ALBERT

Description: Abstract 2378 Epstein-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.

Description: Abstract 595FN2 Introduction: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by aberrant genetic (t(11;14)(q13;q32)) and epigenetic (DNA hypermethylation) dysregulation. 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 B-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). We have previously identified PRMT5 over expression to be relevant to MCL pathogenesis and shown it to work concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. siRNA-mediated knockdown of PRMT5 in MCL cell lines leads to growth arrest and apoptosis, thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for this disease. Methods and Results: 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 from Protein Data Bank (MODELLER9v1 software). We screened a library of 10,000 compounds and eight small molecules were identified for biological investigation based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays using purified PRMT1 (type I PRMT) and PRMT5 (type II PRMT) showed that two compounds (BLL1 and BLL3) were capable of selectively inhibiting PRMT5 and not PRMT1 activity (p

Description: Introduction: Emerging data collected from whole genome and epigenomic studies in solid and blood cancers has pointed toward dysregulation of chromatin remodelers as a unique class of cancer drivers. Next generation sequencing of lymphoma has identified several mutations affecting enzymes that regulate epigenetic control of gene expression. The epigenetic modifier protein arginine methyltransferase 5 (PRMT5) that has been shown to be essential for Epstein-Barr virus-driven B-cell transformation, is overexpressed in several histologic subtypes of B-cell non-Hodgkin's lymphomas (NHL) and is required for the driver activity of oncogenes such as MYC and NOTCH. While these findings suggest that PRMT5 may act as a driver of lymphomagenesis, definitive experiments to address its driver activity have yet to be performed. To address this question, we developed a transgenic mouse model by immunoglobulin m heavy chain enhancer/promoter (Em)-driven PRMT5 over expression in the lymphoid compartment of FVB/N mice. Methods: Eµ-hPRMT5 transgenic mice were created by injecting a vector containing floxed human PRMT5 under the control of the Eµ enhancer/promoter into FVB/N pronuclei that were implanted into pseudo-pregnant FVB/N mice. We obtained 5 founder lines demonstrating the presence of transgene construct by genotype PCR analysis of tail snip DNA. Founder mice were crossed with wild type FVB/N mice to obtain a F1 generation. Mice were followed clinically in standard pathogen-free housing until exhibiting phenotypic features at which time necropsy was performed. Immunophenotypic analysis was performed by flow cytometry, clonality by T cell receptor (TCR) Vb PCR, and pathology by hematoxylin-eosin staining and tissue micro-arrays developed for immunohistochemical staining (IHCS). Statistical significance was determined using a two-tail t-test and survival analysis conducted using Kaplan Meier curves. Results: F1 generation Eµ-hPRMT5 mice significantly overexpressed PRMT5 mRNA in unpurified splenocytes or bone marrow relative to non-transgenic mice (p-value 〈 0.001). Sorting B (CD19), NK (NK1.1) and T-cell (CD3) mononuclear subsets from splenocytes collected from Eµ-hPRMT5 mice (n=3/group) revealed PRMT5 mRNA to be overexpressed 37-fold (p-value

Description: Key PointsEBV infection leads to PRMT5 overexpression and global epigenetic changes that are essential to drive B-lymphocyte transformation. Highly selective PRMT5 inhibitors represent a novel, first-in-class drug that restores critical regulatory checkpoints in lymphoma cells.