enhancer. To investigate the effect of PRMT5 overexpression on CLL pathogenesis, Eμ-PRMT5 mice were crossed with Eμ-TCL1 mice, an aggressive, spontaneous model of CLL, and followed for disease onset, progression, and overall survival. Immunophenotypic analysis was performed using multicolor flow cytometry. Pharmacologic inhibition of PRMT5 was evaluated in freshly isolated CD19+-selected cells isolated from CLL patients. Kaplan-Meier curves were compared using a log-rank test.Results: We observed that Eμ-PRMT5/TCL1 had a shorter time to leukemia onset (defined as >20% CD45+/CD5+/CD19+ in peripheral blood) compared to the Eμ-TCL1 mice. The Eμ-PRMT5/TCL1 had significantly worse overall survival compared to the Eμ-TCL1 mice. In addition to more aggressive disease, Eμ-PRMT5/TCL1 mice developed focal lymphoid tumors and lymphadenopathy in contrast to Eμ-TCL1 mice. We generated an inducible knockout CLL-like cell line using CRISPR guide RNAs targeting PRMT5 . Genomic ablation of PRMT5 led to impaired protein expression and subsequent decrease in viable cells. We next assessed the cytotoxic effect of PRMT5 inhibition in CLL using the pharmacologic agents EPZ015666 (Chan-Penebre et al. 2015 Nat Chem Biol) and an in-house developed PRMT5 inhibitor. Both compounds inhibited growth of CD19+-selected cells isolated from CLL patients as well as CLL-like cell lines (Mec-1 and HG3). Pharmacologic PRMT5 inhibition in vivo was able to efficiently modulate symmetric dimethyl arginine motifs. Studies exploring the impact of in vivo PRMT5 inhibition on disease burden and overall survival in Eμ-PRMT5/TCL1 mice are currently ongoing.Conclusions: Our data indicate that PRMT5 facilitates CLL progression through altered epigenetic programming. PRMT5 overexpression in Eμ-TCL1 mice results in a more aggressive disease model with features of a focal lymphoid accumulation. Studies to further characterize the genomic and phenotypic features of this nodal disease are ongoing. Furthermore, our data support pharmacologic inhibition of PRMT5 as a promising therapeutic paradigm in CLL. DisclosuresByrd:Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics: Research Funding. Baiocchi:Prelude therapeutics: Research Funding; essanex: Research Funding; Theravectys: Consultancy; viracta: Membership on an entity's Board of Directors or advisory committees, Research Funding.

PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression.

The present study reveals a link between protein arginine methyltransferase 5 (PRMT5) and Homebox A9 (HoxA9) in the regulation of cardiomyocyte hypertrophy. In cardiomyocyte hypertrophy induced by β-adrenergic receptor agonist isoprenaline (ISO), PRMT5 expression was decreased while HoxA9 was upregulated. Silencing of PRMT5 or inhibition of PRMT5 by its pharmacological inhibitor EPZ augmented the expressions of cardiomyocyte hypertrophic genes brain natriuretic peptide (BNP) and β-Myosin Heavy Chain (β-MHC), whereas overexpression of PRMT5 inhibited ISO-induced cardiomyocyte hypertrophy, suggesting that PRMT5 ameliorates cardiomyocyte hypertrophy. On the contrary, HoxA9 promoted cardiomyocyte hypertrophy, as implied by the gain-of-function and loss-of-function experiments. HoxA9 was involved in the regulation of PRMT5 in cardiomyocyte hypertrophy, since HoxA9 knockdown prevented si-RPMT5-induced cardiomyocyte hypertrophy, and HoxA9 expression impaired the anti-hypertrophic effect of PRMT5. Co-immunoprecipitation experiments revealed that there were physical interactions between PRMT5 and HoxA9. The symmetric dimethylation level of HoxA9 was decreased by ISO or EPZ treatment, suggesting that HoxA9 is methylated by PRMT5. Additionally, PRMT5 repressed the expression of HoxA9. Chromatin immunoprecipitation (ChIP) assay demonstrated that HoxA9 could bind to the promoter of BNP, and that this binding affinity was further enhanced by ISO or EPZ. In conclusion, this study suggests that PRMT5 symmetric dimethylates HoxA9 and represses HoxA9 expression, thus impairing its binding to BNP promoter and ultimately protecting against cardiomyocyte hypertrophy. These findings provide a novel insight of the mechanism underlying the cardiac protective effect of PRMT5, and suggest potential therapeutic strategies of PRMT5 activation or HoxA9 inhibition in treatment of cardiac hypertrophy.

