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posted on 2023-11-06, 14:20 authored by Jack Carter, Michael Hulse, Monisha Sivakumar, Jessica Burtell, Venkat Thodima, Min Wang, Anjana Agarwal, Komali Vykuntam, Jacob Spruance, Neha Bhagwat, Joseph Rager, Bruce Ruggeri, Peggy Scherle, Koichi Ito C220 dysregulates chromatin- and splicing-mediated regulation of DDR pathways. A, ChIP-qPCR assays using primers against the proximal promoter of indicated DDR genes following capture of chromatin-bound PRMT5 in nontreated MCF7 cells. Data are shown as fold enrichment over IgG (n = 3). B, ChIP-qPCR assays using primers against the proximal promoter of DDR genes following capture of chromatin-bound H4R3me2 from MCF7 cells treated with 250 nmol/L C220 for 4 days. Data are shown as % input (n = 3) C, AS analysis of RNA-seq data from MCF7, HCC1569, and A2780 cells following treatment with 100 nmol/L C220 for 4 days. Total events represent the total number of AS with statistical significance (P < 0.05). RI = retained intron, SE = skipped exon, A3 = alternative 3′ splice-site, A5 = alternative 5′ splice site, MX = mutually exclusive exons, AF = alternative first exon, AL = alternative last exon. D, Violin plot of global intron retention and exon skipping events induced by C220 in A2780 cells annotated with select DDR genes with significant (P < 0.05) events. E and F, qPCR of total ATM or ATM retained intron in A2780 cells treated with 100 nmol/L C220 for 4 days.
Funding
Prelude Therapeutics Incorporated
History
ARTICLE ABSTRACT
Expression of protein arginine methyltransferase 5 (PRMT5) is highly positively correlated to DNA damage repair (DDR) and DNA replication pathway genes in many types of cancer cells, including ovarian and breast cancer. In the current study, we investigated whether pharmacologic inhibition of PRMT5 downregulates DDR/DNA replication pathway genes and sensitizes cancer cells to chemotherapy and PARP inhibition. Potent and selective PRMT5 inhibitors significantly downregulate expression of multiple DDR and DNA replication genes in cancer cells. Mechanistically, PRMT5 inhibition reduces the presence of PRMT5 and H4R3me2s on promoter regions of DDR genes such as BRCA1/2, RAD51, and ATM. PRMT5 inhibition also promotes global alternative splicing changes. Our data suggest that PRMT5 inhibition regulates expression of FANCA, PNKP, and ATM by promoting exon skipping and intron retention. Combining C220 or PRT543 with olaparib or chemotherapeutic agents such as cisplatin demonstrates a potent synergistic interaction in breast and ovarian cancer cells in vitro. Moreover, combination of PRT543 with olaparib effectively inhibits the growth of patient-derived breast and ovarian cancer xenografts. Furthermore, PRT543 treatment significantly inhibits growth of olaparib-resistant tumors in vivo. These studies reveal a novel mechanism of PRMT5 inhibition and suggest beneficial combinatorial effects with other therapies, particularly in patients with tumors that are resistant to therapies dependent on DNA damage as their mechanism of action.
Patients with advanced cancers frequently develop resistance to chemotherapy or PARP inhibitors mainly due to circumvention and/or restoration of the inactivated DDR pathway genes. We demonstrate that inhibition of PRMT5 significantly downregulates a broad range of the DDR and DNA replication pathway genes. PRMT5 inhibitors combined with chemotherapy or PARP inhibitors demonstrate synergistic suppression of cancer cell proliferation and growth in breast and ovarian tumor models, including PARP inhibitor–resistant tumors.
