Additional file 1 of Exposing the DNA methylation-responsive compartment of the leukaemic genome in T-ALL cell lines support its potential as a novel therapeutic target in T-ALL

Additional file 1. Supplementary Fig. 1.a, Overview of our low-coverage whole methylome sequencing approach. DNA was extracted and base pair resolution DNA methylation libraries were prepared by enzymatic methyl-conversion using TET2 and APOBEC. Sequencing libraries were sequenced at low coverage and DNA methylation per covered CpG was assessed from the number of sequenced unconverted (methylated) cytosines (C) and the number of sequenced thymines (T) representing unmethylated cytosine. Global or region-specific DNA methylation was determined by calculating the average across all covered CpGs.b, Average global CpG methylation assessed by low-coverage whole methylome sequencing (black) and DNA methylation at long interspersed nucleotide elements (LINE) (grey) for T-ALL cell lines. Individual points for each replicate (left) and mean of duplicates (right).c,d Average CpG methylation as percentage genome-wide (global) or at CpG islands for 11 T-ALL cell lines assessed by low-coverage whole methylome sequencing. Individual replicates (c) and mean of duplicates (d). e, Percentage of CpG methylation relative to the average over all cell lines for CpG islands and globally. . Supplementary Fig. 2. Expression of genes known to get re-expressed upon DNA demethylation, DAZL, and GAGE12, after treatment with increasing concentrations of 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DAC), and GSK-3685032 (GSK5032) for 3 (left) and 7 days (right). Expression determined by qPCR and shown relative to housekeeping gene GAPDH. Cross indicates that no expression was detected by qPCR. Supplementary Fig. 3.a, Average global CpG methylation relative to an untreated control across four T-ALL cell lines divided based on genomic location. Cells were treated with increasing concentrations of 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DAC), and GSK-3685032 (GSK5032) for 3 or 7 days. b, Average absolute CpG methylation across four T-ALL cell lines treated with 3000 nM GSK5032 for 7 days divided based on genomic location. c, Average global CpG methylation relative to an untreated control across four T-ALL cell lines treated with increasing concentrations of AZA, DAC and GSK5032 for 3 or 7 days divided based on genomic location. SINE, short interspersed nuclear elements; LINE, long interspersed nucleotide elements; ERV, endogenous retrovirus; CGI, CpG island. Supplementary Fig. 4. a, b, Cell viability as percentage of untreated control for eleven T-ALL cells treated with 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DAC), or GSK-3685032 (GSK5032) for 3 (a) and 7 days (b). Cell viability assessed by Alamar Blue assay in triplicates. Dose-response curves and mean and standard error of the mean of triplicates from eleven T-ALL cell lines are shown. Statistical difference in treatment response for all cell lines combined per duration of treatment was assessed by Wilcoxon Rank Sum Test. Supplementary Fig. 5. a, Flow cytometry analysis after staining with propidium iodide (PI) and Annexin V of T-ALL cell lines treated with indicated concentrations of 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DAC), and GSK5-3685032 (GSK5032) for 3 days. Percentages of alive cells (PI-/Annexin V-, green), apoptotic cells (PI-/Annexin V+, blue), and necrotic cells (PI+/Annexin V+, red) are indicated. b, Percentage of dead cells (apoptotic and necrotic) after treatment of JURKAT and SUP-T1 cells with indicated concentrations of AZA, DAC, and GSK5032 for 3 days. Mean and standard deviation of biological triplicates shown. Based on flow cytometry analysis of cells stained with PI and Annexin V (examples shown in panel a). c, Cell cycle analysis with PI staining in JURKAT and SUP-T1 cells treated with indicated concentrations of AZA, DAC, and GSK5032 for 3 days. Gating strategy shown for one representative replicate. G1-phase: blue, G2/M-phase: red; S-phase: green. d, Percentage of cells in each phase of the cell cycle based on PI staining for JURKAT and SUP-T1 cells treated with indicated concentrations of AZA, DAC, and GSK5032 for 3 days. Mean of biological triplicates shown. Fisher’s exact test with p-values adjusted for multiple testing using Benjamini Hochberg. ns, adjusted p-values > 0.05. Supplementary Fig. 6. a-c, Western blot of DNMT1, phosphorylated H2AX (γ-H2AX), and β-actin (BACTIN) for JURKAT (a), LOUCY (b), and SUP-T1 (c) cells treated with increasing concentrations of 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DAC,) and GSK-3685032 (GSK5032) for 3 days. For SUP-T1, protein extracted from untreated JURKAT cells (-AZA) and JURKAT cells treated with 1000 nM AZA (+AZA) were included as antibody controls. d, Expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B after treatment with increasing concentrations of AZA, DAC, and GSK5032 for 3 (top) and 7 days (bottom). Expression determined by qPCR and shown relative to