Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression

Abstract

We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

Figure 1

MTAP is negatively prognostic in MPM (A) Kaplan–Meier survival curve for OS of MTAP positive and MTAP negative patients (n = 79). (B) Kaplan–Meier survival curve for OS of MTAP positive and MTAP negative patients from validation cohort (n = 100). (C) MTAP negative cells (NCI-H2052, NCI-H2591) and MTAP positive cells (MPP89) were transfected with siNT or siPRMT5 20 nM (Q: Qiagen sequence, D: Dharmacon sequence). Cell proliferation was measured by clonogenic assay 5–7 days after transfection. Data were normalized to siNT controls (NCI-H2591: Q p = 0.0001 D p = 0.0001; NCI-H2052: Q p = 0.0001 D p = 0.0001; NCI-H2452: Q p = 0.0001 D p = 0.0001; MPP89: Q p = n.s. D p = n.s.). The levels of PRMT5 expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot 72 h after transfection. These gels have been cropped and full length gels are presented in Supplementary Fig. 3A–C. (D) Cells were transfected with siNT or siWDR77 20 nM. Cell proliferation was measured by clonogenic assay 5–7 days after transfection. Data were normalized to siNT controls (NCI-H2591: p = 0.0001; NCI-H2052: p = 0.0001; NCI-H2452: p = n.s; MPP89: p = n.s.). The levels of PRMT5 expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot 72 h after transfection. These gels have been cropped and full length gels are presented in Supplementary Fig. 3D–F.

Figure 2

PRMT5 silencing mediates growth arrest in MTAP negative mesothelioma. (A) NCI-H2591 cells were transfected with siNT or siPRMT5 (Q: qiagen sequence, D: Dharmacon sequence) 20 nM. Cell proliferation was measured for 168 h with the excelligence real-time cell analyser. Data were normalized to siNT controls (Q p = 0.0015 D p = 0.0019). (B) Venn diagram showing upregulated and downregulated genes comparing the PRMT5 siRNAs versus siNT. (C) Heatmaps and representative GSEA plots showing a significantly enriched up regulated signatures (siPRMT5 vs. siNT).

Figure 3

Identification of Quinacrine Hydrochloride as a PRMT5 perturbagen. (A) Connectivity mapping analysis showing quinacrine as transcriptional suppressor of PRMT5. (B) PRMT5 mRNA expression was evaluated by qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM for 72 h. Data were normalized to untreated control (NCI-H2052 p = 0.0308; NCI-H2591: p = 0.0063; MPP89 p = 0.005). (C) Cells were left untreated or treated with Quinacrine 0.5 µM and 1 µM for 72 h. Cell proliferation was measured by clonogenic assay 5–7 days after treatment. Data were normalized to untreated controls (NCI-H2591: 0.5 µM p = n.s. 1 µM p = 0.0001; NCI-H2052: .5 µM p = n.s. 1 µM p = 0.0017; MPP89: 0.5 µM p = n.s. 1 µM p = n.s.). The levels of PRMT5 expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot 72 h after transfection. These gels have been cropped and full length gels are presented in Supplementary Fig. 3G–I. (D) The PRMT5 promoter activity was measured by a luciferase reporter assay in NCI-H2591 cells transfected with pGL2 basic (EV) or pGL2-PRMT5 and then treated with Quinacrine 1 µM for 72 h. Data were normalized to pGL2 basic (pGL2 basic vs. pGL2-PRMT5 NT p = 0.0162; pGL2 basic vs. pGL2-PRMT5 1 µM p = n.s.; pGL2-PRMT5 NT vs. pGL2-PRMT5 1 µM p = 0.0335). (E) Cells were left untreated or treated with Quinacrine or EPZ015666 10 nM, 100 nM, 1 µM and 10 µM. PRMT5 enzymatic activity was measured after 2 h. (F) NCI-H2591 cells were transfected with GFP empty vector, PRMT5 WT and PRMT5 E444Q and left untreated or treated with Quinacrine 0.5 µM and 1 µM for 72 h. Cell proliferation was measured by clonogenic assay 5–7 days after treatment. Data were normalized to untreated controls (Empty vector: NT vs. 0.5 µM p = 0.0004, NT vs1 µM p < 0.0001; PRMT5 E444Q: NT vs. 1 µM p = 0.0026; Empty vector 1 vs. PRMT5 WT p < 0.0001).

Figure 4

Quinacrine transcriptionally regulates PRMT5 via c-JUN (A) PRMT5 mRNA expression was evaluated by qRT-PCR on RNA extracted from cells transfected with siNT, siCEBP1, sic-JUN, siNF-YA 20 nM, for 72 h. Data were normalized to siNT (siCEBP1 p = n.s.; sic-JUN p = 0.0001; siNF-YA p = n.s). CEBP1, c-JUN and NF-YA mRNA expression was evaluated by qRT-PCR on RNA extracted from cells transfected with siNT, siCEBP1, sic-JUN, siNF-YA 20 nM, for 72 h. Data were normalized to siNT (NCI-H2591: siCEBP1 p = 0.0001; sic-JUN p = 0.0006; siNF-YA p = 0.0033. NCI-H2052: siCEBP1 p = 0.0001; sic-JUN p = 0.0009; siNF-YA p = 0.0001). (B) Cell proliferation was measured by clonogenic assay 5–7 days after treatment. Data were normalized to siNT (NCI-H2591: siCEBP1 p = n.s.; sic-JUN p = 0.0073; siNF-YA p = n.s. NCI-H2052: siCEBP1 p = n.s.; sic-JUN p = 0.0001; siNF-YA p = 0.0001). The levels of PRMT5 and c-JUN expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot. These gels have been cropped and full length gels are presented in Supplementary Fig. 3J–K. (C) C-JUN mRNA expression was evaluated by qRT-PCR on RNA extracted from cells treated for 72 h with Quinacrine 1 µM. Data were normalized to untreated controls (NCI-H2591 p = 0.0001. NCI-H2052 p = 0.0276). (D) PRMT5 and cJUN mRNA expression was evaluated by qRT-PCR on RNA extracted from MPP89 cells transfected with siNT, sic-JUN 20 nM, for 72 h. Data were normalized to siNT (PRMT5 p = n.s. c-JUN p = 0.0205). (E) Cell proliferation was measured by clonogenic assay 5–7 days after treatment. Data were normalized to siNT (sic-JUN p = n.s). The levels of PRMT5 and c-JUN expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot. This gel has been cropped and the full length gel is presented in Supplementary Fig. 3L. (F) C-JUN mRNA expression was evaluated by qRT-PCR on RNA extracted from cells treated for 72 h with quinacrine 1 µM. Data were normalized to untreated control p = n.s.