Protein Arginine Methyltransferase 5 Expression Research Articles

PRMT5 functionally associates with EZH2 to promote colorectal cancer progression through epigenetically repressing CDKN2B expression.

Background: Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that symmetrically di-methylates arginine residues on both histone and non-histone protein substrates. Accumulating evidence suggests that PRMT5 exerts its oncogenic properties in a wide spectrum of human malignancies. However, the underlying mechanisms by which PRMT5 contributes to the progression of colorectal cancer (CRC) remain to be defined.Methods: Western blot and real-time PCR were used to analyze the expression of CDKN2B. Co-immunoprecipitation (Co-IP), immunofluorescence and GST pulldown assays were employed to investigate the interaction between PRMT5 and EZH2. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to validate CDKN2B as a direct target of PRMT5/EZH2. DNA methylation status at the CpG islands of promoter region of CDKN2B gene was analyzed by bisulfite sequencing. The effect of PRMT5/EZH2 on malignant phenotypes was examined through in vitro and in vivo assays. PRMT5 and EZH2 protein expression levels in CRC tissues were analyzed by immunohistochemistry (IHC) staining.Results: We observed that PRMT5-deficient CRC cells exhibit proliferation defects in vitro. PRMT5 was identified as a major transcriptional repressor of CDKN2B (p15INK4b) for determining CRC progression. Mechanistically, PRMT5-mediated histone marks H4R3me2s and H3R8me2s were predominantly deposited at the promoter region of CDKN2B gene in CRC cells. Knockdown of PRMT5 in CRC cells decreased the accumulation of H4R3me2s and H3R8me2s marks and reduced the CpG methylation level of CDKN2B promoter, then re-activated CDKN2B expression. Strikingly, silencing of CDKN2B partially abrogated the proliferation defects caused by PRMT5 depletion in vitro and in vivo. Furthermore, we proved that PRMT5 interacted with Enhancer of zeste homolog 2 (EZH2), leading to enhanced EZH2 binding and H3K27me3 deposition together with decreased transcriptional output of CDKN2B gene. Importantly, we found that the combined interventions exerted a synergistic inhibitory effect of combined treatment with PRMT5i (GSK591) and EZH2i (GSK126) on the growth of CRC cells/xenografts in vitro and in vivo. Moreover, PRMT5 and EZH2 were found to be significantly elevated and associated with poor prognosis in CRC patients.Conclusion: PRMT5 functionally associates with EZH2 to promote CRC progression through epigenetically repressing CDKN2B expression. Thus, our findings raise the possibility that combinational intervention of PRMT5 and EZH2 may be a promising strategy for CRC therapy.

PRMT5 disruption drives antitumor immunity in cervical cancer by reprogramming T cell-mediated response and regulating PD-L1 expression.

Rationale: Protein arginine methyltransferase 5 (PRMT5) is an oncogene that promotes tumor cell proliferation, invasion and metastasis. However, the underlying mechanisms by which PRMT5 contributes to the progression of cervical cancer and especially the tumor microenvironment remain poorly understood.Methods: PRMT5 expression level was analyzed by Q-PCR, western blot, immunohistochemistry, and TCGA database. The role of PRMT5 in tumor growth was observed by transplanted tumor models, and the function of T cells in tumor microenvironment and in vitro co-culture system was investigated through flow cytometry. The transcriptional regulation of PRMT5 was analyzed using luciferase reporter and chromatin immunoprecipitation (ChIP) assay. The therapeutic effect of PRMT5 inhibitor was evaluated in a cervical cancer cell line transplanted tumor model.Results: We observed that the mRNA and protein expression levels of PRMT5 were increased in cervical cancer tissues, and the high expression of PRMT5 was associated with poor outcomes in cervical cancer patients. The absence of PRMT5 significantly inhibited tumor growth in a cervical cancer transplanted tumor model, and importantly, PRMT5 absence in tumors led to increase the number and enhance the function of tumor infiltrating T cells. Mechanistically, PRMT5 enhanced the transcription of STAT1 through symmetric dimethylation of histone H3R2 and thus promoted PD-L1 expression in cervical cancer cells. Moreover, in an in vitro co-culture system, knockdown of PRMT5 in tumor cells could directly enhance the expression of IFN-γ, TNF-α and granzyme B in T cells. These results suggested that PRMT5 promoted the development of cervical cancer by the crosstalk between tumor cells and T cells. Furthermore, the PRMT5 inhibitor EPZ015666 treatment could suppress tumor growth in a cervical cancer transplanted tumor model.Conclusion: Our results clarify a new mechanism which PRMT5 knockdown in cervical cancer cells drives an antitumor function via reprogramming T cell-mediated response and regulating PD-L1 expression. Thus, our study highlights that PRMT5 may be a potential target for cervical cancer therapy.

