Abstract 1137: PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells

Abstract

Abstract PRMT5 (protein arginine methyltransferase 5) is a predominant Type II PRMT that catalyzes symmetric dimethylation of protein arginine residues (sDMA). PRMT5 is overexpressed in many types of cancer and plays roles in multiple essential biological processes to promote cancer growth. Previous studies have shown that PRMT5 is a critical molecule for RNA processing and pre-mRNA splicing. Mechanistically, PRMT5 directly methylates arginine residues of several splicing factors such as Small nuclear ribonucleoprotein (SNRPB and SNRPD3) and Serine and arginine rich splicing factor 1 (SRSF1), which contributes to spliceosome assembly and promotes canonical splicing of many essential genes in cancer cells. In the present study, we examined the effects of PRT543, a potent and selective PRMT5 inhibitor, on alternative splicing in uveal melanoma which frequently express hotspot mutations on Splicing factor 3b subunit 1 (SF3B1). We first confirmed that PRT543-treated MEL202 (SF3B1R625G active mutant) and MEL270 (SF3B1WT) cells show significantly increased global alternative splicing, such as increased retained intron (RI) and skipping exon (SE), determined by delta-PSI (percentage of splice-in) analysis. PRT543 downregulates SF3B1 target genes such as FBXW5, MAP3K7, MBD4 and BRD9 that are associated with increased retention of specific intron sites. Interestingly, downregulation of the SF3B1 target genes are more significant in MEL202 (SF3B1R625G) than MEL270 (SF3B1WT), indicating that PRT543 can regulate the activity of the SF3B1 gain of function mutant. Consistent with previously reported PRMT5 knockout studies in hematological cancer cells, PRT543 also downregulates expression of SRSF1 target genes such as POLD1 and PNKP through increased intron retention in primary and metastatic uveal melanoma cell lines. Furthermore, we uncover that PRT543 strikingly increases retention of a specific intron site of ATM (ex33-34), resulting in a significant reduction of ATM protein levels in PRT543-treated MEL202 and MEL270 cells. Gene set enrichment analysis (GSEA) further reveals that PRT543 significantly and specifically regulates DNA replication and repair pathways in MEL202 cells. Importantly, combining PRT543 with DNA-alkylating agents or PARP inhibitors yields a synergistic reduction in cell viability. In summary, our results suggest that PRMT5 inhibition regulates cancer-associated RNA splicing machinery and the DNA damage response, resulting in synergistic antitumor activity when combined with chemotherapy and/or PARP inhibitors, particularly in cancers with spliceosomal mutations. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Venkat Thodima, Jack Carter, Neha Bhagwat, Monisha Sivakumar, Alexander Grego, Joseph Rager, Mizue Terai, Takami Sato, Omar Abdel-Wahab, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1137.