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Antitumor effects of SpiD3 in patient-derived CLL samples. A, Viability of patient-derived CLL samples (n = 11–15) cocultured ex vivo with 9-15c mouse stroma cells for 48 hours was determined by Annexin-V/PI staining. Ibrutinib (IBR; 1 µmol/L) serves as a control antileukemic drug. Results are normalized to the unstimulated control (dashed line). B, Following ex vivo treatment with SpiD3 (0.125–2 µmol/L), the relative proliferation of patient-derived CLL samples (n = 16–18) under cocurrent CpG ODN 2006 stimulation (3.2 µmol/L) was assessed via MTS assay (48 hours). IBR (1 µmol/L) was used as control antileukemic drug. Results are normalized to the unstimulated control (dashed line). C, UPR induction in patient-derived CLL samples (n = 8) was evaluated following 24 hours ex vivo treatment with SpiD3 or thapsigargin (Thaps; 1 µmol/L) under cocurrent CpG stimulation (3.2 µmol/L) via incubation with TPE-NMI dye. Data are represented as fold change in TPE-NMI MFI compared with the unstimulated control (dashed line). D, Protein synthesis in patient-derived CLL samples (n = 8) was assessed via OPP incorporation following a 24 hours ex vivo treatment with SpiD3, cycloheximide (CHX; 50 µg/mL), or IBR (1 µmol/L) under cocurrent CpG stimulation (3.2 µmol/L). Data are represented as fold change in OPP-Alexa Fluor MFI compared with the unstimulated control (dashed line). E, Representative immunoblot analyses of p65, RELB, p-PRAS (Thr246), total PRAS, p-4E-BP1 (Ser65), total 4E-BP1, and MYC protein in patient-derived CLL samples (n = 6) following a 24-hour treatment with SpiD3 in the presence of CpG (3.2 µmol/L). Black arrows indicate the three isoforms of 4E-BP1. GAPDH served as the loading control. Patient characteristics are tabulated in Supplementary Data S1: Supplementary Table S1. Asterisks denote significance versus stimulated VEH: *, P P P
Eiken et al. (Wed,) studied this question.