Fig. 1. T-ALL cells are dependent on EHMT2.
A Effect of epigenetic modulators BIX01294, C646, CTPB, GSK126, SGC0946, and UNC0638 in T-ALL cell lines (ALL/SIL, CCRF-CEM, DND41, HPB-ALL, HSB2, and PEER). BIX01294 is a diazepin-quinazolin-amine derivative that selectively inhibits the H3K9 di-methylation activity of G9a, and to a lesser extent GLP, without competing for the S-adenosyl-methionine (SAM) cofactor; C646 is a selective small-molecule inhibitor of histone acetyltransferase p300; CTPB is an amide derivative that selectively activates the histone acetyltransferase (HAT) p300; GSK126 is a EZH2 methyltransferase inhibitor; SGC0946 is a highly potent and selective DOT1L methyltransferase inhibitor; UNC0638 is a substrate-competitive small-molecule inhibitor with equal potency for G9a and GLP in cell-based assays. The scatter dot plot represents the effect of small molecules on cellular viability calculated using the area under the curve (AUC) model of log transformed dose-response data using GraphPad V7. A lower AUC corresponds to a greater sensitivity. Statistical significance among groups for treated vs. vehicle (DMSO) (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001) was determined by one-way ANOVA using Bonferroni’s correction for multiple comparison testing. B Graph showing response to the G9a inhibitor, UNC0638, in over 900 cancer cell lines screened as part of the Genomics of Drug Sensitivity in Cancer Project (GDS). T-ALL cell lines are indicated in red, non-T-ALL cell lines in grey. Statistical significance among groups (****P ≤ 0.0001) was determined by a non-parametric t-test (Mann–Whitney). C Effect of epigenetic modulators BIX01294, UNC0638, UNC0642 in T-ALL (n = 10), AML (n = 5) cell lines and primary patients T-ALL cells (n = 4) and primary patients AML cells (n = 4) samples. The scatter dot plot represents the effect of small molecules on cellular viability calculated using the area under the curve (AUC) model of log transformed dose-response (BIX01294 = 0 < [X] < 8 μM; UNC038 = 0 < [X] < 10 μM; UNC042 = 0 < [X] < 10 μM) data using GraphPad V7. A lower AUC corresponds to a greater sensitivity. Statistical significance among groups (**P ≤ 0.01) was determined by a non-parametric t-test (Mann–Whitney). D EHMT2 expression levels in 36 cancer types (1036 cancer cell lines). Data were obtained from the Cancer Cell Line Encyclopedia [16]. A red bar represents the T-ALL cell lines. E G9a and H3K9me2 expression in human thymus and T-ALL lymphoblasts. Formalin-fixed, paraffin embedded tissue sections were stained with anti-G9a and anti-H3K9me2. Scale bars, 20 μm. Hassall’s corpuscles are indicated by arrowheads. F EHMT2 expression levels in T cells lymphocytes (n = 5) or in T-ALL lymphoblasts (n = 33). The line in the box-and-whisker diagram represents the Log2 EHMT2 median expression calculated according to the ΔΔCT method. The upper edge (hinge) of the box indicates the 75th percentile of the data, and the lower hinge the 25th percentile. The end of the vertical line represents the minimum and the maximum data values. Statistical significance among groups (*P ≤ 0.05) was determined by a non-parametric t-test (Mann–Whitney). G Western blot showing expression of G9a (l long isoform, s short isoform), GLP, and ICN1 in a panel of T-ALL cell lines. Actin was used as a loading control. H Effect of G9a inhibitors BIX01294, UNC0638, and UNC0642 in primary T-ALL blasts (n = 3) or isolated T cells (n = 6). Cells were grown at increasing concentrations of G9a/GLP inhibitors (BIX01294, UNC0638, and UNC0642) and viability evaluated at day 3 by an ATP-based assay and plotted as the percentage of viable cells relative to a DMSO control. Shown is the mean ± standard deviation (SD) of a minimum of two replicates. I Effect of the G9a inhibitors BIX01294, UNC0638, and UNC0642 in primary T-ALL cells (n = 3) or isolated T cells (n = 6). The scatter dot plot represents the effect of small molecules on cellular viability calculated using the AUC model of log transformed dose-responses data using GraphPad V7. The line in the box-and-whisker diagram represents the AUC median. The upper edge (hinge) of the box indicates the 75th percentile of the data, and the lower hinge the 25th percentile. The ends of the vertical line indicate the minimum and the maximum data values. Statistical significance (*P ≤ 0.05) was determined by a non-parametric t-test (Mann–Whitney). J Effect of G9a inhibitors BIX01294, UNC0638, and UNC0642 in CD25+ thymic cells isolated from CD1 mice or PDLX-0122 T-ALL cells. Cells were grown at increasing concentrations of G9a/GLP inhibitors (BIX01294, UNC0638, and UNC0642) and viability evaluated at day 3 by an ATP-based assay and plotted as the percentage of viable cells. Shown is the mean ± standard deviation (SD) of a minimum of three replicates.