Overexpression Of Protein Arginine Methyltransferase 5 Research Articles

PRMT5 Targets Tumor Suppressor Micro RNAs to Regulate Cyclin D1 and c-MYC in Mantle Cell Lymphoma

Protein arginine methyltransferase-5 (PRMT5), a major type II arginine methyltransferase, is an important epigenetic modifier with oncogene-like properties due to its transcriptional repressive activity. When over-expressed, PRMT5 has been shown to target and silence the expression of multiple regulatory and tumor suppressor genes. Global symmetric dimethylation of arginine residues within the N-terminal of histones (H2A(Me2)R3, H3(Me2)R8, H4(Me2)R3) plays a critical role in B cell transformation, correlates with increased tumor cell proliferation and survival. PRMT5 expression is enhanced in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma (MCL) and supports constitutive CYCLIN D1/CDK4/6 activity leading to inactivation of the RBL2/E2F tumor suppressor pathway. Other work has identified PRMT5 as a vital contributor to MYC-driven oncogenesis. While PRMT5 has been characterized as a transcriptional repressor, few micro-RNAs (miRs) have been identified as direct targets.Here we utilize next generation sequencing and whole genome mapping to identify miRs targeted by PRMT5 in mantle cell lymphomas. ChIP-seq analysis revealed genome-wide recruitment of the PRMT5-specific epigenetic mark H3(Me2)R8 in B-cell lymphoma cell lines (Jeko, Pfeiffer, SUDHL2) with minimal enrichment on chromatin from normal B cells. PRMT5 was found to target as many as 8593 genes in each lymphoma cell line examined with 4662 genes that are common targets between three different lymphoma cell lines. Comparing Chip-Seq to RNA-Seq data of each lymphoma cell line treated with PRMT5 shRNA identified miR-33b, miR-96 and miR-503 as direct targets. Predicted 3' untranslated region (UTR) targets of these miRs included CYCLIN D1 (miR-33b, miR-96, miR-503), MYC (miR-33b) and PRMT5 (miR-96), three gene products that have been shown to be highly relevant to the malignant phenotype of aggressive MCL. Validation studies with ChIP-real-time polymerase chain reaction (RT-PCR) and quantitative-RT-PCR demonstrated that CRISPR-CAS9-mediated PRMT5 deletion led to transcriptional derepression of miR-33b, miR-96 and miR-503 in MCL lines (CC-MCL, Jeko, and SP53) and in primary MCL patient samples. Inhibition of PRMT5 led to loss of recruitment of an epigenetic repressor complex at the miR-96 promoter containing PRMT5 and HDAC3, gain of p65 and enrichment of hyperacetylated lysine epigenetic marks H4K8, H3K14 and H2BK12, changes consistent with restored transcriptional activity of miR96. Promoter regions of miR33b and miR503 showed loss of recruitment of a repressive complex consisting of SP1, HDAC2 and gain of p300, CBP and hyperacetylated lysine epigenetic marks H3K9 and H3K14 following PRMT5 inhibition. Restored expression of miR-33b led to simultaneous down-modulation of CYCLIN D1 and c-MYC, whereas miR-96 re-expression led to loss of CYCLIN D1 and PRMT5. Re-expression of miR-503 on the contrary only impacted CYCLIN D1 expression. Furthermore, luciferase reporter assays with wild-type and mutant 3' UTRs showed that binding sites of miR-33b, miR-96 and miR-503 were critical for translational regulation of CYCLIN D1 and C-MYC. Inducible re-expression of miR-33b, miR-96 and miR-503 inhibited proliferation of MCL cells as determined by MTS assay and promoted cellular apoptosis as measured by staining with Annexin V/PI staining and flow cytometry. In vivo studies with the MCL cell line CC-MCL1 designed to conditionally express miR-96 and or miR-33b are currently underway.These results link dysregulated PRMT5 expression to transcriptional silencing of tumor suppressor miRs capable of regulating critical drivers of aggressive histologic subtypes of MCL and indicate that multiple mechanisms are involved in the regulation of CYCLIN D1 expression in MCL. Furthermore, this data supports PRMT5 overexpression as a critical factor involved with maintenance of the malignant phenotype of MCL, and supports strategies to selectively inhibit this promising therapeutic target in this disease. DisclosuresBaiocchi:Essanex: Research Funding.

Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia.

Imatinib-insensitive leukemia stem cells (LSCs) are believed to be responsible for resistance to BCR-ABL tyrosine kinase inhibitors and relapse of chronic myelogenous leukemia (CML). Identifying therapeutic targets to eradicate CML LSCs may be a strategy to cure CML. In the present study, we discovered a positive feedback loop between BCR-ABL and protein arginine methyltransferase 5 (PRMT5) in CML cells. Overexpression of PRMT5 was observed in human CML LSCs. Silencing PRMT5 with shRNA or blocking PRMT5 methyltransferase activity with the small-molecule inhibitor PJ-68 reduced survival, serial replating capacity, and long-term culture-initiating cells (LTC-ICs) in LSCs from CML patients. Further, PRMT5 knockdown or PJ-68 treatment dramatically prolonged survival in a murine model of retroviral BCR-ABL-driven CML and impaired the in vivo self-renewal capacity of transplanted CML LSCs. PJ-68 also inhibited long-term engraftment of human CML CD34+ cells in immunodeficient mice. Moreover, inhibition of PRMT5 abrogated the Wnt/β-catenin pathway in CML CD34+ cells by depleting dishevelled homolog 3 (DVL3). This study suggests that epigenetic methylation modification on histone protein arginine residues is a regulatory mechanism to control self-renewal of LSCs and indicates that PRMT5 may represent a potential therapeutic target against LSCs.

The E3 ubiquitin ligase CHIP mediates ubiquitination and proteasomal degradation of PRMT5

Protein arginine methyltransferase 5 (PRMT5) is an important member of the protein arginine methyltransferase family that regulates many cellular processes through epigenetic control of target gene expression. Because of its overexpression in a number of human cancers and its essential role in cell proliferation, transformation, and cell cycle progression, PRMT5 has been recently proposed to function as an oncoprotein in cancer cells. However, how its expression is regulated in cancer cells remains largely unknown. We have previously demonstrated that the transcription of PRMT5 can be negatively regulated by the PKC/c-Fos signaling pathway through modulating the transcription factor NF-Y in prostate cancer cells. In the present study, we demonstrated that PRMT5 undergoes polyubiquitination, possibly through multiple lysine residues. We also identified carboxyl terminus of heat shock cognate 70-interacting protein (CHIP), an important chaperone-dependent E3 ubiquitin ligase that couples protein folding/refolding to protein degradation, as an interacting protein of PRMT5 via mass spectrometry. Their interaction was further verified by co-immuoprecipitation, GST pull-down, and bimolecular fluorescence complementation (BiFC) assay. In addition, we provided evidence that the CHIP/chaperone system is essential for the negative regulation of PRMT5 expression via K48-linked ubiquitin-dependent proteasomal degradation. Given that down-regulation of CHIP and overexpression of PRMT5 have been observed in several human cancers, our finding suggests that down-regulation of CHIP may be one of the mechanisms underlying PRMT5 overexpression in these cancers.

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing.

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

PRMT5 is required for lymphomagenesis triggered by multiple oncogenic drivers.

Protein arginine methyltransferase 5 (PRMT5) has been implicated as a key modulator of lymphomagenesis. Whether PRMT5 has overt oncogenic function in the context of leukemia/lymphoma and whether it represents a therapeutic target remains to be established. We demonstrate that inactivation of PRMT5 inhibits colony-forming activity by multiple oncogenic drivers, including cyclin D1, c-MYC, NOTCH1, and MLL-AF9. Furthermore, we demonstrate that PRMT5 overexpression specifically cooperates with cyclin D1 to drive lymphomagenesis in a mouse model, revealing inherent neoplastic activity. Molecular analysis of lymphomas revealed that arginine methylation of p53 selectively suppresses expression of crucial proapoptotic and antiproliferative target genes, thereby sustaining tumor cell self-renewal and proliferation and bypassing the need for the acquisition of inactivating p53 mutations. Critically, analysis of human tumor specimens reveals a strong correlation between cyclin D1 overexpression and p53 methylation, supporting the biomedical relevance of this pathway. We have identified and functionally validated a crucial role for PRMT5 for the inhibition of p53-dependent tumor suppression in response to oncogenic insults. The requisite role for PRMT5 in the context of multiple lymphoma/leukemia oncogenic drivers suggests a molecular rationale for therapeutic development.

