FIGURE 2 from Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects

SpiD3 modifies transcriptional profiles and subverts oncogenic pathways in CLL cells. A–D, RNA sequencing of OSU-CLL cells treated with SpiD3 (1, 2 µmol/L; 4 hours) or equivalent DMSO vehicle (VEH; n = 3 independent experiments). A, Hierarchical clustering of the top 500 differentially expressed genes (DEG) in SpiD3-treated cells (FDR B, GSEA of the top 500 DEGs in 2 µmol/L SpiD3-treated cells. C, Heat map of correlation between WGCNA module and the indicated treatment conditions. Each heat map cell displays the correlation coefficient (top) and corresponding P value (bottom). D, Volcano plot of SpiD3-treated cells (2 µmol/L) with select CLL-relevant genes labeled. Genes meeting both the statistical significance (FDR 2 FC| > 1) were used for downstream analysis (red). Genes meeting only statistical significance (blue), only fold-change (green), or neither threshold (gray) are shown for comparison. E, Representative immunoblots (n = 3 independent experiments) of the indicated proteins in SpiD3-treated OSU-CLL cells (4 hours). F, Cell cycle analysis of OSU-CLL cells treated with increasing amounts of SpiD3 (48 hours). BET inhibitor, JQ-1 (1 µmol/L), served as a positive control for cell cycle arrest (n = 5 independent experiments). Insert depicts a representative immunoblot for p21 expression following SpiD3 treatment (24 hours; n = 3 independent experiments). β-ACTIN served as the loading control. G, OSU-CLL cells were pretreated with 5 mmol/L N-acetylcysteine (NAC, 1 hour) followed by SpiD3 or VEH (24 hours). Pyocyanin (PYO, 1 mmol/L) served as a control ROS inducer (n = 3 independent experiments). Percent viability per condition is denoted below. Data are represented as mean ± SEM. Asterisks denote significance versus VEH: *, P P P