KPT-185 induces selective cytotoxicity in CLL cells. (A) CD19⁺ cells from CLL patients (N = 13) and normal donors (N = 12) were examined for XPO1 expression by immunoblot. Results are shown from 1 of 2 identical experiments. (B) Data analysis of band intensities measured in 2 immunoblots of CLL patient and normal B-cell samples (XPO1/actin ratio). (C) RNA was extracted from CD19⁺ cells from CLL patients (N = 8) or normal donors (N = 8). XPO1 expression was determined by real-time RT-PCR analysis. Ct values are relative to actin. Higher relative Ct values represent lower gene expression. (D) KPT-185 induces a time and dose-dependent cytotoxicity of CLL cells as measured by MTS assay (N = 10 per timepoint). (E) KPT-185 and KPT-251 induce comparable level of cytotoxicity of CLL cells at 72-hour time-point as measured by MTS assay (N = 6 each). (F) KPT-185 is not cytotoxic to normal PBMC and isolated B cells as measured by annexin-V/PI flow cytometry (N = 6 each). (G) Comparison of the cytotoxic effect of KPT-185 on CLL versus normal B cells as measured by MTS assay (N = 8 each). (H-J) Cytogenetic abnormalities and IVGH mutational status were examined for differences in response to KPT-185 of CLL cells. (K-I) Treatment with SINEs promotes cell death through a caspase-dependent pathway. CLL patient cells were treated with various concentrations of KPT-251 for 12 or 24 hours in presence or absence of the caspase inhibitor Q-VD-OPH. Lysates derived from these cells (12 hours) were assessed for cleavage of PARP and caspase 3 by immunoblot analysis. (L) Apoptosis was measured at 24 hours by annexin-V/PI flow cytometry.