Dysregulated YY1/PRMT5 axis promotes the progression and metastasis of laryngeal cancer by targeting Hippo pathway

Metastases lead to high mortality in laryngeal cancer, but the regulation of its underlying mechanisms remains elusive. We identified Protein arginine methyltransferase 5 (PRMT5) was significantly up‐regulated in laryngeal cancer tissues, which predicts poor patient prognosis. Functional assays demonstrated that PRMT5 overexpression promoted the invasive capacity and lymph node metastasis in vitro and in vivo. Mechanistic experiments suggested that LATS2 was a downstream target of PRMT5. PRMT5 inhibition increased the expression of LATS2 and YAP phosphorylation in laryngeal cancer cells, thereby promoting laryngeal cancer metastasis. Furthermore, informatics and experimental data confirmed that PRMT5 gene was transcriptionally activated by YY1. Collectively, our results unravelled the important role of PRMT5 in laryngeal cancer tumorigenesis and metastasis. The dysregulation YY1/PRMT5/LATS2/YAP axis may contribute to laryngeal cancer progression; thus, PRMT5 may be a potential therapeutic strategy for patients with laryngeal cancer.

Trichostatin A Induces Autophagy in Cervical Cancer Cells by Regulating the PRMT5-STC1-TRPV6-JNK Pathway

Objective: The aim of this study was to investigate the effects of trichostatin A (TSA) on cervical cancer and the related mechanisms. Methods: The HeLa and Caski cervical cancer cell lines were treated with different concentrations of TSA. Cell viability was measured by MTT assays. Cell apoptosis was analysed using flow cytometry. Expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6), protein arginine methyltransferase 5 (PRMT5), and stanniocalcin 1 (STC1) was determined by qRT-PCR and Western blotting. Protein levels of LC3 II/I, beclin1, p62, JNK, and p-JNK were detected by Western blotting. Results: Treatment with TSA significantly decreased HeLa and Caski cell viability and enhanced the apoptosis rate in a dose-dependent manner. TSA markedly elevated beclin1 protein levels and the LC3 II/I ratio and significantly reduced p62 levels in a dose-dependent manner. In addition, TSA (1 μM) significantly suppressed PRMT5 and TRPV6 levels and enhanced STC1 and p-JNK levels. The lysosomal inhibitor bafilomycin-A1 synergistically enhanced the TSA-mediated increase in autophagic flux. Either the overexpression of TRPV6 or the inhibition of JNK signalling markedly enhanced cell viability, inhibited apoptosis, and autophagy and reduced p-JNK levels in TSA-treated cells. The inhibition of STC1 significantly increased TRPV6 protein levels and reduced p-JNK levels. Overexpression of PRMT5 dramatically decreased STC1 and p-JNK protein levels and increased TRPV6 levels. Conclusion: TSA suppresses cervical cancer cell proliferation and induces apoptosis and autophagy through regulation of the PRMT5/STC1/TRPV6/JNK axis.

PRMT5/Wnt4 axis promotes lymph-node metastasis and proliferation of laryngeal carcinoma

