Selinexor synergizes in vitro and in vivo with ibrutinib. (A) CD19⁺ cells from CLL patients (n = 6) were isolated from peripheral blood and incubated with vehicle, 0.5 μM selinexor (SEL), 1 μM ibrutinib (Ibr) or selinexor + ibrutinib. Ibr was given as a 1-hour pulse exposure followed by washout, and SEL was given continuously for 24 hours. Viability was determined by annexin-V/PI flow cytometry and is shown relative to time-matched dimethylsulfoxide controls for each group. Horizontal bars represent averages. Each agent alone (SEL or Ibr) significantly decreased cell viability compared with vehicle (P < .03). The combination produced a synergistic effect on viability (P = .041). (B) CD19⁺ cells from CLL patients (n = 6) were unstimulated or 3.2 µM CpG-stimulated in the presence of vehicle, 0.5 μM SEL (24-hour continuous exposure), 1 μM Ibr (1-hour pulse exposure with washout), or SEL + Ibr. Cytotoxicity was measured by annexin/PI. Horizontal bars represent averages. Each agent alone (SEL or Ibr) significantly decreased cell viability compared with vehicle (P = .001). The combination produced a synergistic decrease in viability (P = .005). (C) CD19⁺ cells from CLL patients were incubated with 0.5 μM SEL (24-hour continuous exposure), 1 μM Ibr (1-hour pulse exposure with washout), or SEL + Ibr on an HS5 human bone marrow stromal cell layer for 24 hours. Cytotoxicity was measured by annexin-V/PI flow-based assay. Horizontal bars represent averages. SEL and Ibr together resulted in significantly more cytotoxicity than either agent alone (P < .001). (D) Overall survival (OS) curves for C57BL/6 mice engrafted with spleen lymphocytes derived from the Eμ-TCL1 transgenic mouse. Mice with active leukemia (defined as ≥10% CD5⁺/CD19⁺ cells in the leukocyte population) were randomized to treatment with ibrutinib (∼30 mg/kg/day via drinking water) or ibrutinib + selinexor (15 mg/kg on 2 consecutive days each week via oral gavage; n = 6 per group). (E) Persistent lymphocytes collected at baseline and 9 months after beginning ibrutinib from the same patients were treated in vitro with selinexor at 0.5 μM (n = 13). Cytotoxicity was measured by annexin-V/PI flow cytometry after 72 hours.