PRMT1 expression predicts sensitivity to platinum-based chemotherapy in patients with ovarian serous carcinoma.

Patients with ovarian serous carcinoma are generally diagnosed at an advanced disease stage. The standard treatment for these patients is maximal debulking surgery followed by platinum-taxane combination chemotherapy. Despite initially responding well, more than half of patients become refractory to first-line chemotherapy. Upregulation of protein arginine methyltransferase 1 (PRMT1) expression has been demonstrated to methylate apoptosis signal-regulated kinase 1 and inhibit its activity, thereby contributing to chemoresistance. The present study investigated the association between PRMT1 expression and sensitivity to platinum-based chemotherapy in 51 patients with ovarian serous carcinoma (International Federation of Gynecology and Obstetrics stages III and IV), and the effect of RNA interference-mediated downregulation of PRMT1 on the sensitivity of ovarian cancer cells to cisplatin and carboplatin in vitro. Immunohistochemistry of tumor specimens was used to compare the expression levels of PRMT1, a Cell Counting Kit-8 assay and small interfering RNA transfection were performed for chemosensitivity assays, and reverse transcription-quantitative PCR was used to examine PRMT1 mRNA expression. Patients were divided into platinum-sensitive (n=26) and platinum-resistant (n=25) groups. PRMT1 expression was significantly lower in the platinum-sensitive group than in the platinum-resistant group (P=0.019). When patients were categorized according to PRMT1 expression, those in the low PRMT1 expression group were more sensitive to platinum-based chemotherapy than those in the high PRMT1 expression group (P=0.01). Additionally, in vitro experiments revealed that suppression of PRMT1 expression by siRNA significantly increased the sensitivity of human ovarian serous carcinoma cells to cisplatin and carboplatin (P<0.05). In conclusion, PRMT1 expression could predict sensitivity to platinum-based chemotherapy in patients with ovarian serous carcinoma.

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.

Protein Arginine Methyltransferase 5 Promotes pICln-Dependent Androgen Receptor Transcription in Castration-Resistant Prostate Cancer.

The majority of advanced prostate cancer therapies aim to inhibit androgen receptor (AR) signaling. However, AR reactivation inevitably drives disease progression to castration-resistant prostate cancer (CRPC). Here we demonstrate that protein arginine methyltransferase 5 (PRMT5) functions as an epigenetic activator of AR transcription in CRPC, requiring cooperation with a methylosome subunit pICln. In vitro and in xenograft tumors in mice, targeting PRMT5 or pICln suppressed growth of CRPC cells. Full-length AR and AR-V7 transcription activation required both PRMT5 and pICln but not MEP50. This activation of transcription was accompanied by PRMT5-mediated symmetric dimethylation of H4R3 at the proximal AR promoter. Further, knockdown of PRMT5 abolished the binding of pICln (but not vice versa) to the AR proximal promoter region, suggesting that PRMT5 recruits pICln to the AR promoter to activate AR transcription. Differential gene expression analysis in 22Rv1 cells confirmed that PRMT5 and pICln both regulate the androgen signaling pathway. In addition, PRMT5 and pICln protein expression positively correlated with AR and AR-V7 protein expression in CRPC tissues and their expression was highly correlated at the mRNA level across multiple publicly available CRPC datasets. Our results suggest that targeting PRMT5 or pICln may be explored as a novel therapy for CRPC treatment by suppressing expression of AR and AR splice variants to circumvent AR reactivation. SIGNIFICANCE: This study provides evidence that targeting PRMT5 can eliminate expression of AR and can be explored as a novel therapeutic approach to treat metastatic hormone-naïve and castration-resistant prostate cancer.