Exploration of cyanine compounds as selective inhibitors of protein arginine methyltransferases: synthesis and biological evaluation.

Protein arginine methyltransferase 1 (PRMT1) is involved in many biological activities, such as gene transcription, signal transduction, and RNA processing. Overexpression of PRMT1 is related to cardiovascular diseases, kidney diseases, and cancers; therefore, selective PRMT1 inhibitors serve as chemical probes to investigate the biological function of PRMT1 and drug candidates for disease treatment. Our previous work found trimethine cyanine compounds that effectively inhibit PRMT1 activity. In our present study, we systematically investigated the structure–activity relationship of cyanine structures. A pentamethine compound, E-84 (compound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity, and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound.

Identification of a First-in-Class PRMT5 Inhibitor with Potent in Vitro and in Vivo Activity in Preclinical Models of Mantle Cell Lymphoma

Protein Arginine Methyltransferase-5 (PRMT5) has been reported to play a role in multiple diverse cellular processes including tumorigenesis. Overexpression of PRMT5 has been demonstrated in cell lines and primary patient samples derived from lymphomas, particularly Mantle Cell Lymphoma (MCL). Furthermore, knockdown of PRMT5 expression inhibits the proliferation of MCL cell lines. The mechanisms behind the oncogenic potential of PRMT5 are unclear, but the protein has been postulated to regulate processes such as cell death, cell cycle progression, and RNA processing through the dimethylation of arginine residues within a variety of cytoplasmic and nuclear target proteins. Epizyme developed small molecule inhibitors of PRMT5 enzyme activity in order to understand the role of PRMT5-mediated arginine methylation in tumorigenesis and to develop PRMT5-targeted cancer therapeutics. Here, we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with anti-proliferative effects in both in vivo and in vitro models of MCL. A diverse compound library was screened for inhibitors of arginine methylation by purified recombinant PRMT5:MEP50 complex and multiple hits were identified. The inhibitors are SAM uncompetitive, peptide competitive and bind with the PRMT5:MEP50 complex in a unique binding mode not previously observed. Further optimization yielded YQ36286, an orally available inhibitor of PRMT5 with enzymatic activity in biochemical assays with an IC50 in the low nM range and broad selectivity against a panel of other histone methyltransferases. YQ36286 demonstrated potent cellular activity as measured by its ability to inhibit symmetric dimethylation of SmD3, a cytoplasmic PRMT5 substrate in a time- and concentration-dependent manner. Treatment of MCL cell lines with YQ36286 led to inhibition of SmD3 methylation and cell killing, with IC50s in the nM range. Oral dosing of YQ36286 demonstrated dose-dependent anti-tumor activity in multiple MCL xenograft models. In xenograft studies with the Z138 MCL cell line, near 95% tumor growth inhibition was observed after 21 days of dosing with a corresponding decrease in symmetrically dimethylated levels of PRMT5 substrates. In summary, we have developed the first potent and selective small molecule inhibitor of PRMT5 that has cellular activity and in vivo efficacy. MCL cells are dependent on PRMT5 activity for their survival as demonstrated with YQ36286. This small molecule represents a starting point for the development of PRMT5 inhibitors as potential cancer therapeutics. DisclosuresPenebre:Company stock options: Equity Ownership; Epizyme Inc.: Employment; GSK Research Funding: Research Funding. Kuplast:GSK research funding: Research Funding; Company Stock options: Equity Ownership; Epizyme Inc.: Employment. Majer:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Johnston:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Rioux:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Munchhof:Epizyme Inc.: Employment; GSK research funding: Research Funding. Jin:Epizyme Inc.: Employment; GSK research funding: Research Funding; Company stock options: Equity Ownership. Boriak-Sjodin:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Wigle:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Jacques:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. West:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Lingaraj:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Stickland:GSK research funding: Research Funding; Company Stock options: Equity Ownership; Epizyme Inc.: Employment. Ribich:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Raimondi:Epizyme: Employment, Equity Ownership; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Porter-Scott:Company stock options: Equity Ownership; GSK research funding: Research Funding; Epizyme Inc.: Employment. Waters:Epizyme, Inc: Employment, Equity Ownership; GSK research funding: Research Funding. Pollock:Epizyme: Employment, Equity Ownership; GSK research funding: Research Funding. Smith:GSK research funding: Research Funding; Epizyme: Employment, Equity Ownership. Barbash:GlaxoSmithKline Pharmaceuticals: Employment. Kruger:GlaxoSmithKline Pharmaceuticals: Employment, Equity Ownership. Copeland:Mersana: Membership on an entity’s Board of Directors or advisory committees; Epizyme, Inc: Employment, Equity Ownership; Celgene, Inc: Research Funding; Eisai Inc: Research Funding; Glaxo Smith Kline, Inc: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; New Enterprise Associates: Ad hoc consultant, Ad hoc consultant Other. Moyer:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Chesworth:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Duncan:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding.

