Identification of PRMT5 as a therapeutic target in cholangiocarcinoma

Abstract

Background: Cholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified.

Objective: We characterised the enzyme protein arginine-methyltransferase 5 (PRMT5) as a novel therapeutic target in CCA.

Design: We evaluated the expression of PRMT5, its functional partner MEP50 and methylthioadenosine phosphorylase (MTAP)-an enzyme that modulates the sensitivity of PRMT5 to pharmacological inhibitors-in human CCA tissues. PRMT5-targeting drugs, currently tested in clinical trials for other malignancies, were assessed in human CCA cell lines and organoids, as well as in two immunocompetent CCA mouse models. Transcriptomic, proteomic and functional analyses were performed to explore the underlying antitumoural mechanisms.

Results: PRMT5 and MEP50 proteins were correlatively overexpressed in most CCA tissues. MTAP was absent in 25% of intrahepatic CCA. PRMT5-targeting drugs markedly inhibited CCA cell proliferation, synergising with cisplatin and gemcitabine and hindered the growth of cholangiocarcinoma organoids. PRMT5 inhibition blunted the expression of oncogenic genes involved in chromatin remodelling and DNA repair, consistently inducing the formation of RNA loops and promoting DNA damage. Treatment with PRMT5-targeting drugs significantly restrained the growth of experimental CCA without adverse effects and concomitantly induced the recruitment of CD4 and CD8 T cells to shrinking tumourous lesions.

Conclusion: PRMT5 and MEP50 are frequently upregulated in human CCA, and PRMT5-targeting drugs have significant antitumoural efficacy in clinically relevant CCA models. Our findings support the evaluation of PRMT5 inhibitors in clinical trials, including their combination with cytotoxic and immune therapies.

Keywords: CHOLANGIOCARCINOMA; MOLECULAR MECHANISMS; PHARMACOTHERAPY.

Figure 1. PRMT5 and MEP50 gene expression in human cholangiocarcinoma (CCA). (A) PRMT5 and MEP50 mRNA levels in intrahepatic CCA (iCCA) compared with normal bile ducts (NBDs) (transcriptomic dataset GSE32225), analysis of their mutual gene expression correlation and correlation with the indicated genes. (B) PRMT5 and MEP50 mRNA levels in eCCA compared with NBDs (transcriptomic dataset GSE132305), analysis of their mutual gene expression correlation and correlation with the indicated genes. (C) PRMT5 and MEP50 gene expression in an integrated dataset of iCCA samples (n=276) according to the levels of expression of the S100P gene, a marker of highly aggressive tumours. * p<0.05, ** p<0.01.

Figure 2. Immunohistochemical analysis of PRMT5, MEP50 and MTAP proteins in human cholangiocarcinoma (CCA) tissues. (A) Representative images of PRMT5 and MEP50 immunohistochemical analyses in human iCCA and eCCA tissue samples, including iCCA samples obtained by needle biopsies. Graphs show the immunohistochemical scores for each protein in intrahepatic CCA (iCCA) and extrahepatic CCA (eCCA) tissue samples. (B) Analysis of the correlation between PRMT5 and MEP50 protein levels in iCCA tissue samples. (C) Graphs showing the distribution of PRMT5 and MEP50 scores according to tumour grade (G1–G3). (D) Representative images of MTAP immunohistochemical analyses in human iCCA and eCCA tissue samples. Graphs show the immunohistochemical scores for MTAP in iCCA and eCCA tissue samples.

Figure 3. Impact of PRMT5 targeting on the growth of cholangiocarcinoma (CCA) cells. (A) CCA cell lines viability (fitness score) on CRISPR/Cas9 drop-out screen for PRMT5 and MEP50. Negative values indicate reduced survival on gene knockout. Data were retrieved from https://score.depmap.sanger.uk/. (B) GI₅₀ values for the PRMT5 inhibitors GSK3326595 and JNJ64619178 in the indicated CCA cell lines. (C) Immunoblot analysis of PRMT5-dependent symmetric dimethylarginine (SMDA) protein marks in control and GSK3326595 or JNJ64619178 treated HuCCT-1 and TFK-1 cells at the indicated doses for 5 days. (D) Upper panel: GI₅₀ values for MRTX1719 in HuCCT-1 and TFK-1 cells and analysis of MTAP protein levels by immunoblot. Lower panel: GI₅₀ values for MRTX1719 in TFK-1 control cells (TFK-1 WT) and TFK-1 cells expressing MTAP (TFK-1 MTAP), and immunoblot analyses for MTAP and SMDA protein marks in these cells. (E) Colony formation assays in the indicated cell lines treated with GSK3326595 or JNJ64619178. (F) Combination studies of the growth inhibitory effects of GSK3326595 or JNJ64619178 with cisplatin or gemcitabine in HuCCT-1 and TFK-1 cells. Dark grey bars denote the existence of synergism at the indicated doses (combination index, CI, <1). CI was calculated as described in online supplemental materials and methods. *p<0.05, **p<0.01 and ***p<0.001 vs controls.

Figure 4. Pharmacological targeting of PRMT5 inhibits the growth of human CCA tumouroids. Effect of GSK3326595 on the growth of human CCA tumouroids. Representative images of control and treated tumouroids at the end of treatments are shown along the quantification of tumouroids growth in the indicated conditions. **p<0.01 and ***p<0.001 vs controls (DMSO, vehicle-treated cultures). CCA, cholangiocarcinoma; DMSO, dimethyl sulfoxide.