Metastasis is the main cause of laryngeal cancer-related death; its molecular mechanism remains unknown. Here we identify protein arginine methyltransferase 5 (PRMT5) as a new metastasis-promoting factor in laryngeal carcinoma, and explore its underlying mechanism of action in regulating laryngeal cancer progression. We illustrated that PRMT5 expression was positively correlated with tumor stages, lymphatic metastasis, and unfavorable outcome. Functional assays revealed that PRMT5 promoted laryngeal carcinoma cell proliferation, migration, and invasive capacity in vitro, as well as lymph-node metastasis in vivo. The ectopic expression of PRMT5 induced EMT with downregulation of E-cadherin and upregulation of N-cadherin, snail, and MMP9. Mechanistic results revealed that the metastatic effects could be attributed to PRMT5-mediated activation of Wnt signaling, and Wnt4 is an important driver of Wnt/β-catenin signaling pathway. Wnt4 silencing could reverse PRMT5-induced cell proliferation, migration, and invasion capacities. Furthermore, inhibition of the Wnt/β-catenin signaling pathway abolished the effect of PRMT5-induced proliferation, whereas activation of the pathway enhanced the effect of PRMT5 overexpression on cell proliferation. These results demonstrated that the oncogenic role of PRMT5 could be attributed to PRMT5/Wnt4 axis-mediated activation of the Wnt/β-catenin signaling pathway. PRMT5 may serve as a novel prognostic marker and a therapeutic target for lymphatic metastasis of laryngeal carcinoma.

Abstract 652: Exploiting PRMT5, a metabolic and epigenetic regulator, as a novel therapeutic target in mantle cell lymphoma

Abstract Distinct genetic and epigenetic alterations as well as metabolic reprogramming are among the hallmarks of mantle cell lymphoma (MCL), an incurable cancer. Deletion of MTAP, encoding 5-Methylthioadenosine phosphorylase (MTAP), results in the accumulation of metabolite methylthioadenosine (MTA), a selective inhibitor of the catalytic activity of protein arginine methyltransferase 5 (PRMT5). MTAP loss therefore presents a passenger vulnerability to PRMT5 inhibition. PRMT5 is a type II protein arginine methyltransferase that is involved in cellular processes via the regulation of the active status of transcription by symmetric dimethylation of arginine in histones, and the post-translational modification by symmetric dimethylation of arginine in nuclear and cytoplasmic oncoproteins and tumor suppressors. We recently reported that CDKN2A and MTAP were commonly co-deleted in ibrutinib-resistant MCL tumors. Consistently, PRMT5 was found to be selectively over expressed in ibrutinib-resistant MCL cells. Metabolomics analysis revealed that both the substrate (S-Adenosylmethionine) and the product (S-adenosylhomocysteine) of the PRMT5 enzyme are elevated in ibrutinib-resistant MCL, suggesting an increase in PRMT5 enzymatic activity. Importantly, our clinical data analysis indicates that the expression levels of PRMT5 in MCL patients are inversely correlated with patient responses to ibrutinib treatment. Moreover, pharmacological blockade with the PRMT5 specific inhibitor, GSK3226595, significantly inhibited tumor growth in ibrutinib-resistant MCL PDX models, demonstrating that PRMT5 is an actionable target in MCL. Mechanistically, PRMTs play an important role in mRNA splicing, among which MDM4 mRNA was identified as one of the key mRNAs that senses defects in the spliceosomal machinery during PRMT5 deficiency. Alternative splicing of MDM4 mRNA reduces the expression of Mdm2 and Mdm4 E3 ligases, leading to the upregulation of p53. Selective overexpression of PRMT5 in MCL may drive repressive epigenetic marks on chromatin to silence regulatory and tumor suppressor genes. Inhibition of PRMT5 reverses PRMT5-catalyzed epigenetic marks, restores regulatory pathways and induces cell death in ibrutinib-resistant primary MCL. Taken together, sustained PRMT5 expression and activity presents a vulnerability in ibrutinib-resistant MCL where MTAP is frequently deleted. As metabolic reprogramming towards OXPHOS is one of the hallmarks of ibrutinib-resistant MCL, combinatory inhibition of OXPHOS and PRMT5 may represent a rational therapeutic strategy that ultimately eradicates MCL. Citation Format: Yixin Yao, Lingzhi Li, Liang Zhang, Shaojun Zhang, Hui Guo, Hui Zhang, Maria Badillo, Zhihong Chen, Kimberly Hartig, Preetesh Jain, Krystle Nomie, Linghua Wang, Michael Wang. Exploiting PRMT5, a metabolic and epigenetic regulator, as a novel therapeutic target in mantle cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 652.