CSIG-17. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA MODELS TO TRAMETINIB TREATMENT

Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor. With limited effective therapeutic strategies, prognosis for GBM is very poor. Our previous study shows that the expression of Protein Arginine Methyltransferase 5 (PRMT5) is upregulated in GBM; its inhibition promotes anti-GBM effect through apoptosis and senescence of mature and immature tumor cells, respectively. In GBM, RAS-RAF- MEK-ERK signaling is aberrantly activated and promotes tumor growth. Therefore, MEK inhibitors, including trametinib are currently under investigation for GBM therapy. In this study, we tested whether inhibition of PRMT5 can enhance the anti-tumor efficacy of trametinib in GBM. METHODS Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, and western blot analysis were conducted. In vivo, PRMT5-intact and -depleted GBMNS were intracranially implanted in NSG mice and treated with trametinib by daily oral gavage, and tumor progression and mice survival rate were analyzed by MRI and Kaplan-Meier survival curve, respectively. RESULTS Trametinib treatment upregulated the expression of PRMT5 in GBMNS. Depletion of PRMT5 increased the cytotoxic effect of trametinib in GBMNS. In concurrence with the trametinib-induced PRMT5 upregulation, trametinib treatment increased the activity of AKT that was blocked with PRMT5 knockdown. In vivo, PRMT5-depletion extended the survival of the tumor bearing mice that further increased in combination with trametinib treatment. Interestingly, trametinib treatment alone had no survival benefit. CONCLUSION Trametinib treatment induces PRMT5 expression. Depletion of trametinib-induced PRMT5 expression sensitizes GBMNS for trametinib by inhibiting AKT activity.

Targeting the Synthetic Lethality Interaction of MTAP and PRMT5 to Overcome Drug Resistance and Enhance Anti-Cancer Immunity in Mantle Cell Lymphoma