Figure 5. PRMT5 inhibition markedly alters the expression of genes involved in chromatin regulation, DNA damage repair, lipid metabolism and immune response and induces aberrant mRNA splicing in CCA cells. (A) Most relevant categories of differentially expressed genes identified by Gene Ontology Biological Process (GO-BP) functional classification in HuCCT-1 cells treated with JNJ64619178 (3 nM, 3 days treatment). (B) Number of alternative splicing events in protein-coding genes differentially affected by PRMT5 inhibition in HuCCT-1 cells. (C) GO-BP functional classification of the aberrant splicing events in HuCCT-1 cells treated with JNJ64619178. (D) Volcano plot representing all the significant (p<0.05) splicing events. Selected genes from the indicated GO-BP functional categories are highlighted. The chromatin modifiers and DNA replication and repair genes KAT5, KMT5C and POLD1 are identified.

Figure 6. The effects of PRMT5 inhibition on the proteome of cholangiocarcinoma (CCA) cells are consistent with the observed transcriptional alterations. (A) Most relevant categories of differentially expressed proteins identified by Gene Ontology Biological Process (GO-BP) functional classification in HuCCT-1 cells treated with JNJ64619178 (6 nM, 4 days treatment). (B) Volcano plot representing all the significant (p<0.05) differentially expressed proteins. Selected proteins included in the indicated GO-BP functional categories are identified. (C) Validation of the effects of PRMT5 inhibition on the expression of the indicated genes and proteins identified in the transcriptomic and proteomic analyses. HuCCT-1 and TFK-1 cells were treated with the indicated concentrations of JNJ64619178 for 3 or 4 days for qPCR and Western blot analyses, respectively. a, p<0.001; b, p<0.01; c, p<0.05 vs controls.

Figure 7. PRMT5 inhibition induces DNA damage in cholangiocarcinoma (CCA) cells. (A) Representative images of comet assays showing levels of overall DNA strands breaks in HuCCT-1 and TFK-1 cells treated with JNJ64619178 (3 nM) for 3 days. Graph shows the quantification of the comet tail length at the level of individual cells in the number of cells indicated. (B) HuCCT-1 and TFK-1 cells were probed for R-loops using the S9.6 antibody after 3 days of treatment with JNJ64619178. Graph shows the quantification of RNA–DNA hybrids (R-loops) per nucleus. (C) Effect of JNJ64619178 on H2AX protein levels in HuCCT-1 and TFK-1 cells. Cells were treated for 3 days with the indicated JNJ64619178 concentrations. ***p<0.001 vs controls (DMSO, vehicle-treated cultures). DMSO, dimethyl sulfoxide.

Figure 8. Antitumoural effects of PRMT5 inhibition in a mouse model of cholangiocarcinoma (CCA) development in the context of liver injury. (A) Diagram showing the Jnk^Δhepa+DEN+CCl₄ (Jnk^Δhepa+DC) experimental CCA model and the treatments applied (n=6 mice per group). (B) Immunohistochemical detection of PRMT5, MEP50 and CK19 proteins in liver tissue sections harbouring CCA lesions developed in this model. Representative images are shown. (C) Graphs show tumour burden, estimated as the liver-to-body weight ratio (liver index), and the quantification of nodules in the surface of the livers from the different groups of mice. Representative images of the livers of control Jnk^Δhepa+DEN+CCl₄, and GSK3326595-treated mice are shown. (D) Immunohistochemical analysis of CK19 and H&E stainings showing the extent of CCA lesions in control Jnk^Δhepa+DEN+CCl₄ and GSK3326595-treated mice. Representative images are shown. (E) Representative images showing the immunohistochemical detection of CD4⁺ and CD8⁺ T cells (yellow arrows), and quantification of tumour-infiltrating CD8⁺ and CD4⁺ T cells in control Jnk^Δhepa+DEN+CCl₄ and GSK3326595-treated mice. (F) Body weights and serum liver parameters (AST, ALT and ALP) in the different groups of mice at the end of treatments. *p<0.05, **p<0.01 and ***p<0.001.

Figure 9. Antitumoural effects of PRMT5 inhibition in an aggressive cholangiocarcinoma (CCA) model triggered by hydrodynamic tail vein injection (HTVI) of activated forms of TAZ and AKT. (A) Diagram showing the TAZ-AKT model and the treatments applied (n=6 mice per group). Lower panels show representative images of the immunohistochemical analysis of PRMT5, and MEP50 in the lesions developing in TAZ-AKT mice at 2 and 3 weeks after plasmids injection, along with the demonstration of TAZ and p-AKT (Ser473) expression. Representative images are shown. (B) Serum transaminases (AST and ALT) levels in the different groups of mice. (C) Graphs show tumour burden, estimated as the liver-to-body weight ratio (liver index), and representative macroscopic images of the livers from control, TAZ-AKT mice and TAZ-AKT mice treated with JNJ64619178. (D) Representative H&E-stained liver tissue sections showing the extent of CCA lesions in TAZ-AKT and JNJ64619178-treated TAZ-AKT mice. Graph shows the quantification of tumourous areas. (E) Representative images showing the immunohistochemical detection of CD4⁺ and CD8⁺ T cells and quantification of tumour-infiltrating CD8⁺ and CD4⁺ T cells (yellow arrows) in control TAZ-AKT and JNJ64619178-treated TAZ-AKT mice. (F) Most relevant categories of differentially expressed genes identified by Gene Ontology Biological Process (GO-BP) functional classification of transcriptomic data from TAZ-AKT and JNJ64619178-treated TAZ-AKT mice livers. (G) qPCR analysis of the expression of DNA damage response (DDR)-related genes in control, TAZ-AKT and JNJ64619178-treated TAZ-AKT mice livers. *p<0.05, **p<0.01 and ***p<0.001. ALT, alanine transaminase; AST, aspartate aminotransferase.