Protein Arginine Methyltransferase 5 Regulates WNT/β-Catenin Target Gene Expression in at Multiple Levels

Introduction: It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promotes lymphomagenesis. Over-expression of the type II protein arginine methyltransferase (PRMT5) enzyme has been associated with increased cell proliferation and survival in both solid and blood cancers. We have reported that PRMT5 is essential for EBV-driven B cell transformation and that it regulates the EZH2, EED and SUZ12, components of the Polycomb-repressive complex 2 (PRC2) complex via transcriptional silencing of RBL2and hyper-phosphorylation of RB1 in aggressive lymphomas. Here we report a mechanistic assessment of how PRMT5 over-expression supports the WNT/β-CATENIN pathway at multiple levels and drives oncogenic β-CATENIN target genes in lymphoma.

Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells was achieved by infecting with sh-PRMT5 lentivirus (or sh-GFP control), a selective small molecule PRMT5 inhibitor (Alinari et al, Blood 2015, CMP5) or CRISPR/CAS9 PRMT5 deletion. Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by MTS proliferation assay and FACS analysis. WIF1 protein detection in cell culture media was performed by ELISA.

Results: PRMT5 regulated WNT/β-CATENIN signaling by direct transcriptional repression of AXIN2 and WIF1, two proteins that negatively regulate this pathway. PRMT5 inhibition with shRNA, CRISPR/CAS9 deletion, or CMP5 led to restored expression of AXIN2 and WIF1 transcript and protein that was associated with transcriptional repression of WNT/β-CATENIN target genes CYCLIN D1, MYC, and SURVIVIN. With PRMT5 inhibition, we observed differential enhanced recruitment of co-repressors LSD and HDAC2 and loss of associated epigenetic marks H3K4Me3 and H3K9Me (associated with the LSD demethylase) and H3K9Ac and H3K14Ac (associated with HDAC2) at each b-CATENIN target promoter. PRMT5 inhibition was found to reduce recruitment of co-activators CBP and MLL1 and respective epigenetic mark H3K4me3. The de-repression of AXIN2 and WIF1 was associated with loss of phospho-AKT (S450, S473) and down-stream survival pathways. Reduction in phospho-AKT was attributed to physical association between AXIN2 and the down-stream activity of secreted WIF1 in media of lymphoma cells treated with PRMT5 inhibition or CRISPR/CAS9 PRMT5 deletion. Furthermore, reduction of phospho-AKT prevented dimerization of MLL1 leading to dissociation of the BCL9-Pygopus TCF1/b-CATENIN transcriptional activation complex at MYC, CYCLIND1, and SURVIVIN promoters.

Our observations show that PRMT5 is an important epigenetic regulator that governs the expression WNT/β-CATENIN-driven oncogenes c-MYC, CYCLIND D1 and SURVIVIN. PRMT5 inhibition restores regulation at several levels that converge on AKT signaling; (i) AXIN2-AKT interaction and (ii) WIF1 inhibition of WNT signaling. The restored regulation occurs via modulation of the downstream transcriptional machinery that is supported by constitutive AKT activity. This data identifies a novel pathway to interfere with WNT/β-CATENIN signaling and validates the driver role orchestrated by PRMT5 in lymphoma.