Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.

Keywords: Leukemia; PRMT7; RUNX1; T-ALL; arginine methylation.

Figure 1

PRMT7 expression in hematological malignancies and healthy T-cells. (a) Expression of PRMT7 in leukemias (n = 4430), lymphomas (n = 1306), and healthy cells (n = 428) in Hemap dataset (p-values presented in Table S1); (b) summary of expression of all PRMT family genes in T-ALL; (c) expression of PRMT7 in subgroups of T-ALL (HOXA vs. TAL1/LMO1 p = 0.003, HOXA vs. LYL1/LMO2 p = 0.026, HOXA vs. TLX3 p = 0.010, HOXA vs. TLX1 p = 0.006, HOXA vs. NKX2-1 p = 0.845, LYL1/LMO2 vs. HOXA p = 0.025, LYL1/LMO2 vs. TAL1/LMO1 p = 1.098 × 10⁻⁹, LYL1/LMO2 vs. TLX3 p = 2.368 × 10⁻⁷, LYL1/LMO2 vs. TLX1 p = 9.859 × 10⁻⁸, and LYL1/LMO2 vs. NKX2-1 p = 0.198, TAL1/LMO2 n = 171, HOXA n = 56, LYL1/LMO2 n = 54, TLX3 n = 35 and NKX2-1 n = 11); (d) expression of PRMT7 mRNA in T- and B-ALL cell lines as measured by qRT-PCR (n = 3).; and (e) expression of PRMT7 and the selected essential T-cell developmental genes during the T-cell differentiation and maturation process at single-cell resolution. Color indicates the mean expression and size the proportion of cells expressing the gene relative to the absolute number of cells detected in the dataset.

Figure 2

Association of PRMT7 expression with patient outcome. EFS of patients in (a) the TARGET and (b) the Japanese patient cohorts. Patients were classified into two groups by using the median expression value of PRMT7 as a cut-off.

Figure 3

Functional consequences of genetic deletion of PRMT7 in T-ALL cells. (a) Immunohistochemical staining (scale bar 50 µm) showing PRMT7 expression in J-EV, J-KO3, M-WT and M-KO11 cell lines. Brown color indicates positivity to the PRMT7 protein and blue marks PRMT7 negative cells. (b) Western blotting in unmodified (J-EV and M-WT) and knockout cell lines (marked with KO) by using the PRMT7 antibody. Quantitation in Western blot is relative to J-EV or M-EV control cell lines. J stands for Jurkat and M for Molt4 cell line, EV for empty vector and WT for wild type. The uncropped Western blots have been shown in Figure S6. Effect of the PRMT7 knockout on (c) colony formation (n = 4) and (d) cell viability (n = 3). Cell viability results are presented as line plots for all the examined time points and as scatter plots for the 72 h time point. Asterisks denote samples with significant p-values (<0.05) when compared to the unmodified (J-EV and M-WT) cells.

Figure 4

Effects of PRMT7 knockout on arginine monomethylation in T-ALL cells. (a) A heatmap of top differentially regulated peptides with altered arginine monomethylation level in PRMT7 knockout cells (J-KO3 and J-KO5) compared to unmodified cells (J-EV) (Benjamini-Hochberg adjusted p-value ≤ 0.01). Color key indicates the average arginine monomethylation in log2 abundance. Duplicate image with row names is presented in Figure S4c. Impact of PRMT7 knockout to arginine monomethylation by (b) CORUM protein complexes (Benjamini-Hochberg adjusted log2 p-value < 0.05) and (c) KEGG pathways (Benjamini-Hochberg adjusted log2 p-value < 0.05), with both cell lines (J-KO3 and J-KO5) combined for the analysis. (d) Tabulation of arginine monomethylation changes in peptides belonging to proteins associated with T-ALL pathogenesis in PRMT7 knockout cell lines (J-KO3 and J-KO5). Red color indicates increased and blue decreased level of arginine monomethylation compared to unmodified cells.

Figure 5

Effects of PRMT7 deletion on gene expression in T-ALL cells. Volcano plots displaying the differentially expressed genes (log2FC ≥ ±1.5, FDR < 0.05) in (a) J-KO3 and (b) J-KO5 cell lines. (c) A volcano plot displaying gene expression changes (log2FC ≥ ±1.5, FDR < 0.05) in genes known to be associated with T-ALL pathogenesis and altered in all six PRMT7 knockout cell lines labeled with different symbols. Red color indicates genes with increased and blue with decreased expression compared to unmodified cells. Three biological replicates were combined for the analysis for each cell line. J stands for Jurkat and M for Molt4 cell line, EV for empty vector and WT for wild type.