Misregulated GCR and EZH2 signaling as a consequence of NR3C1 alterations in BPDCN. (A) Luciferase reporter assay for glucocorticoid transcriptional transactivation activity in COS7 cells transduced with empty pcDNA3.1 vector, pcDNA3.1-GCR, and pcDNA3.1-GCR-FP, as indicated, after a 6-hour treatment with either dimethyl sulfoxide (DMSO) or 100 nM dexamethasone (n = 3). (B) Heatmap representation of the common differentially expressed genes among CAL-1-GCR-FP (GCR-FP[+]), CAL-1 control cells (GCR-FP[−]), and shGCR-transduced cells (shGCR[+]) compared with controls (shGCR[−]) after treatment with 100 nM dexamethasone for 6 hours (n = 3 for each group). Black arrows indicate genes cited in the text. (C) GSEA plots showing gene regulatory circuits that are differentially expressed between dexamethasone-treated CAL-1-GCR-FP or shGCR compared with respective control CAL-1 cells (CAL-1-GFP or CAL-1 shCtrl). (D) Western blot analysis and quantification of global H3K27me3 levels in CAL-1-GCR-FP (GCR-FP[+]) and CAL-1 shGCR (upper) and respective CAL-1 control cells (CAL-1 GFP [GCR-FP(−)] and CAL-1 shCtrl) (lower) upon 6-hour or 24-hour treatment with 100 nM dexamethasone (n = 3). (E) ChIP for H3K27me3 followed by qPCR analysis for enrichment on HOXA gene promoters, as indicated, in CAL-1-GCR-FP (GCR-FP[+]), CAL-1 shGCR, and respective CAL-1 control cells (CAL-1 GFP [GCR-FP(−)] and CAL-1 shCtrl) treated for 24 hours with 100 nM dexamethasone (n = 3). (F) GSEA plots showing gene regulatory circuits that are differentially expressed between BPDCN patients presenting, or not, del(5q) abnormalities that target NR3C1 (bone marrow or skin, as indicated; unmutated EZH2 and ASXL1 cases only). (G) Tumor bioluminescence (7 and 14 days postinjection) for xenotransplanted nude mice bearing tumors derived from CAL-1 shCtrl and shGCR cell lines (left). P value determined by Wilcoxon test; n = 6 for each group. Bioluminescence imaging for representative mouse (right). D, day; GSEA, gene set enrichment analysis.