BCR-independent expression of BCL2A1 in CARD11 mutated cells. (A) BCR signaling triggers the NF-κB1 pathway through the activation of several proteins such as BTK, which can be inhibited by ibrutinib, and complexes such as CBM. Several previously described CBM substrates are mentioned including HOIL1, which is used in the following experiments as a marker of CBM activity. (B) NF-κB luciferase assay (left panel) and BCL2A1 promoter LightSwitch luciferase assay (right panel) in HEK293 cells transfected with a pUNO1 (empty), pUNO1-hCARD11-WT, pUNO1-hCARD11-G123S or pUNO1-hCARD11-G123D vector (48 hours). Two-way ANOVA; ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05. The mean and standard deviation of 3 independent experiments are represented. (C) BCL2A1 gene expression was measured by RT-qPCR in CARD11^WT MCL (SP53, NTS3, and REC-1) and DLBCL (U2932) cell lines and CARD11^MUT MCL cells (001-024, DFBL) and DLBCL (OCILY3, SUDHL16) cell lines treated with ibrutinib (500 nM) for 6 hours. Detailed CARD11 mutational status is described in supplemental Table 4. Mann-Whitney test; ∗P < .05.