LUBAC is an effective target for the treatment of DLBCL. (A) Elevated phosphorylation and degradation of IκBα in unstimulated HM876 cells were suppressed by thiolutin. (B) Diagram of allogeneic transplantation model. s.c., subcutaneously. (C) Chemical structure of aureothricin. (D) Inhibition of LUBAC ligase activity by aureothricin and thiolutin in vitro. ATP, adenosine triphosphate; IB, immunoblotting. (E) Chemical structure of thiolutin. (F) Thiolutin inhibited linear polyubiquitination mediated by HOIP (amino acids 699-1072). (G) Upon stimulation of DLBCL2 cells with CD40 ligand, thiolutin suppressed phosphorylation and degradation of IκBα in a dose-dependent manner. DLBCL2 cells were exposed to thiolutin or DMSO for 2 hours and then stimulated with CD40 ligand (30 ng/mL) for the indicated times. (H) Levels of LUBAC components in DLBCL2 (upper panel) and HM876 (lower panel) cells treated with thiolutin were reduced in a dose-dependent manner. (I) Cell lysates of DLBCL2 (left panel) and HM876 (right panel) cells treated with or without thiolutin (0.1 μM) for 2 hours were analyzed by immunoblotting. Samples were probed with anti-linear ubiquitin specific antibody (LUB9). (J) Viability of DLBCL lines after 48 hours treatment with the indicated concentrations of thiolutin, normalized against that of control (DMSO-treated) cells. Data are means ± SD from three experiments. (K) Viability of HM876 cells after 48 hours treatment with the indicated concentrations of thiolutin, normalized against that of control (DMSO-treated) cells. Data are means ± SD from three experiments. (L and M) Thiolutin suppressed growth of lymphomas in vivo. (L) Gross appearance of engrafted tumors. (M) Tumor weight. Data represent means ± SD. *P < .05, **P < .01, and *** P < .001, 2-tailed unpaired Student t test (J and K) or one-way ANOVA with Turkey’s post hoc tests (M). See also supplemental Figure 6.