Background: Complex genetic, epigenetic alterations and metabolic reprogramming are among the hallmarks of mantle cell lymphoma, an aggressive B-cell type of non-Hodgkin's lymphoma. CDKN2A and MTAP are frequently co-deleted in human cancers including MCL. Deletion of MTAP gene results in the accumulation of methylthioadenosine (MTA), a selective inhibitor of protein arginine methyltransferase 5 (PRMT5), signifying a dependence of MTAP deleted tumors on PRMT5. PRMT5 plays an important role in normal cellular processes via epigenetic and post-translational modification of RNA splicing, DNA repair and cell cycle regulators. These pathways may be activated in response to targeted therapies to maintain tumor growth and survive chemotherapy. Sustained PRMT5 expression and activity could be therapeutically targeted in MTAP-deficient MCL. Mutations in RNA splicing factors are common in lymphoma and increase the susceptibility to perturbations in splicing events. Deregulated PRMT5 might induce alternative splicing, leading to loss of expression of proteins or epitopes, and subsequent immune evasion and failure of tumor specific immune therapy. Multiple mechanisms may co-exist. Results: CDKN2A and MTAP were commonly co-deleted in ibrutinib-resistant MCL tumors (71%, p = 0.01). To corroborate with this finding, we found that PRMT5 was overexpressed in both primary ibrutinib-resistant MCL and cell lines, with its expression levels inversely correlated with the patient responses to ibrutinib treatment (p = 0.004). Metabolomics analysis revealed elevated S-Adenosyl-methionine (SAM) and S-Adenosyl-homocysteine (SAH) (t-test p &amp;lt;0.001 and p &amp;lt;0.05), PRMT5 cofactor and product, respectively, in ibrutinib-resistant MCL cell lines. PRMT5 plays an important role in the regulation of cell cycle progression. Consistently, treatment with PRMT5 inhibitors induced cell cycle arrest in MCL cell lines (20% and 47% reduction in S phase for JVM13 and JVM2 respectively). Interestingly, treatment with the catalytic inhibitors (GSK3226595 and EPZ015666) led to S-phase stasis in which MCL cells seem incapable of replication. Meanwhile, PRMT5 inhibition with these inhibitors prevented the growth of MCL cell lines as indicated by remarkable decreases in the cell viability (IC50 = 1-1.4 µM and 0.58-0.95µM, respectively). PRMT5 enzymatic activity is required for its function in promoting cell cycle progression and proliferation, as treatment with its inhibitors leads to dramatic reduction in the level of SDMA-containing proteins, especially that of H4R3me2s. More importantly, pharmacological blockade of PRMT5 with single agent GSK3226595 significantly inhibited tumor growth in ibrutinib-resistant MCL PDX models (n = 5, p &amp;lt;0.05), implicating PRMT5 as an actionable target in MCL. PRMT5-mediated alternative splicing may contribute to immune escape and immune suppression. Indeed, our preliminary results revealed that T cells from MCL specimen with high expression of PRMT5 displayed decreased glucose uptake (fold MFI &amp;lt;1-1.23 vs. 2.2), mitochondria mass (p &amp;lt;0.01) and ΔΨm (fold MFI &amp;lt;1-1.26 vs. 2.3), by flow cytometry. Strikingly, the same patient-derived cytotoxic T-lymphocytes (CTL) exhibit reduced cytotoxicity towards autologous lymphoma cells, compared to those derived from healthy donors (p &amp;lt;0.05), strongly implicating impaired T cell metabolic fitness plus immune escape and suppression diminished T cell effector function. Conclusion: We presented evidence that MCL with MTAP deletion confers synthetic lethal dependence on PRMT5, making it an attractive therapeutic target. As an important metabolic and epigenetic regulator, loss of PRMT5 leads to malfunction of the constitutive splicing machinery that induces alternative splicing events, genetic instability and cell cycle arrest. Sustained PRMT5 expression and activity confers tumor aggressiveness and therapeutic resistance in MTAP-deficient MCL. Pharmacological blockade of PRMT5 may dramatically enhance T-cell immunity, while suppressing MCL cell proliferation and tumor growth in vivo. Further successful mechanistic studies would facilitate the identification of novel therapeutics and strategies to overcome resistance to targeted therapies and significantly improve the efficacy of immunotherapy for much better clinical outcomes of MCL. Disclosures Wang: Oncternal: Consultancy, Research Funding; Lu Daopei Medical Group: Honoraria; Juno: Consultancy, Research Funding; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; MoreHealth: Consultancy; Targeted Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; Verastem: Research Funding; OncLive: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; Molecular Templates: Research Funding; Nobel Insights: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; InnoCare: Consultancy.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Targeting methyltransferase PRMT5 retards the carcinogenesis and metastasis of HNSCC via epigenetically inhibiting Twist1 transcription