PRMT5 enhances generation of induced pluripotent stem cells from dairy goat embryonic fibroblasts via down-regulation of p53.

Protein arginine methyltransferase 5 (PRMT5), is thought to play a role in epigenetic reprogramming of mouse germ cells. However, up to now there has been little information concerning its expression profile and effects on generation of induced pluripotent stem cells (iPSCs) from somatic cells, in livestock. Here, we have explored PRMT5 expression profiles in dairy goats and its consequences to derivation of iPSCs from dairy goat embryonic fibroblasts (GEFs). We investigated effects of PRMT5 on iPS-like cells production in vitro. alkaline phosphatase (AP) staining, QRT-PCR and western blotting analysis of expression of related markers were used to evaluate efficiency of generation of iPSCs derived from GEFs. These showed PRMT5 to be a conservative gene widely expressed in various tissues and different-aged testes. PRMT5 overexpression in combination with OCT3/4, SOX2, KLF4 and C-MYC (POSKM) significantly increased number of AP positive iPS-like colony-derived GEFs compared to OSKM alone, in our dairy goats. Moreover, our results demonstrated that PRMT5 overexpression stimulated GEF proliferation and down-regulated p53, p21 (a target gene of p53) and the apoptotic marker caspase 3, to enhance somatic cell reprogramming. This study provides an efficient model for future studies on mechanisms underlying goat somatic cell reprogramming and differentiation.

Transcriptional activation of PRMT5 by NF-Y is required for cell growth and negatively regulated by the PKC/c-Fos signaling in prostate cancer cells

Protein arginine methyltransferase 5 (PRMT5) symmetrically methylates arginine residues of histones and non-histone protein substrates and regulates a variety of cellular processes through epigenetic control of target gene expression or post-translational modification of signaling molecules. Recent evidence suggests that PRMT5 may function as an oncogene and its overexpression contributes to the development and progression of several human cancers. However, the mechanism underlying the regulation of PRMT5 expression in cancer cells remains largely unknown. In the present study, we have mapped the proximal promoter of PRMT5 to the −240bp region and identified nuclear transcription factor Y (NF-Y) as a critical transcription factor that binds to the two inverted CCAAT boxes and regulates PRMT5 expression in multiple cancer cell lines. Further, we present evidence that loss of PRMT5 is responsible for cell growth inhibition induced by knockdown of NF-YA, a subunit of NF-Y that forms a heterotrimeric complex with NF-YB and NF-YC for function. Significantly, we have found that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) in LNCaP prostate cancer cells down-regulates the expression of NF-YA and PRMT5 at the transcription level in a c-Fos-dependent manner. Given that down-regulation of several PKC isozymes is implicated in the development and progression of several human cancers, our findings suggest that the PKC-c-Fos-NF-Y signaling pathway may be responsible for PRMT5 overexpression in a subset of human cancer patients.

