Fig 2: In vitro and in vivo antitumor effects of CRD-1441551 on ATL cell lines with MALT1 activation(A) In vitro antigrowth effect of CRD-1441551 on three ATL cell lines with MALT1 activation. Cell viability was measured after a 6-day exposure. GI50 values were calculated. Data represent mean ± SD (n = 3), which were confirmed in two independent experiments.(B) Western blot analysis of cleaved caspase-3 and cleaved caspase-7 in LM-Y1 and LM-Y2 cells. Cells were treated with 10-5 M CRD-1441551 for the indicated durations.(C) Tumor growth in an S1T xenograft model during CRD-1441551 treatment. Mice were treated for 14 consecutive days with CRD-1441551 at dosages of 60 or 100 mg/kg twice daily, lenalidomide at a dosage of 30 mg/kg once daily, or vehicle. Data represent mean ± SD (n = 6 per group). See also Figure S6.(D) Protein expression of cBCL10, components of the CBM complex, RelB, and IRF4 in S1T xenograft tumors. Tumor lysates were prepared 8 h after a single administration of CRD-1441551 and subjected to western blotting. Band intensities were quantified, normalized to control levels, and presented as percentage values in the accompanying bar graph. Data represent mean ± SD (n = 3 per group).

Fig 3: CRD-1441551 suppresses ATL PDX tumors(A) Overview of somatic mutations in established five PDX tumors. Each column and row correspond to a PDX model and mutated gene, respectively. Detailed plots of TCR pathway mutations are shown in Figure S5.(B) Inhibition of MALT1 paracaspase activity by CRD-1441551 and related effects on PDX tumors. Tumor lysates were prepared after the efficacy study and subjected to western blotting. Representative results of western blotting are shown (n = 2 per treatment group in each PDX model). The band intensities of IRF4 were quantified (n = 2 per treatment group in each PDX model), and the percent IRF4 inhibition by CRD-1441551 treatment is shown in bar charts. Reduction of cBCL10 and IRF4 and accumulation of RelB were observed following CRD-1441551 treatment in three models (M67, M143, and M156).(C) Suppression of PDX tumors by CRD-1441551. Recipient mice with 300‑600 mm3 tumors were treated twice daily with 60 mg/kg CRD-1441551 or vehicle for 21 days or 12 days for the M153 model. Tumor growth in each PDX model was assessed every 3–4 days during CRD-1441551 treatment. Growth suppression was observed in PDX tumors, M67, M143, and M156. Data represent mean ± SD (n = 5 per group). Two-way ANOVA followed by Tukey’s post hoc test was performed, p < 0.05 or p < 0.01 are shown by ∗ or ∗∗, respectively.(D) Histology of tumor tissue in the five PDX models. Microscopic views of samples stained with Ki-67 are shown at high magnification (×400). Scale bars, 50 μm. The frequency of Ki-67-positive cells was assessed (five high-power fields per tumor) and shown in a bar chart. Data represent mean frequency ±SD (n = 5 per treatment group in each PDX model). A paired Student’s t test was performed, and p < 0.05 is shown by ∗.(E) Serum LDH and human sIL-2R levels after treatment. Recipient mice transplanted with the M67 PDX clone showed decreased levels of LDH and human sIL-2R after CRD-1441551 treatment. Data represent mean ± SD (n = 5 per group). A paired Student’s t test was performed, and p < 0.05 or p < 0.01 are shown by ∗ or ∗∗, respectively.See also Figure S7, S8, and Table S4.