Figure 3.
Figure 3. Subsets of MCLs are addicted to MALT1. (A) Effect of MALT1 shRNA #1 and #2 on MALT1 mRNA level in MALT1-activated (Jeko-1 and Rec-1) and MALT1-inactive (Maver-1 and Z-138) MCLs 48 hours after shRNA induction measured by quantitative PCR. MALT1 mRNA levels were normalized to expression of GAPDH. Error bars indicate SDs. (B) Effect of MALT1 shRNA #1 and #2 on MALT1 protein in MALT1-activated (Jeko-1 and Rec-1) and MALT1-inactive (Maver-1 and Z-138) MCLs 48 hours after shRNA induction measured by western blotting. (C) Effect of MALT1 knockdown by 2 independent shRNAs on viability of MCL cell lines. A previously described, nontoxic shRNA against MSMO1 did not induce toxicity in any cell line. Data are shown as means ± SDs of at least 3 independent experiments. (D) Rescue of Jeko-1 and Rec-1 cells from MALT1 shRNA-induced toxicity by exogenous expression of a MALT1 cDNA. Data are shown as means ± SDs of at least 3 independent experiments. (E) Western blot analysis of MALT1 knockdown in Jeko-1 mouse xenograft tumor biopsies from cells transduced with MALT1 shRNA #2 compared with control shRNA-transduced cells (shRNA against MSMO1). (F) Tumor growth curve of Jeko-1 xenograft mouse models that inducibly express MALT1 shRNA #2 (blue) or a control shRNA against MSMO1 (red). MALT1 knockdown significantly reduced in vivo tumor growth (P = 1.9 × 10−5, MALT1 shRNA vs control shRNA on day 12; 1-tailed 2-sample t test). Error bars indicate SDs. (G) Western blot analysis of MCL cell lines, treated with z-VRPR-fmk for 48 hours, for cleavage of CYLD, RelB, A20, and BCL10 in MALT1-activated MCL cell lines (Mino and SP53) vs MALT1-inactive MCLs (Maver-1 and Z-138). (H) Cell viability of MCL cell lines after incubation with the MALT1 inhibitor z-VRPR-fmk. Representative results from at least 3 independent replicates are shown. Error bars indicate SDs. (I) Carboxyfluorescein diacetate succinimidyl ester staining after treatment with z-VRPR-fmk or DMSO was measured on day 0 and after 2, 4, and 6 days. In Z-138 cells, no difference in cell proliferation was detectable (P = .4 on day 6). In contrast, Mino cells showed reduced proliferation after treatment with z-VRPR-fmk (P < 10−15 on day 6). Representative results from at least 3 independent replicates are shown. (J) Western blotting for FLAG and BTK following transduction of Mino cells with either a BTKC481S cDNA or an empty vector. (K) Determination of cell viability of Mino cells expressing either an empty vector (red) or a BTKC481S cDNA (blue) following treatment with ibrutinib or z-VRPR-fmk. Representative results from at least 3 independent replicates are shown. Error bars indicate SDs. *P < .05, **P < .01, ***P < .001.

Subsets of MCLs are addicted to MALT1. (A) Effect of MALT1 shRNA #1 and #2 on MALT1 mRNA level in MALT1-activated (Jeko-1 and Rec-1) and MALT1-inactive (Maver-1 and Z-138) MCLs 48 hours after shRNA induction measured by quantitative PCR. MALT1 mRNA levels were normalized to expression of GAPDH. Error bars indicate SDs. (B) Effect of MALT1 shRNA #1 and #2 on MALT1 protein in MALT1-activated (Jeko-1 and Rec-1) and MALT1-inactive (Maver-1 and Z-138) MCLs 48 hours after shRNA induction measured by western blotting. (C) Effect of MALT1 knockdown by 2 independent shRNAs on viability of MCL cell lines. A previously described, nontoxic shRNA against MSMO1 did not induce toxicity in any cell line. Data are shown as means ± SDs of at least 3 independent experiments. (D) Rescue of Jeko-1 and Rec-1 cells from MALT1 shRNA-induced toxicity by exogenous expression of a MALT1 cDNA. Data are shown as means ± SDs of at least 3 independent experiments. (E) Western blot analysis of MALT1 knockdown in Jeko-1 mouse xenograft tumor biopsies from cells transduced with MALT1 shRNA #2 compared with control shRNA-transduced cells (shRNA against MSMO1). (F) Tumor growth curve of Jeko-1 xenograft mouse models that inducibly express MALT1 shRNA #2 (blue) or a control shRNA against MSMO1 (red). MALT1 knockdown significantly reduced in vivo tumor growth (P = 1.9 × 10−5, MALT1 shRNA vs control shRNA on day 12; 1-tailed 2-sample t test). Error bars indicate SDs. (G) Western blot analysis of MCL cell lines, treated with z-VRPR-fmk for 48 hours, for cleavage of CYLD, RelB, A20, and BCL10 in MALT1-activated MCL cell lines (Mino and SP53) vs MALT1-inactive MCLs (Maver-1 and Z-138). (H) Cell viability of MCL cell lines after incubation with the MALT1 inhibitor z-VRPR-fmk. Representative results from at least 3 independent replicates are shown. Error bars indicate SDs. (I) Carboxyfluorescein diacetate succinimidyl ester staining after treatment with z-VRPR-fmk or DMSO was measured on day 0 and after 2, 4, and 6 days. In Z-138 cells, no difference in cell proliferation was detectable (P = .4 on day 6). In contrast, Mino cells showed reduced proliferation after treatment with z-VRPR-fmk (P < 10−15 on day 6). Representative results from at least 3 independent replicates are shown. (J) Western blotting for FLAG and BTK following transduction of Mino cells with either a BTKC481S cDNA or an empty vector. (K) Determination of cell viability of Mino cells expressing either an empty vector (red) or a BTKC481S cDNA (blue) following treatment with ibrutinib or z-VRPR-fmk. Representative results from at least 3 independent replicates are shown. Error bars indicate SDs. *P < .05, **P < .01, ***P < .001.

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