Inhibition of Cardiomyocyte Hypertrophy by Protein Arginine Methyltransferase 5

Protein arginine methyltransferase 5 (PRMT5), a protein arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues within target proteins, has been implicated in many essential cellular processes ranging from the regulation of gene expression to cell proliferation and differentiation. PRMT5 is highly expressed in the heart; the functional role of PRMT5 in the heart, however, remains largely elusive. In the present study, we show that PRMT5 specifically interacts with GATA4 in both co-transfected HEK293T cells and neonatal rat cardiomyocytes by co-immunoprecipitation. Importantly, this interaction leads to the arginine methylation of GATA4 at positions of 229, 265, and 317, which leads to an inhibition of the GATA4 transcriptional activity, predominantly through blocking the p300-mediated acetylation of GATA4 in cardiomyocytes. Moreover, overexpression of PRMT5 substantially inhibited the acetylation of GATA4 and cardiac hypertrophic responses in phenylephrine-stimulated cardiomyocytes, whereas knockdown of PRMT5 induced GATA4 activation and cardiomyocyte hypertrophy. Furthermore, in response to phenylephrine stimulation, PRMT5 translocates into the cytoplasm, thus relieving its repression on GATA4 activity in the nucleus and leading to hypertrophic gene expression in cardiomyocytes. These findings indicate that PRMT5 is an essential regulator of myocardial hypertrophic signaling and suggest that strategies aimed at activating PRMT5 in the heart may represent a potential therapeutic approach for the prevention of cardiac hypertrophy and heart failure.

Protein arginine methyltransferase 6 enhances ligand-dependent and -independent activity of estrogen receptor α via distinct mechanisms

Recent studies reported that protein arginine methyltransferase 6 (PRMT6) enhances estrogen-induced activity of estrogen receptor α (ERα) and dysfunction of PRMT6 is associated with overall better survival for ERα-positive breast cancer patients. However, it is unclear how PRMT6 promotes ERα activity. Here we report that PRMT6 specifically interacts with ERα at its ligand-binding domain. PRMT6 also methylates ERα both in vitro and in vivo. In addition to enhancing estrogen-induced ERα activity, PRMT6 over-expression up-regulates estrogen-independent activity of ERα and PRMT6 gene silencing in MCF7 cells inhibits ligand-independent ERα activation. More interestingly, the effect of PRMT6 on the ligand-independent ERα activity does not require its methyltransferase activity. Instead, PRMT6 competes with Hsp90 for ERα binding: PRMT6 and Hsp90 bindings to ERα are mutually exclusive and PRMT6 over-expression reduces ERα interaction with Hsp90. In conclusion, PRMT6 requires its methyltransferase activity to enhance ERα's ligand-induced activity, but its effect on ligand-independent activity is likely mediated through competing with Hsp90 for binding to the C-terminal domain of ERα. PRMT6-ERα interaction would prevent ERα-Hsp90 association. Since Hsp90 and associated chaperones serve to maintain ERα conformation for ligand-binding yet functionally inactive, inhibition of ERα-Hsp90 interaction would relieve ERα from the constraint of chaperone complex.

Cellular Localization of Protein Arginine Methyltransferase-5 Correlates With Grade of Pulmonary Neuroendocrine Tumors

Epigenetic regulation plays an important role in oncogenesis. Histone modification is recognized as one strategy for modifying epigenetic control and cancer therapies. Cell culture and animal models show that protein arginine methyltransferase-5 (PRMT5) is an important epigenetic modifier of histone and nonhistone proteins in lymphomas, breast, colorectal, and lung cancer. Overexpression of PRMT5 is associated with malignant phenotype in …

Abstract 1128: Preclinical and pharmacological activities of protein arginine methyltransferase 5(PRMT5) enzyme inhibition in acute myeloid leukemia .

