Developing a Novel Class of Drug to Inhibit Protein Arginine Methyltransferase 5 (PRMT5) Enzyme Dysregulation in Mantle Cell Lymphoma

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<0.0001). PRMT methylation assays were also performed with SWI/SNF complexes containing PRMT5, PRMT7 (type II) or PRMT4 (type I) and both BLL1 and BLL3 demonstrated selective PRMT5 inhibition. Both drugs interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in MCL cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 (10uM - 100uM) showed a dose-dependent response of inhibition of cellular proliferation, induction of cell cycle arrest, and promotion of caspase-independent cell death in 7 MCL cell lines. BLL1 treatment of MCL cells resulted in down modulation of cyclin D1 and Mcl1, critical molecules involved in the pathogenesis of MCL. The loss of cyclin D1 and Mcl1 expression occurred as early as 1 hour after treatment with BLL1 (50uM). PRMT5 associates with the co-repressors HDAC2, MBD2 and DNMT3a on target gene promoters, thus we next evaluated the effect of BLL1 on transcriptional repression of known target anti cancer genes. The association with other co-repressors provided rationale for examining PRMT5 inhibition alone and in combination with agents that target epigenetic processes. Combination treatment of MCL cells with subtoxic doses of BLL1 (25uM), hypomethylating agent (5-azacitidine, 500nM) and HDAC inhibitor (TSA 75nM) showed synergistic induction of cell death and loss of S2Me-H4R3. Analysis of the ST7 tumor suppressor, a target repressed by PRMT5, showed mRNA levels to increase 5–7-fold following treatment with BLL1. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for both BLL1 and BLL3. In vivo evaluation of BLL1 in a preclinical, xenograft model of human MCL are currently in progress. Primary tumors of 46 patients with MCL (common, blastoid or pleomorphic histology) demonstrated abundant PRMT5 expression in both cytoplasmic and nuclear compartments (87% PRMT5 pos). Conclusions: We have successfully developed a new class of drug to selectively target PRMT5 enzymatic activity. PRMT5 over expression is linked with post translational modification of both histone and non histone proteins that contribute to key oncogenic pathways in MCL. Inhibition of type II PRMT enzymes reverses transcriptional repression of anti cancer genes and restores important regulatory cellular checkpoints of cell growth and survival. We are currently developing drugs with improved selectivity and potency. The anti tumor activity of this novel class of drug and PRMT5 expression profiles seen in MCL primary tumor specimens, supports further exploration of targeting this pathway in hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Emerging epigenetic-modulating therapies in lymphoma

“…Notably, PRMT5 is also required for formation of GCBs, which are the cell of origin in most DLBCLs 122 . PRMT5 overexpression has also been identified in MCL, and PRMT5 inhibition was shown to have cytotoxic effects on MCL cell lines that were associated with downregulation of the genes encoding cyclin D1 ( CCND1 ) and the differentiation protein MCL1 (REFS 123–124 ). Oral administration of the PRMT5 inhibitor EPZ015666 has been shown to induce dose-dependent reductions in tumour volume in mouse xenograft models of MCL 125 .…”