UNC1999, and not UNC2400, derepresses the PRC2 gene targets. (A) Summary of the upregulated (blue) and downregulated (red) transcripts in 2 independent MLL-AF9–transformed leukemia lines after a 5-day treatment with 3 μM of compounds or after knockdown of EED vs Renilla, as identified by microarray analysis with a cutoff of FC of >1.5 and a P value of <.01. (B) Scatter plot to compare the global gene expression pattern in MLL-AF9–transformed leukemia cells following DMSO (x-axis) vs UNC1999 treatment (y-axis). Plotted are Log10 values of the signal intensities of all transcripts on gene microarrays after normalization. The flanking lines in green indicate 1.5-fold change in gene expression. (C) Boxplots showing the expression levels of upregulated transcripts in the compound- vs DMSO-treated samples. Y-axis represents the Log10 value of signal intensities detected by microarray. (D) Venn diagram of the upregulated transcripts shown in panel A. (E) Summary of GSEA using the MSIgDB. Green and red indicate the positive and negative correlation to UNC1999-treated cells, respectively. (F-H) GSEA revealing significant enrichment of the EZH2-repressed (F) or EED-repressed gene signatures (G) and those negatively associated with hematopoietic stem cells (H) in the UNC1999- vs DMSO-treated cells. (I) RT-qPCR detects relative expression levels of the indicated genes in MLL-AF9–transformed leukemia cells following treatment with 3 μM of compounds or EED knockdown (shEED) for 5 days. Y-axis represents fold-change after normalization to GAPDH and to control (DMSO treatment or Renilla knockdown [shRen]), and error bars represent SD of triplicates. *P < .05; **P < .01; ***P < .001. FC, fold-change; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MSIgDB, Molecular Signatures Database; NES, normalized enrichment score; Ren, Renilla; shEED, shRNA against EED; shREN, shRNA against Renilla.