Abstract With standard cytotoxic chemotherapy the majority of patients with Acute Myeloid Leukemia (AML) fail to achieve long term remission. Protein Arginine Methyltransferase 5 (PRMT5) can catalyze methylation of arginine residues in target proteins like histones and P53. The epigenetic marks of PRMT5 on histones H3 (S2Me-H3R8) and H4 (S2Me-H4R3), directly lead to transcriptional silencing of genes. Overexpression of PRMT5 has been reported in hematologic and solid malignancies and represents a promising therapeutic target. Thus, we hypothesized that inhibition of PRMT5 enzymatic activity using a first in class, selective small molecule inhibitor “BLL1” would result in antileukemic activity and novel methods to treat AML. PRMT5 was upregulated in AML-derived cell lines and primary patient samples. PRMT5 in complex with co-repressors like histone deacetylase (HDAC)s and DNA methyltrasferase (DNMT)s is involved in epigenetic silencing of tumor suppressor genes. Using Co-IP assay we captured PRMT5 in association with DNMT3a and HDAC2 in AML cells, in which treatment with BLL1 (25μM) resulted in disruption of this interaction. We confirmed the ability and specificity of BLL1 by detecting the loss of unique epigenetic mark of PRMT5, S2Me-H3R8. We previously reported that pharmacologic inhibition of DNMTs (by decitabine) and HDACs (by AR42) or the disruption of their repressor complexes (by bortezomib) can inhibit critical oncogenes in AML, KIT and FLT3, by upregulating miR-29b and consequently lead to better clinical response in AML. Similar to these epigenetic-targeting compounds, BLL1 upregulated miR-29b (>7 fold; MV411, FLT3 ITD+ cells) and resulted in downregulation of FLT3 RNA (>4 fold) and protein (3.8 fold). We examined the in vitro antileukemic activity of BLL1 in AML and demonstrated a dose-dependent inhibition of cell proliferation (MTS assay) in AML cell lines (MV411; IC50=6μM & Kasumi1, KITmut, IC50=11μM) and primary patient blasts (#1, FLT3 ITD+, NPM1+, CD34−; IC50=8μM & #2, FLT3 ITD+, CD34+; IC50=7.5μM). Colony forming ability was significantly decreased (p<0.001) in BLL1 (2μM) treated AML cells compared to controls. We observed a BLL1-dependent increase in apoptosis measured by AnnexinV staining (25 & 50μM, MV411; 8.6 fold & Kasumi1; 4 fold) within 48 hrs. BLL1 treatment promoted cell maturation measured by increased monocyte differentiation marker CD11b in AML cells (50μM, MV411; 11.5 fold & Kasumi1; 6.8 fold) and primary patient blasts (25μM, #1; 4 fold). In conclusion, our data suggest that inhibiting PRMT5 activity interferes with the production or activity of oncoproteins and, subsequently, disrupts leukemogenic and proliferation pathways in AML. Given the substantial antileukemic potential of PRMT5 inhibitor compound on AML cells and primary patient blasts, an assessment of therapeutic implications of this novel drug in vivo is highly warranted. Citation Format: Somayeh S. Tarighat, Kiran Mahasenan, Danilo Perrotti, Ramiro Garzon, Michael Caligiuri, Chenglong Li, William Blum, Guido Marcucci, Robert A. Baiocchi. Preclinical and pharmacological activities of protein arginine methyltransferase 5(PRMT5) enzyme inhibition in acute myeloid leukemia . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1128. doi:10.1158/1538-7445.AM2013-1128

Abstract 4637: Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme.

Abstract Glioblastoma Multiforme (GBM) is associated with a dismal prognosis despite intensive multimodal therapy, highlighting the need for novel therapeutic approaches. Chromatin remodeling complexes and associated co-repressors such as histone deacetylases (HDAC), DNA methyltransferases (DNMT) and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor and regulatory gene expression and may contribute to glial cell transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. We have previously identified PRMT5 over-expression in GBM cell lines, primary GBM tumors, and GBM that spontaneously develop in a pre-clinical mouse model. The degree of PRMT5 over-expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p<0.0001), PRMT5 works concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. PRMT5 silending in GBM cell lines leads to growth arrest, apoptosis and loss of HDAC2, DNMT3a and PRMT5 co-repressor complex recruitment at chemokine gene promoters. These findings provide evidence that PRMT5 is a central repressive element required for maintenance of target gene silencing. Thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for GBM. A rational design of small molecule compounds to inhibit PRMT5 activity led us to construct an in silico model of the human PRMT5 catalytic domain based on available homologous crystal structures. We screened a library of 10,000 compounds and eight small molecules were identified based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays showed that a lead compound (BLL1) was capable of selectively inhibiting PRMT5 and not PRMT1 or PRMT7 activity (p<0.0001). BLL1 interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in GBM cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 showed a dose-dependent response of inhibition of cellular proliferation in GBM cell lines. Combination treatment of GBM cells with subtoxic doses of BLL1, hypomethylating agent 5-azacitidine, and HDAC inhibitor TSA showed synergistic induction of cell death, loss of the epigenetic mark S2Me-H4R3, and de-repression of the immune modulating chemokine CXCL10. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for BLL1. We have successfully developed a first in class drug to selectively target dysregulated PRMT5 enzymatic activity in GBM. We are currently developing drugs with improved selectivity and potency. Citation Format: Fengting Yan, Kate Gordon, Kiran Mahasenan, Mark Lustberg, Lapo Alinari, Christian T. Earl, Balveen Kaur, Chenglong Li, Robert A. Baiocchi. Developing a first in class of drug to inhibit protein arginine methyltransferase 5 (PRMT5) enzyme dysregulation in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4637. doi:10.1158/1538-7445.AM2013-4637

Methyltransferase PRMT1 Is a Binding Partner of HBx and a Negative Regulator of Hepatitis B Virus Transcription

The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.

