Selinexor is active in the setting of acquired resistance to ibrutinib. (A) DT40 BTK-null cells with WT or C481S BTK were exposed to 1 μM ibrutinib for 1 hour, 0.5 μM selinexor for 24 hours, or dimethylsulfoxide (vehicle) for 24 hours. Cytotoxicity after 24 hours was measured by annexinV/PI flow cytometry. Viable populations were calculated as a percent of viability of vehicle control. Three biological replicates were performed. Selinexor induced significantly more cell death compared with vehicle in cells expressing C481S (P = .042), WT (P = .027), or empty vector (P = .011). (B) C57BL/6 mice were engrafted with spleen lymphocytes derived from an Eμ-TCL1 transgenic mouse with acquired resistance to ibrutinib. Mice were followed for leukemia development (defined as ≥10% CD5⁺/CD19⁺ cells in the leukocyte population), and once leukemic, randomized to treatment with ibrutinib alone (∼30 mg/kg/day via drinking water), selinexor alone (15 mg/kg on 2 consecutive days each week via oral gavage), or vehicle. As expected, mice treated with ibrutinib did not show any survival advantage compared with vehicle control, whereas mice treated with selinexor showed improved survival (n = 12-14 per group). (C) In vivo EdU labeling was performed in a cohort of mice engrafted as described in B. Mice were treated for 2 days with vehicle, SEL, or Ibr (n = 5 for each group). EdU was injected on day 3. Spleens were analyzed by flow cytometry for percentage of Edu-positive cells within the leukemic population (CD45⁺/CD19⁺/CD5⁺ cells). (D) CLL cells derived from ibrutinib resistant patients (n = 3) were treated in vitro with selinexor at 0.5 μM. Cytotoxicity was measured by annexin-V/PI after 48 hours.