DEX sensitivity and GC pathway activation in CCRF-CEM cells and JDP2 expression in paired diagnostic/relapsed human pediatric T-ALLs. (A) Cell viability assay was assessed by Hoechst live/dead stain using flow cytometry. Individual data points for all the S1/S19 clones are shown. JDP2 overexpression caused moderate DEX resistance (JDP [blue] vs EV [gray], P < .0001 by ANCOVA). KDM6A inactivation significantly increased DEX sensitivity (EV/KDM [pink] vs EV [gray]; P <.0001) and JDP2 overexpression reversed this phenotype and conferred profound resistance (JDP/KDM [green] vs EV/KDM [pink]; P <.0001). See supplemental Table 2 for additional statistical analyses. (B) NR3C1 mRNA expression in untreated CCRF-CEM S1 and S19 cells and in cells exposed to 1 μM of DEX for 18 hours. NR3C1 is upregulated in the presence of DEX in all genotypes (P < .0001 by ANCOVA). JDP2 overexpression in the background of KDM6A inactivation resulted in a marked reduction in responsiveness to DEX (JDP/KDM vs EV/KDM; P <.0001). See supplemental Table 2 for additional statistical analyses. (C) Western blot showing glucocorticoid receptor protein levels in cells exposed to either control vehicle or DEX (500 nM) for 24 hours in all CCRF-CEM S1 and S19 subclones. (D) Change in JDP2 expression from diagnosis to relapse in 9 paired human T-ALL samples, including 2 with a KDM6A mutation. As shown, T-ALL with a V1112 frameshift KDM6A mutation had a unique ∼10-fold increase in JDP2 expression, and leukemia with a V1113_S1114 frameshift KDM6A mutation acquired an NR3C1 mutation at relapse.