Abstract 1584: Targeting protein arginine methyltransferase 5 (PRMT5) enzyme over expression in high grade astrocytomas

Abstract

Abstract High grade astrocytomas are aggressive brain tumors that are associated with a dismal prognosis and are incurable with a median survival of less than 15 months despite intensive multimodal therapy. The linability to effectively target grade III and grade IV (glioblastoma multiforme, GBM) astrocytomas highlights the need for novel therapeutic approaches. Recent studies have shown that epigenetic regulation of chromatin plays a central role in the control of cell growth, differentiation, and survival. Chromatin remodeling enzymes like histone deacetylases, DNA methyltransferases and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor gene (TSG) expression and may contribute towards cellular transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. Eight patient-derived GBM cell lines and 45 primary GBM tumors showed abundant expression of PRMT5 protein. Confocal microscopy and immunohistochemical staining showed the PRMT5 signal to localize primarily to the nucleus. Normal brain tissue, normal human astrocytes and low/intermediate grade astrocytomas failed to show PRMT5 expression. The degree of PRMT5 over expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p<0.0001). Elevated PRMT5 expression was observed in high grade astrocytomas that spontaneously develop in a pre-clinical mouse model of GBM employing conditional Nf1, TP53 and PTEN haploinsufficiency. Small inhibitory RNAs (siRNA) specific for PRMT5 led to loss of PRMT5 protein expression and S2Me of H4R3, a histone target of PRMT5. PRMT5 inhibition led GBM cell lines to cell cycle arrest, apoptosis, and complete loss of cell migratory activity. Apoptosis occurred independent of caspase and p53 pathways. Furthermore, PRMT5 knockdown led GBM cell lines to become sensitized to the toxic effects of temazolomide, a drug frequently used to manage patients with GBM. We utilized gene microarray analysis of cDNA isolated from siRNA (or control RNA) treated GBM cells to identify potential targets of PRMT5 and identified the TSG ST7 and three chemokine transcripts (RANTES, IP10, CXCL11) to be upregulated with PRMT5 knockdown. We used chromatin immuno precipitation to show that siRNA treatment led to loss of PRMT5 recruitment on ST7 and chemokine gene promoters that coincided with restoration of transcriptional and translational activity leading to marked elevation in protein expression. PRMT5 knock-down led to secretion of all chemokines into growth medium. These findings identify PRMT5 as an independent prognostic factor for GBM and an attractive therapeutic target for high grade astrocytomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1584.