Protein arginine methyltransferase 5 (PRMT5) is an important type II arginine methyltransferase that can play roles in cancers in a highly tissue-specific manner, but its role in the carcinogenesis and metastasis of head and neck squamous cell carcinoma (HNSCC) remains unclear. Here, we detected PRMT5 expression in HNSCC tissues and performed series of in vivo and in vitro assays to investigate the function and mechanism of PRMT5 in HNSCC. We found that PRMT5 was overexpressed in dysplastic and cancer tissues, and associated with lymph node metastasis and worse patient survival. PRMT5 knockdown repressed the malignant phenotype of HNSCC cells in vitro and in vivo. PRMT5 specific inhibitor blocked the formation of precancerous lesion and HNSCC in 4NQO-induced tongue carcinogenesis model, prevented lymph node metastasis in tongue orthotopic xenograft model and inhibited cancer development in subcutaneous xenograft model and Patient-Derived tumor Xenograft (PDX) model. Mechanistically, PRMT5-catalyzed H3R2me2s promotes the enrichment of H3K4me3 in the Twist1 promoter region by recruiting WDR5, and subsequently activates the transcription of Twist1. The rescue experiments indicated that overexpressed Twist1 abrogated the inhibition of cell invasion induced by PRMT5 inhibitor. In summary, this study elucidates that PRMT5 inhibition could reduce H3K4me3-mediated Twist1 transcription and retard the carcinogenesis and metastasis of HNSCC.

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.

Non-Histone Arginine Methylation by Protein Arginine Methyltransferases.

Protein arginine methyltransferase (PRMT) enzymes play a crucial role in RNA splicing, DNA damage repair, cell signaling, and differentiation. Arginine methylation is a prominent posttransitional modification of histones and various non-histone proteins that can either activate or repress gene expression. The aberrant expression of PRMTs has been linked to multiple abnormalities, notably cancer. Herein, we review a number of non-histone protein substrates for all nine members of human PRMTs and how PRMT-mediated non-histone arginine methylation modulates various diseases. Additionally, we highlight the most recent clinical studies for several PRMT inhibitors.

Abstract 1886: Role of PRMT1 and p120 catenin in mediating EMT and Osimeritinib resistance in NSCLC

Abstract Osimeritinib, a Tyrosine Kinase Inhibitor (TKI) has become a first-line therapy in EGFR-mutant NSCLC patients. Resistance to Osimeritinib treatment may occur due to acquired C797S mutations, MET amplifications and ALK rearrangements and other mechanisms not clearly defined. EMT may be one of the reasons for TKI resistance. EMT is a process by which epithelial cells acquire mesenchymal characteristics that increase invasiveness. We hypothesize that loss of E-cadherin during EMT, results in abnormal expression and localization of p120 catenin, an E-cadherin regulating protein, and Kaiso factor, a transcription repressor of Wnt signaling, promoting oncogenesis. Protein arginine methyl transferase 1 (PRMT1) an EMT regulator catalyzes the methylation of Twist1, which represses E-cadherin. However, the role of PRMT1, p120 catenin, and Kaiso factor in Osimeritinib resistance is still unknown. We earlier studied PRMT1/p120 catenin in NSCLC cell lines with and without EGFR mutations and determined their role in Erlotinib resistance. Recently, to study the resistance mechanisms, we have made parental (P) NSCLC cells, H2170, H358, H1975 and H3255 cells resistant to Osimeritinib. MTT cell viability assays were performed on the parental cell lines and Osimeritinib resistant cell lines (OR) which showed that the H2170OR, H3255OR, H358OR, H1975OR cell lines had IC50 for Osimeritinib, 140, 2.8, 4.6, 10 fold higher in comparison to H2170P, H3255P, H358P, and H1975P cell lines, respectively. We further performed qPCR to study the modulation of EMT biomarkers in Osimeritinib resistant NSCLC cells as compared to parental cells and found that there was upregulation of PRMT1, p120 catenin and Kaiso factor by 1.5 fold to 3.67 fold respectively in H1975 cells and 1 fold to 5.73 fold in H3255 cells. Immunofluorescence studies in H1975OR and H3255OR cells for expression of PRMT1 showed that there was 3.18 fold increase as compared to 2.21 fold increase in Erlotinib resistant H1975 (H1975ER) and 4.77 fold increase in H3255OR as compared to 2.27 fold increase in H3255ER. We further investigated the cancer stem cell (CSC) markers BMI and Oct-4 expression in Erlotinib resistant cells and found an increase in CSC by 1.1 fold and 1.2 fold in H358ER cells. Studies with these CSC markers in Osimeritinib resistant cell lines are being conducted. Furthermore, we corelated the expression of PRMT1 with smoking in NSCLC tumors from Winnebago county area. Our results showed a trend that smokers had higher expression and that 29% of smokers with less than a 30 pack-year history show high expression of PRMT1 compared to 44% in smokers with greater than a 30 pack-year history. In conclusion, biomarkers like PRMT1 may mediate the process of EMT by methylation of transcriptional factors such as Twist1 and upregulation of p120 that prevents transcriptional repression of Kaiso factor target genes, promoting the EMT process in Osimeritinib and Erlotinib resistant cells. Citation Format: Kavya Sri Racherla, Nabiha H. Khan, Sanjana Singh, Neelu Puri. Role of PRMT1 and p120 catenin in mediating EMT and Osimeritinib resistance in NSCLC [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 1886.