PRMT6 overexpression upregulates TSP-1 and downregulates MMPs: Its implication in motility and invasion

In this study, we investigate the molecular mechanism by which protein arginine methyltransferase 6 (PRMT6) exerts anti-invasiveness effect against breast cancer cells and prostate cancer cells. PRMT6 has been known to be responsible for asymmetric dimethylation of histone H3 at R2 (H3R2me2a). To investigate the biological role of PRMT6, we first established stable cell lines expressing GFP-PRMT6 with MCF7 and PC3 cells. Growth rates and colony forming abilities of PRMT6-overexpressing cells were significantly retarded compared to control GFP expressing cells. This growth retardation seems to be associated with p21WAF1 induction. In addition, our data show that migration and invasion of prostate cancer cells was strongly suppressed by PRMT6 overexpression. In parallel, the levels of thrombospondin-1 (TSP-1), a potent natural inhibitor of angiogenesis, were highly up-regulated in both PRMT6-overexpressing cells. Furthermore, this suppression of migration and invasion by PRMT6 overexpression was significantly rescued by specific knock-down of TSP-1. Concomitantly, down-regulations of MMP-2 and -9 were observed in PRMT6-overexpressing cells. Taken together, our data demonstrate that PRMT6 overexpression is associated with regulation of motility and invasion through up-regulation of TSP-1 and down-regulation of MMPs in human cancer cells.

Protein Arginine Methyltransferase 5 (PRMT5) Over-Expression Is Essential for Epstein-Barr Virus-Driven B-Cell Transformation.

Abstract 2378Epstein-Barr virus (EBV) is a ubiquitous, gamma herpes virus that infects human epithelial cells and B lymphocytes. Over 90% of adults worldwide are infected with EBV and, collectively, this virus is associated with a broad spectrum of benign and malignant disease. EBV is a potent oncogenic virus and is capable of efficiently transforming B cells in both in vitro and in vivo models. While signaling cascades contributing toward B cell immortalization and transformation following EBV infection have been described, epigenetic events that contribute toward the B cell transformation process remain poorly characterized. EBV-transformed lymphoblastoid lines (LCL) and spontaneous B cell lymphomas that arise in the hu-PBL-SCID model of EBV-induced lymphomagenesis show abundant expression of the protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme that catalyzes symmetric dimethylation of arginine residues on histones and non histone proteins (P53, CYCLIN D1). PRMT5 partners with multiple co-repressor proteins such as HDAC2, MBD2 and DNMT3a to silence multiple regulatory and tumor suppressor gene products. All EBV-transformed B cell lines and primary tumors showed cytoplasmic and nuclear staining for PRMT5 and its associated epigenetic marks symmetric dimethyl histone 3, arginine 8 (S2Me-H3R8) and S2Me-H4R3. Resting and activated B cells did not demonstrate PRMT5 over expression or associated global epigenetic marks. Infection of primary human B cells with the B95.8 strain (but not mutant P3HR1 or inactivated EBV) led to dysregulated expression of PRMT5 as early as day 4 post infection. By day 8 post EBV infection, PRMT5 location had transitioned to the nucleus and this localization coincided with acquisition of S2Me-H4R3 and S2Me-H3R8 and loss of the asymmetric epigenetic mark 2Me-H4R3,a type I PRMT histone mark. PRMT5 over-expression was dependent on LMP-1-driven NFkB activity and transcriptional silencing of miR96 expression, a micro RNA that targets the PRMT5 3'UTR. To determine if PRMT5 over expression was essential for induction and maintenance of the transformed phenotype, we infected resting B cells with EBV and, at various time points (day 4, 7, 14 or 21), added a novel, highly selective small molecule inhibitor of PRMT5 activity, an inactive small molecule control, shRNA specific for PRMT5 or control shRNA. Absolute CD19+ cell counts and confocal microscopy experiments to monitor PRMT5 and its epigenetic marks were performed and showed that PRMT5 activity was critical for EBV-driven B cell transformation to proceed. PRMT5 inhibition of resting or activated B cells did not result in any loss of viability. Global transcriptome analysis identified several tumor suppressor genes, including the protein tyrosine phosphatase PTPROt, were silenced during EBV-driven B cell transformation. Chromatin immune precipitation (ChIP)-sequencing using monoclonal antibodies specific for PMRT5 and S2Me-H3R8 (or control IgG) confirmed the PTPROt promoter to be directly targeted by PRMT5 and PTPROt transcript was found to become silenced during EBV-driven B cell transformation. Real time PCR and RNA-seq showed PTPROt transcript to become restored with PRMT5 inhibition. PTPROt expression led to dephosphorylation and inhibition of the LYN, SYK, and Bruton's Tyrosine kinase (BTK) kinase proteins, critical proteins involved in regulation of the B cell receptor (BCR). This model provided us with direct evidence that PRMT5 activity is critical to EBV-driven B cell transformation and supports our hypothesis that PRMT5 dysregulation drives epigenetic events that directly contribute to key initiating events during B cell transformation as well as to the maintenance of the malignant phenotype. We believe this is the first example of oncogenic virus driving over expression of an epigenetic modifier that catalyzes placement of global repressive epigenetic marks that silence of regulatory and tumor suppressor genes. This data justifies pursuit of experimental therapeutic strategies focused on selective PRMT5 inhibition in cancer. Disclosures:No relevant conflicts of interest to declare.

