ABSTRACT
The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.
Subject(s)
Enzyme Inhibitors/therapeutic use , Lung Neoplasms/drug therapy , Protein-Arginine N-Methyltransferases/drug effects , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Animals , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Humans , Lung Neoplasms/pathology , Mice , Pyrimidines/pharmacology , Pyrroles/pharmacologyABSTRACT
Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.
Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Animals , Apoptosis , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Cycle , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Discovery , Epigenomics , Female , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Mice , Neoplastic Stem Cells/metabolism , Protein-Arginine N-Methyltransferases/drug effects , Protein-Arginine N-Methyltransferases/genetics , RNA Splicing , Xenograft Model Antitumor AssaysABSTRACT
In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Homologous Recombination/drug effects , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Antineoplastic Agents/therapeutic use , DNA Breaks, Double-Stranded/drug effects , DNA End-Joining Repair/drug effects , Enzyme Inhibitors/therapeutic use , Female , Humans , Ovarian Neoplasms/genetics , Protein-Arginine N-Methyltransferases/drug effects , Recombinational DNA Repair/drug effectsABSTRACT
Cadmium (Cd) is considered as a carcinogenic chemical with potential to endanger normal cellular functioning. The present study was aimed to investigate the impact of Cd on the expression of two oncogenic epigenetic regulators, viz., protein arginine methyltransferase 5 (PRMT5) and the polycomb repressive complex 2 (PRC2) member enhancer of Zeste homolog 2 (EZH2). Our results indicate that Cd at 1⯵M concentration increases the viability of HepG2 and MCF7 cells and significantly upregulates the expression of PRMT5 and EZH2, leading to an increased global level of symmetric dimethylarginine (SDMA), H4R3me2s, and H3K27me3. The luciferase reporter assay showed that the promoter activity of PRMT5 and EZH2 is significantly enhanced in both cell lines. Furthermore, Cd exposure induces global DNA hypomethylation due to a decrease in DNA methyltransferases (DNMTs) expression. Methylation-specific and bisulfite sequencing PCR reveal that the proximal promoters of PRMT5 and EZH2, which harbour CpG islands, are almost demethylated when exposed to Cd. The Cd exposure also increases the protein level of transcription factors NFYA and E2F1; consistently, the two transcription factors are found to be enriched at the PRMT5 and EZH2 promoter in chromatin immunoprecipitation experiments. The alterations induced by Cd in the two cancer cell lines were also observed in a non-cancerous cell line (HEK-293). In conclusion, we propose that Cd increases the expression of two oncogenic methyltransferases, possibly with a DNA methylation-dependent mechanism. Further studies focused on the epigenetic alterations induced by Cd would provide mechanistic insights on the carcinogenicity of this metal toxicant at the molecular level.
Subject(s)
Cadmium/pharmacology , DNA Methylation/drug effects , Enhancer of Zeste Homolog 2 Protein/metabolism , Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Transcription Factors , CCAAT-Binding Factor/drug effects , CCAAT-Binding Factor/metabolism , Cadmium/metabolism , Cell Survival/drug effects , CpG Islands , E2F1 Transcription Factor/drug effects , E2F1 Transcription Factor/metabolism , Enhancer of Zeste Homolog 2 Protein/drug effects , Enhancer of Zeste Homolog 2 Protein/genetics , HEK293 Cells , Humans , MCF-7 Cells , Methyltransferases/drug effects , Methyltransferases/genetics , Promoter Regions, Genetic/drug effects , Protein-Arginine N-Methyltransferases/drug effects , Protein-Arginine N-Methyltransferases/genetics , Transcription Factors/metabolismABSTRACT
Aging-related ED is predominantly attributed to neurovascular dysfunction mediated by NO suppression and increased oxidative stress in penis. The alterations of protein arginine methyltransferases 1 (PRMT1)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetrical dimethylarginine (ADMA)/NO synthase (NOS) pathway regulate NO production in the vascular endothelium. Epigallocatechin-3-gallate (EGCG) is one of the most abundant and antioxidative ingredients isolated from green tea. In the present study, 40 Sprague-Dawley rats were randomly distributed into four groups: one young rat group and three aged rat groups treated with daily gavage feedings of EGCG at doses of 0, 10 mg kg-1 and 100 mg kg-1 for 12 weeks, respectively. Erectile function was assessed by electrical stimulation of the cavernous nerves with intracavernous pressure (ICP) measurement. After euthanasia, penile tissue was investigated using Western blot and ELISA to assess the PRMT1/DDAH/ADMA/NOS metabolism pathway. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected by colorimetry. We also evaluated smooth muscle contents. The ratio of maximal ICP and mean systemic arterial pressure (MAP) was markedly higher in EGCG-treated aged rats than in untreated aged rats. We found that DDAH1 and DDAH2 were expressed in cavernosal tissue, and they were downregulated in corpora of aged rats. The administration of EGCG upregulated the expression and activity of DDAH. In contrast, EGCG treatment downregulated the expression of PRMT1 and ADMA content. Moreover, EGCG-treated rats showed an improvement in smooth muscle expression, the ratio of smooth muscle cell/collagen fibril, SOD activity, and MDA levels when compared with untreated aged rats.
Subject(s)
Amidohydrolases/drug effects , Antioxidants/therapeutic use , Arginine/analogs & derivatives , Catechin/analogs & derivatives , Erectile Dysfunction/drug therapy , Nitric Oxide Synthase/drug effects , Protein-Arginine N-Methyltransferases/drug effects , Aging , Animals , Arginine/drug effects , Arterial Pressure/drug effects , Catechin/therapeutic use , Cyclic GMP/metabolism , Male , Muscle, Smooth/drug effects , Muscle, Smooth/growth & development , Penile Erection/drug effects , Penis/drug effects , Penis/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Superoxide Dismutase-1/drug effects , Superoxide Dismutase-1/metabolismABSTRACT
Nerve growth factor (NGF)-specific signal transduction leads to changes in protein methylation during neuronal differentiation of PC12 cells (Cimato et al. [1997] J. Cell Biol. 138:1089-1103). In the present work, we demonstrate that, among NGF-regulated proteins, arginine methylation is more prevalent than carboxylmethylation. Type I protein arginine methyltransferase (PRMT) activity produces asymmetric dimethylation of the terminal guanidinonitrogen of arginines in substrate proteins, particularly glycine and arginine-rich (GAR) segments of proteins. Several GAR peptides were used to assay for methyltransferase activity and to compete with endogenous cellular proteins for the PRMT activity in PC12 cell extracts. Peptides derived from fibrillarin and nucleolin, as well as a synthetic GAR peptide containing a repetitive GRG motif, are each extremely effective at blocking in vitro methylation of the NGF-regulated PC12 cell methylated proteins. Myelin basic protein, a substrate for type II PRMT, selectively inhibits a 45 kDa protein but is a much less effective inhibitor of total methylation at an equimolar concentration. In addition, the fibrillarin- and nucleolin-derived peptides were used to detect elevated PRMT activity in homogenates of NGF-treated PC12 cells. Finally, immunoprecipitation of PRMT1 from PC12 cells provides the first demonstration of an NGF-activated methyltransferase and implicates PRMT1 in NGF signal transduction.