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.

Discovery of First-in-Class Protein Arginine Methyltransferase 5 (PRMT5) Degraders.

The aberrant expression of protein arginine methyltransferase 5 (PRMT5) has been associated with multiple cancers. Using the proteolysis targeting chimera technology, we discovered a first-in-class PRMT5 degrader 15 (MS4322). Here, we report the design, synthesis, and characterization of compound 15 and two structurally similar controls 17 (MS4370) and 21 (MS4369), with impaired binding to the von Hippel-Lindau E3 ligase and PRMT5, respectively. Compound 15, but not 17 and 21, effectively reduced the PRMT5 protein level in MCF-7 cells. Our mechanism studies indicate that compound 15 degraded PRMT5 in an E3 ligase- and proteasome-dependent manner. Compound 15 also effectively reduced the PRMT5 protein level and inhibited growth in multiple cancer cell lines. Moreover, compound 15 was highly selective for PRMT5 in a global proteomic study and exhibited good plasma exposure in mice. Collectively, compound 15 and its two controls 17 and 21 are valuable chemical tools for exploring the PRMT5 functions in health and disease.

PRMT5, regulated by lncRNA ZFAS1/miR-150-5p, promoted androgen-independent prostate cancer migration and invasion

IntroductionProstate cancer (PCa) is the most common male genitourinary malignancy in the world. The protein arginine methyltransferase 5 (PRMT5) is one of the main members of the type II PRMT family, which was reported to regulate androgen-dependent PCa cell proliferation. However, the upstream regulators of PRMT5 and its effects on androgen-independent PCa metastasis remained unclear. In the present study, we investigated whether PRMT5 could be a novel diagnostic marker and be used as a therapeutic target in PCa, to explore the possible molecular mechanism, and to understand the clinical importance of PRMT5 in PCa.Material and methodsThe present study evaluated PRMT5 expression levels in PCa and normal prostate samples using public datasets, including TCGA, GEPIA and GSE21032. Furthermore, CCK-8 assay, flow cytometer assay, and transwell assay were conducted to detect the roles of PRMT5. Luciferase reporter assay was used to determine the relationship among ZFAS1/miR-150-5p/PRMT5.ResultsOur results showed that PRMT5 was overexpressed in PCa samples. PRMT5 significantly promoted androgen-dependent PCa proliferation and cell cycle progression and suppressed cell apoptosis. However, PRMT5 did not affect androgen-independent PCa proliferation but it could significantly induce androgen-independent PCa metastasis. Knockdown of PRMT5 suppressed, whereas overexpression of PRMT5 induced, cell migration and invasion in androgen-independent DU145 and PC-3 cells. Moreover, our results showed that the ZFAS1/miR-150-5p axis regulated PRMT5 expression in PCa cells. Furthermore, the study showed that ZFAS1 and PRMT5 were overexpressed and miR-150-5p was down-regulated in PCa samples. Higher expression levels of ZFAS1 and PRMT5 were correlated with shorter disease-free survival time in PCa patients.ConclusionsThese results showed that PRMT5 may be a therapeutic target for PCa.

