Protein arginine methyltransferase 2 controls inflammatory signaling in acute myeloid leukemia

Abstract

Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.

Fig. 1. Patients with AML displaying a low PRMT2 expression show a higher inflammatory signature.

a PRMT2 gene expression within the Leucegene cohort (non-APL AML, n = 371) at diagnosis with the PRMT2^low patients in red (n = 37) and PRMT2^high patients in blue (n = 37). b Kaplan-Meier survival curves of the patients depending on their PRMT2 expression: above or below the median of expression of the cohort. c Mutation profile of the PRMT2^low (n = 10) and PRMT2^high (n = 10) patients for a subset of frequently mutated genes in AML. Blue: mutated gene; gray: non mutated gene. d The bar graph shows the most significantly overrepresented hallmarks after a gene set enrichment analysis (GSEA) of PRMT2^low compared to PRMT2^high patients with normalized enrichment scores on the right. All gene sets are FDR < 25% and p < 0.01. e GSEA plot for the “TNFA signaling via NFKB” hallmark with FDR and nominal p value indicated. f Heatmap representing the top 20 enriched genes of the “TNFA signaling via NFKB” hallmark. NF-κB-related genes appear in bold.

Fig. 2. PRMT2 is associated with inflammatory factors’ expression.

Gene expression of IL6 (a), TNF (b), FTH1 (c) and FTL (d) within the PRMT2^low patients in red (n = 37) and PRMT2^high patients in blue (n = 37). ImmuCellAI analysis showing monocyte (e) and NKT cell (f) abundance within the PRMT2^low patients in red (n = 37) and PRMT2^high patients in blue (n = 37). Error bars represent the mean ± SD. Paired t tests with p values indicated in the graphs: *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 3. The loss of PRMT2 increases the inflammatory phenotype of leukemic cells in vitro.

a Western Blot analysis on WT HL-60 or three different clones of PRMT2^KO (KO) cells for PRMT2 and other PRMT (-1, -3, CARM1, and PRMT5) protein expressions. GAPDH is used as loading control. The presented blot is a representative figure from three independent experiments. b Assessment of cell proliferation of the 3 PRMT2^KO clones compared to WT HL-60 cells by cell counting, shown as mean ± SD of three independent experiments (n = 9). c The bar graph represents the most significantly overrepresented hallmarks after a gene set enrichment analysis (GSEA) of #2 and #3 PRMT2^KO clones compared to WT with normalized enrichment scores on the right. All gene sets are FDR < 25% and p < 0.01. d GSEA plot for the “TNFA signaling via NFKB” hallmark with FDR and nominal p indicated. e Heatmap representing the top 20 enriched genes of the “TNFA signaling via NFKB” hallmark. NF-κB-related genes appear in bold.

Fig. 4. The absence of PRMT2 exacerbates the inflammatory phenotype of mouse bone marrow-derived macrophages.

a Schematic representation for the obtention of BMDMs from purified monocytes from Prmt2^–/– or control (Prmt2^+/+) mouse BM. b Morphological analysis of monocytes after 0 and 5 days of differentiation into macrophages. Images from phase-contrast microscopy using an Incucyte® S3 Live-Cell Analysis System at magnification x20. Il6 (c) and Tnf (d) expressions measured by RT-QPCR on total RNA from mouse BMDMs after stimulation by 100 ng/mL LPS for 8 h. ELISA on BMDM culture media for IL6 (e) and TNF (f) after stimulation by 100 ng/mL LPS for 8 h. All error bars represent the mean ± SD of three independent experiments. One sample t test with p values indicated in the graphs: *p < 0.05.

Fig. 5. PRMT2 is involved in the human inflammatory response through the control of STAT3 activation in vitro upon stress.

a RT-QPCR on total RNA measuring IL6 in PRMT2^KO and WT cells after stimulation by 100 ng/mL LPS during 4, 8, or 24 h. b ELISA on human IL6 in PRMT2^KO and WT cell culture medium after stimulation by LPS (100 ng/mL) during 24 h. Western Blot analysis (c) and quantitation (d) of phosphorylated (Y705) and total forms of STAT3 after stimulation by LPS (100 ng/mL) during 4, 8, 24 or 48 h. GAPDH is used as a loading control. Western Blot analysis (e) and quantitation (f) of phosphorylated and total forms of STAT3 after stimulation by LPS (100 ng/mL) during 8 h of HL-60 WT, KO, or KO cells infected with viruses containing DNA coding for either an empty vector (EV), exogenous PRMT2 (PRMT2) or a catalytically dead mutant (E220Q). GAPDH is used as a loading control. All the presented blots are representative figures from at least three independent experiments. All error bars represent the mean ± SD of three independent experiments. Ctrl: Control. One-way ANOVA (a, d, f) or one sample t test (b) with p values indicated in the graphs: *p < 0.05; **p < 0.01.

Fig. 6. The absence of PRMT2 deregulates the NF-κB signaling pathway in AML upon stress.

Western Blot analysis (a) and quantitation (b) of phosphorylated (Y705) and total forms of STAT3 after stimulation by IL6 (40 ng/mL) during 0.5, 2, 8 or 24 h, shown as mean ± SD of three independent experiments and analyzed by one-way ANOVA. c Western Blot analysis of phosphorylated and total forms of the SAPK/JNK, ERK1/2, and p38 MAPKs on PRMT2^KO and WT cells after stimulation by 100 ng/mL LPS during 4, 8, 24 or 48 h. d Western Blot analysis of NF-κB p65 and phosphorylated (Y705) STAT3 nuclear translocation after cellular fractionation of PRMT2^KO and WT HL-60 cells. SP1 and ACTB correspond to nuclear and cytoplasmic controls, respectively. Ctrl: Control. All presented blots are representative figures from three independent experiments.