NFS-06. Targeting PRMT5 in MTAP-deleted NF1 high grade gliomas.

Abstract Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome that is characterized by hyperactivation of the RAS/MAPK signaling pathway. Our analysis of NF1-associated high grade gliomas revealed that 57% of these tumors harbor homozygous deletions in chromosome 9p21.3, encompassing the tumor suppressor genes CDKN2A, CDKN2B, and MTAP. Methylthioadenosine phosphorylase (MTAP) is critical for the methionine salvage pathway and its loss leads to increased vulnerability to PRMT5 inhibition. PRMT5 methylation activity is also associated with multiple kinases in the RAS/MAPK signaling pathway, warranting further investigation on a combined PRMT5 and RAS/MAPK inhibition therapy. Cell lines (n = 4) with loss-of-function mutations in the NF1, CDKN2A/B, and MTAP genes and CRIPSR-Cas9 MTAP-knockout (MTAP-KO) cells were first validated for MTA accumulation using liquid chromatography-mass spectrometry (LC-MS). Cell viability assays were then performed using a brain-penetrant, MTA-cooperative PRMT5 inhibitor, TNG908. Specific inhibition of PRMT5 was confirmed by immunoblotting for symmetric dimethylarginine (SDMA) expression and genes involved in the RAS/MAPK signaling pathway. Additionally, the impact of TNG908 on cell cycle progression and apoptosis was assessed using flow cytometry. Combination of TNG908 with MEK inhibitors was also assessed. MTAP-deleted and MTAP-KO cells showed increased MTA accumulation compared to wild-type MTAP (MTAP-WT) cells, resulting in significant sensitivity towards TNG908. PRMT5 inhibition was confirmed by reduced SDMA expression in MTAP-KO cells but not in MTAP-WT cells, indicating TNG908’s MTA-dependent inhibition activity. PRMT5 inhibition also significantly increased (p 0.05) the proportion of cells in the G2/M phase of the cell cycle and enhanced apoptotic activity (p 0.05). Combinatorial inhibition using TNG908 and Binimetinib demonstrated significant synergy (ZIP score= 14.36) in MTAP-KO cells and additive activity in TM31 cells (ZIP score= 0.272). These findings highlight PRMT5 inhibition as a promising therapeutic strategy for MTAP-deleted NF1 HGGs. Moreover, the combination of TNG908 and Binimetinib shows strong potential and warrants further investigation in vivo.