Protein Arginine Methyltransferase 5 as a Therapeutic Target for KRAS Mutated Colorectal Cancer.

Nearly 45% of colorectal cancer (CRC) patients harbor a mutation in their KRAS gene for which, despite many years of research, there are still no targeted therapies available. Protein Arginine Methyltransferase 5 (PRMT5) is a transcription regulator for multiple cellular processes that is currently being tested as a potential target in several cancer types. PRMT5 has been previously shown to be overexpressed in approximately 75% of CRC patient tumor samples, as well as negatively correlated with CRC patient survival. Here, we provide evidence that PRMT5 can act as a surrogate target for mutated KRAS in CRC. Our findings show that PRMT5 expression is upregulated, as well as positively correlated with KRAS expression, in CRC patient datasets. Moreover, our results reveal that PRMT5 is further overexpressed in KRAS mutant CRC cells when compared to KRAS wild type (WT) CRC cells at both the transcriptional and translational levels. Additionally, our data demonstrate that this further overexpression of PRMT5 in the KRAS mutant CRC cells affects an even greater degree of growth inhibition, apoptosis, and cell cycle arrest, following treatment with PRMT5 inhibitor, when compared to the KRAS WT CRC cells. Our research therefore suggests for the first time that PRMT5 and KRAS may crosstalk, and thus, PRMT5 can potentially be used as a surrogate target for mutated KRAS in CRC.

Targeting G6PD reverses paclitaxel resistance in ovarian cancer by suppressing GSTP1

Ovarian cancer is one of the leading causes of mortality in women worldwide. Currently, paclitaxel is one of the most effective chemotherapies. However, resistance to paclitaxel is a major cause of therapy failure and the precise mechanism of paclitaxel resistance remains unclear. In this study, we demonstrated that the oxidative pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) promotes paclitaxel resistance. We showed that G6PD expression was higher in paclitaxel-resistant cancer cells than in their paclitaxel-sensitive counterparts. Furthermore, we demonstrated that suppressing G6PD using shRNA, or an inhibitor, either as single agents or in combination, sensitized paclitaxel-resistant cancer cells to paclitaxel treatment and thereby improving the therapeutic efficacy of paclitaxel. Interestingly, we found that the upregulation of G6PD in paclitaxel-resistant cells was due to the decreased expression of protein arginine methyltransferase 6 (PRMT6), which targets the promoter of G6PD. We further identified that G6PD promotes paclitaxel resistance by regulating the expression of glutathione S-transferase P1 (GSTP1), which confers resistance to chemotherapy by detoxifying several anticancer drugs. Taken together, our results suggest that G6PD is a novel potential target to overcome paclitaxel resistance.

PRMT1 Is Critical for the Transcriptional Activity and the Stability of the Progesterone Receptor.

SummaryThe progesterone receptor (PR) is an inducible transcription factor that plays critical roles in female reproductive processes and in several aspects of breast cancer tumorigenesis. Our report describes the type I protein arginine methyltransferase 1 (PRMT1) as a cofactor controlling progesterone pathway, through the direct methylation of PR. Mechanistic assays in breast cancer cells indicate that PRMT1 methylates PR at the arginine 637 and reduces the stability of the receptor, thereby accelerating its recycling and finally its transcriptional activity. Depletion of PRMT1 decreases the expression of a subset of progesterone-inducible genes, controlling breast cancer cells proliferation and migration. Consistently, Kaplan-Meier analysis revealed that low expression of PRMT1 predicts a longer survival among the subgroup with high PR. Our study highlights PR methylation as a molecular switch adapting the transcription requirement of breast cells during tumorigenesis.

The polymorphism rs975484 in the protein arginine methyltransferase 1 gene modulates expression of immune checkpoint genes in hepatocellular carcinoma

Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPβ-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.

PRMT1 promotes neuroblastoma cell survival through ATF5

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.