FIGURE 5 from Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects

SpiD3 synergizes with ibrutinib and elicits cytotoxic effects in ibrutinib-resistant CLL cells. A–D, Combination assays to test synergy between SpiD3 and ibrutinib (IBR; BTK inhibitor) in preclinical CLL models. HG-3 cells (A–B; n = 3 independent experiments) or CpG ODN 2006 (CpG; 3.2 µmol/L)-stimulated patient-derived CLL cells (C–D; n = 6) were treated with SpiD3, IBR, or both (1:1 ratio) for 72 hours. MTS assay was performed to detect the dose effect. A and C, Dose-effect of the single drugs and their combination (1:1 ratio). B and D, CI of the combined doses (0.5 µmol/L SpiD3 + 0.5 µmol/L IBR = 1 µmol/L on graph). CI values were calculated using the Chou-Talalay method by the software Compusyn. CI values >1 are antagonistic, CI values = 1 are additive, and CI values <1 are synergistic. E, Mitochondrial activity in parental wild-type (WT) and ibrutinib-resistant (IR) HG-3 cell lines were assessed by MTS assay following treatment with increasing concentrations of SpiD3 or IBR (72 hours; n = 6 independent experiments/cell line). IC₅₀ values (mean ± SEM) are noted for each cell line. F and G, Representative immunoblot analyses of p-BTK (Tyr223), total BTK, p-PRAS (Thr246), total PRAS, p-ERK1/2 (Thr202/Tyr204), total ERK1/2, MYC, PARP (total and cleaved), IKKα, IKKβ, p65, and RELB in WT- and IR-HG3 cells treated with SpiD3 (1–2 µmol/L) or IBR (1 µmol/L) for 4 hours. BCR activation was induced by adding soluble α-IgM (10 µg/mL) for the last 15 minutes of treatment (n = 5 independent experiments). GAPDH served as the loading control. H, Spleen-derived malignant B cells from terminally diseased Eµ-Myc/TCL1 mice (n = 8) were stimulated ex vivo with 1X PMA/Ionomycin and treated with SpiD3 (0.25–2 µmol/L), IBR (1 µmol/L), or JQ-1 (1 µmol/L) for 48 hours. Proliferation was assessed via MTS assay and normalized to the unstimulated vehicle (dashed line). Asterisks indicate significance versus stimulated vehicle. *, P < 0.05; **, P < 0.01; ***, P < 0.001.