PRMT5 Is a Key Epigenetic Regulator That Promotes Transcriptional Activation in Mantle Cell Lymphoma By Regulating the Lysine Methyltransferase SETD7 and MLL1 Activity

Introduction: Post-translational histone modifications directly modify chromatin structure to influence a wide variety of cellular events including gene expression, DNA replication and repair, and cell cycle control. While histone lysine methylation can confer either transcriptionally active or repressive states, the symmetric dimethylation of arginine residues on histone tails is generally associated with transcriptional repression. Overexpression and dysregulation of PRMT5, the major type II protein arginine methyltransferase, has been shown to drive cellular proliferation and survival of multiple cancer types including mantle cell lymphoma (MCL), a subtype of non-Hodgkin lymphoma associated with poor prognosis. Our work has shown that PRMT5 drives the symmetric dimethylation of arginine (histones H3R8 and H4R3) and attenuates RB/E2F regulatory pathways leading to the expression and activation of the PRC2 lysine methylation programs. We have previously shown that PRMT5 promotes constitutive expression of the WNT/β-CATENIN target genes (MYC, CCND1, and SURVIVIN) via the epigenetic repression of AXIN2 and WIF1 regulatory genes. This PRMT5 mediated epigenetic program allows for constitutive activation of WNT and PI3/AKT downstream pathways relevant to MCL growth and survival. The inhibition of PRMT5 triggers histone deacetylation and H3K4me3 demethylation on promoters of β-CATENIN target genes. H3K4 is modified by several enzymes containing SET domains including the SETD7 and MLL1 proteins. We hypothesized that PRMT5 inhibition, through its ability to repress AKT phosphorylation, would regulate SETD7 and MLL1 activity and regulate downstream pathways relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived MCL cell lines and primary lymphoma tumor cells was achieved with sh-PRMT5 lentivirus (or sh-GFP control) and utilization of a selective small molecule, SAM-competitive PRMT5 inhibitor (PRT382). Gene and protein expression was monitored by reverse transcription (RT) real time PCR and western immunoblotting, respectively. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. Interactions within the transcriptional complex were examined by co-immunoprecipitation. Cellular growth and apoptosis was assessed by proliferation assays and FACS analysis. Results: Inhibition of PRMT5 by shRNA-mediated knock down or treatment with PRT382 (Prelude Therapeutics) reduced H3K4me2 in MCL cells via indirect epigenetic repression of SETD7. ChIP-PCR studies showed PRMT5 to be recruited to the SETD7 promoter, suggesting that the activity of PRMT5 promoted the transcription of this lysine methyltransferase. Our findings show that reduced phosphor-AKT by PRMT5 inhibition inhibits dimerization of the MLL1 complex leading to dissociation of MLL1 from the transcriptional activation complex and decreased H3K4me1 and H3K4me3. PRMT5 inhibition led to dissociation of the BCL9- Pygopus-MLL1 transcriptional activating complex and to assembly of repressive LSD1 (the histone demethylase affecting H3K4me3)-HDAC2 (the histone lysine deacetylase) containing complexes. Furthermore, PRMT5 inhibition led to differentially enhanced recruitment of this repressive LSD1/HDAC2 complex and decreased H3K9Ac and H3K14Ac epigenetic marks on promoters of β-CATENIN target genes. PRMT5 inhibition regulates lysine methylation at H3K4 through epigenetic silencing of SETD7 and MLL1 activity, as well as histone H3 acetylation driven by histone acetyltransferase KAT2A. ChIP-Seq studies examining SETD7 and PRC2 recruitment in context of PRMT5 inhibition are currently underway. Conclusions: Our observations show that dysregulated PRMT5 activity acts as a master epigenetic regulator affecting arginine and lysine histone marks. PRMT5 can act directly to repress target tumor suppressor genes while simultaneously indirectly activating SETD7 and MLL1 to drive transcriptional activation of key oncogenes that promote the proliferation and survival of MCL. These results support the notion that inhibiting PRMT5 leads to erasure of repressive histone arginine and lysine marks and promote the restoration of normal growth and survival checkpoints in this disease. Scherle: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.