HDAC6 inhibition increases CD20 levels in a panel of B-cell tumor cell lines, primary CLL cells and in vivo. (A) EBV-positive Burkitt lymphoma cell lines (Daudi, HS-Sultan), EBV-negative Burkitt lymphoma cell line Ramos, EBV-negative diffuse large B-cell lymphoma cell lines (Pfeiffer, Ly-1, Ly-7, Karpas 422), as well as EBV-transformed lymphoblastoid cell lines (A1, A2) were pretreated with 5 or 10 µM tubacin (depending on tubacin toxicity) for 48 hours. The levels of surface CD20 were analyzed with flow cytometry on staining with FITC-conjugated anti-CD20 antibody. Cell viability was assessed with PI staining. The results are presented as a percentage of MFI of control cells (± SD). Statistical significance was determined with Mann-Whitney test, *P < .05, ***P < .001 vs control. The experiments were repeated independently twice. (B) Peripheral blood mononuclear cells from patients with CLL were treated with increasing concentrations of tubacin (n = 40) or ricolinostat (n = 27) for 48 hours. The cells were stained with anti-CD20-FITC, anti-CD19-phycoerythrin antibodies, and 7-aminoactinomycin D. Primary cells were gated according to side scatter and forward scatter, followed by doublet discrimination. CD20 expression was assessed in a population of CD19-positive, alive (7-aminoactinomycin D–negative) cells. The results are presented as a percentage of MFI of control cells (± SD). Statistical significance was determined with Kruskal-Wallis test, *P < .05, **P < .01, ****P < .0001, vs controls. (C) BALB/c SCID mice were inoculated subcutaneously with Raji cells stably transduced to express luciferase (Raji-LUC). Mice (n = 5-9) were then injected intraperitoneally with rituximab (10 mg/kg) and/or ricolinostat (100 mg/kg) 3 times a week for 3 consecutive weeks. Survival of mice was compared by Kaplan-Meier analysis. The experiment was performed once. Statistical significance was determined using log-rank survival analysis.