The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization, Stress Granules, and Detergent-Insoluble Aggregates of FUS/TLS

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.

Suppression of PRMT6-mediated arginine methylation of p16 protein potentiates its ability to arrest A549 cell proliferation

The tumor suppressor p16INK4A (p16) blocks the cell cycle progression by inhibiting phosphorylation of the retinoblastoma protein. We describe here a novel aspect of the posttranslational control that has an important functional consequence on p16 protein. We first discovered that the p16 protein was methylated in various cell lineages. We then determined that the arginine 22, 131 and 138 of p16 were the main methylation sites. Western blotting and TUNEL analyses revealed that the p16 protein bearing these point mutations induced a higher apoptosis ratio than wild-type p16 in A549 cells. Furthermore, co-immunoprecipitation assays suggested that decrease of p16 arginine methylation level promoted the association of p16 with CDK4. Additionally, we determined that the protein arginine methyltransferase 6 (PRMT6) was responsible for the p16 arginine methylation. Results from flow cytometric analysis demonstrated that PRMT6 overexpression counteracted the cell cycle arrest at G1 phase induced by wild-type p16 in A549 cells. We also provided evidence that PRMT6 was able to interact with p16, and that the intensity of p16-CDK4 association was reduced upon PRMT6 overexpression. Together, data presented in this report establish that methylation at specific arginine residues of p16 protein by PRMT6 may be critical for the activity of p16.

The arginine methyltransferase PRMT6 regulates cell proliferation and senescence through transcriptional repression of tumor suppressor genes

The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression and executes its repressing as well as activating function by asymmetric dimethylation of histone H3 at R2 (H3 R2me2a). Given that elevated expression levels of PRMT6 have been reported in various cancer types, we explore here its role in cell proliferation and senescence. We find that knockdown of PRMT6 results in proliferation defects of transformed as well as non-transformed cells, causes G1-phase arrest and induces senescence. This phenotype is accompanied by transcriptional upregulation of important cell cycle regulators, most prominently the cyclin-dependent kinase (CDK) inhibitor gene p21 (p21CIP1/WAF1, CDKN1A) and p16 (p16INK4A, CDKN2A). Chromatin immuno-precipitation analysis reveals that the p21 gene is a direct target of PRMT6 and the corresponding histone mark H3 R2me2a. Using a cell model of oncogene-induced senescence (OIS), in which p21 is an essential activator of the senescent phenotype, we show that PRMT6 expression declines upon induction of senescence and conversely p21 gene expression increases. Moreover, overexpression of PRMT6 leads to reduced levels of OIS. These findings indicate that the transcriptional repressor activity of PRMT6 facilitates cell proliferation and blocks senescence by regulation of tumor suppressor genes and that this might contribute to the oncogenic capacity of PRMT6.