Inhibition of PRMT5 Disrupts Cell Cycle Progression and DNA Damage Signaling, Revealing a Potential Novel Combination Therapy for Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and remains a therapeutic challenge. There is an urgent need for novel therapeutic strategies for PDAC, and the targeting of pathways highly relevant to its pathobiology is critical. PDAC cells often possess defects in DNA damage repair (DDR) pathways that are not present in healthy cells, which can be exploited for therapeutic strategies. Protein arginine methyltransferase 5 (PRMT5) is a type II histone arginine methyltransferase involved in various cellular processes, such as cell cycle progression and DDR. In PDAC tumors, PRMT5 expression is elevated and predicts shorter overall survival and disease‐free patient survival than those with lower PRMT5 expression. Here, we examined the effect of PRMT5 inhibition on PDAC in vitro, the impact of its inhibition on cell cycle and DDR, as well as potential synergy between PRMT5 inhibition and DNA damage‐inducing ionizing radiation (IR) as a novel therapeutic strategy for PDAC. Treatment of PDAC cells withthe PRMT5 inhibitor (PRMT5i), EPZ015938, resulted in a dose‐dependent decrease of cell growth by time‐lapse live‐cell imaging and clonogenic survival assays. Flow cytometry‐based cell cycle analysis showed that PDAC cells treated with EPZ015938 had increased numbers in G2/M, suggesting that PRMT5 inhibition triggers a G2/M arrest. Using a phospho‐protein array of cell cycle control proteins, we found higher levels of WEE1, P‐S216‐CDC25C, and P‐Y15‐CDC2, supporting G2/M arrest following treatment with EPZ015938. The phospho‐protein array also revealed an increase in P‐S15‐TP53, a signal of DNA damage mediated by the DNA damage sensors, ATM/ATR, along with a decrease in ATM. We confirmed the ATM downregulation at the transcript level by quantitative RT‐PCR. Pathway‐specific qPCR array analysis displayed differential expression of DDR genes related to ATM and G2/M DNA damage checkpoint regulation, further implicating a mechanistic role for these pathways in response to PRMT5i. The downregulation of ATM combined with the presence of G2/M arrest and signals of DNA damage led us to examine the related ATR pathway. In protein lysates from PDAC cells treated with PRMT5i followed by western blot, we found increased phosphorylation of ATR at S1981 and H2A.X at S139. This suggests that PRMT5 inhibition activates DDR in PDAC cells via ATR. To harness the potential vulnerability presented by PRMT5i‐mediated G2/M arrest, we combined PRMT5i treatment with ionizing radiation (IR) exposure to find that the combination of EPZ015938 and IR displayed a greater reduction in PDAC cell growth in vitro over individual treatments. In conclusion, we find that PRMT5 is a critical player in DNA damage via modulation of the ATM/ATR pathways, and combined PRMT5i and IR have beneficial anti‐tumor effects in vitro, revealing an exciting avenue for further exploration as a potential new therapeutic strategy in PDAC.