Abstract
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.
Disclosures
Baiocchi: Essanex: Research Funding.
Protein arginine methyltransferase 5 represses tumor suppressor miRNAs that down-regulate CYCLIN D1 and c-MYC expression in aggressive B-cell lymphoma