housekeeping gene GAPDH. Supplementary Fig. 7. a, Venn diagram showing the overlap between genes whose promoters keep ≥ 80% DNA methylation after treatment with 300 nM GSK-3685032 (GSK5032) for 7 days in LOUCY (blue) and SUP-T1 cells (red). b, Pathway enrichment analysis for the 60 genes whose promoters retain ≥ 80% DNA methylation after treatment with 300 nM GSK5032 in both LOUCY and SUP-T1 cells (overlap shown in panel a). List of genes can be found in Supplementary Table 9. c, Number of genes in LOUCY and SUP-T1 cells that are up or downregulated after treatment with 10 nM 5-aza-2′-deoxycytidine (DAC) for 3 days or 300 nM GSK5032 for 3 and 7 days. Genes were considered downregulated with a fold change (FC) < -2 and an adjusted p-value < 0.05. Genes were considered upregulated with FC > 2 and an adjusted p-value < 0.05. Otherwise, the expression of genes was considered to not change. d, Euler plot showing the overlap between upregulated genes (FC > 2 and adjusted p-value < 0.05) in red and promoters that lost DNA methylation (DNA methylation difference ≥ 25% and adjusted p-value < 0.01) in blue in LOUCY and SUP-T1 cells treated with 10 nM DAC for 3 days or 300 nM GSK5032 for 3 and 7 days. e, Venn diagram showing the overlap between methylation-sensitive genes defined after treatment with 300 nM GSK5032 for 7 days in LOUCY (blue) and SUP-T1 cells (red). Definition of methylation-sensitivity for each analysed gene can be found in Supplementary Table 13. Supplementary Fig. 8. a, -log10 of the adjusted p-value for enrichment analysis (phyper) of immune-related genes, cancer/testis antigens, tumour suppressors, and oncogenes among upregulated genes in LOUCY and SUP-T1 cells after treatment with 10 nM 5-aza-2′-deoxycytidine (DAC) for 3 days or 300 nM GSK-3685032 (GSK5032) for 3 or 7 days and genes defined as methylation-sensitive after treatment with 300 nM GSK5032 for 7 days. Genes included in each gene list can be found in Supplementary Table 14. b, c, Volcano plots showing change in expression of selected gene sets between treated and control cells as log2 of fold change and -log10 of the adjusted p-value for LOUCY (b) and SUP-T1 (c) cells treated with 10 nM DAC for 3 days or 300 nM GSK5032 for 3 or 7 days. Significantly up or downregulated genes shown as red dots. Total number of upregulated genes and percentage of all covered genes in gene set written in red. Written p-values represent Benjamini Hochberg adjusted p-values of enrichment analysis. Supplementary Fig. 9. a, -log10 of the adjusted p-value for tissue enrichment analysis on upregulated genes in LOUCY and SUP-T1 cells after treatment 300 nM GSK-3685032 (GSK5032) for 3 or 7 days. b, -log10 of the adjusted p-value for tissue enrichment analysis on methylation-sensitive genes in LOUCY and SUP-T1 cells defined after treatment 300 nM GSK5032 for 7 days. Definition of methylation-sensitivity for each analysed gene can be found in Supplementary Table 13. c, Pathway enrichment analysis for methylation-sensitive genes defined after treatment with 300 nM GSK5032 for 7 days in LOUCY (left) and SUP-T1 cells (right). Pathways related to tissue development are highlighted in bold. Definition of methylation-sensitivity for each analysed gene can be found in Supplementary Table 13. Supplementary Fig. 10. a, Circos representation of genome-wide methylation levels of transposable elements (TEs; including ERV, LINE, and SINE) in LOUCY (left) and SUP-T1 (right) cells before and after treatment with 10 nM 5-aza-2′-deoxycytidine (DAC) for 3 days or 300 nM GSK-3685032 (GSK5032) for 3 or 7 days. Average level of methylation of all TEs in 500kb windows is shown and displayed by chromosome. The colour gradient indicates DNA methylation level. b, c, Violin plots of DNA methylation levels for LOUCY (b) and SUP-T1 (c) cells untreated and treated with 10 nM DAC for 3 days or 300 nM GSK5032 for 3 or 7 days. Percentages of TEs within each class (ERV, LINE, and SINE) that had methylation levels below 0.3 or above 0.7 are shown on top. Wilcoxon signed-ranked test; *** p < 0.001. d, Bar graphs of percent of ERV, LINE and SINEs that showed no change (grey), loss (blue), or gain of methylation (red) after treatment with DAC and GSK5032 in LOUCY (left) and SUP-T1 (right) cells. Loss and gain of methylation were based on a methylation difference of ± 25% (compared to untreated) and adjusted p-value < 0.01. Percent of TEs gaining methylation was below 0.4% in both LOUCY and SUP-T1 for all treatments. e, Number of differentially expressed ERV, LINEs, and SINEs in LOUCY (left) and SUP-T1 (right) cells that were upregulated (red; fold change > 2, adjusted p-value < 0.05) or downregulated (blue; fold change < -2, adjusted p-value < 0.05) after treatment with 10 nM DAC for 3 days or 300 nM GSK5032 for 3 or 7 days compared to untreated cells. Total number of TEs detected are depicted on top of each graph for each cell line, respectively. f,g, Euler diagrams showing the number of TEs with significant loss of methylation (left), significant upregulation of expression (middle), an