Gain-of-function mutations in CARD11 promote enhanced aggregation and idiosyncratic signalosome assembly

Abstract

BENTA (B cell Expansion with NF-κB and T cell Anergy) is a novel lymphoproliferative disorder caused by germline, gain-of-function (GOF) mutations in the lymphocyte-restricted scaffolding protein CARD11. Similar somatic CARD11 mutations are found in lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL). Normally, antigen receptor (AgR) engagement converts CARD11 into an active conformation that nucleates a signalosome required for IκB kinase (IKK) activation and NF-κB nuclear translocation. However, GOF CARD11 mutants drive constitutive NF-κB activity without AgR stimulation. Here we show that unlike wild-type CARD11, GOF CARD11 mutants can form large, peculiar cytosolic protein aggregates we term mCADS (mutant CARD11 dependent shells). MALT1 and phospho-IKK are reliably colocalized with mCADS, indicative of active signaling. Moreover, endogenous mCADS are detectable in ABC-DLBCL lines harboring similar GOF CARD11 mutations. The unique aggregation potential of GOF CARD11 mutants may represent a novel therapeutic target for treating BENTA or DLBCL.

Keywords: Aggregates; B cell lymphoma; BENTA; CARD11; MALT1; NF-kB; Signalosome.

Fig. 1.

Gain-of-function CARD11 mutants form mCADS. (A) Cartoon of CARD11 protein indicating approximate locations of BENTA-associated GOF mutations described. L = LATCH domain, CC = coiled-coil. (B) Confocal immunofluorescent (IF) microscopy analysis of BJAB B cells transfected with WT and GOF CARD11-FLAG mutants, detected using anti-FLAG-AlexaFluor 647 Ab. Merge image includes DAPI staining. Scale bar = 5 μm. (C) JPM50.6 T cells were transfected with WT or C49Y CARD11 and fixed for TEM analysis 24 hrs post-transfection. Numbers indicate magnification, squares denote putative mCADS that are not found in WT cells (n = 30 cells analyzed for each). Data are representative of two independent experiments performed with WT, C49Y, E134G, or G123D CARD11. (D) ImmunoGold staining and TEM analysis of JPM50.6 T cells transfected with E134G CARD11 as prepared in Supp. Fig. 2A. Data are representative of 2 independent experiments. (E) TEM micrographs of recombinant CARD11 (aa 8–302) showing heterogeneous clusters of WT (20–40 nm), E134G (60–120 nm), and G123D (20–40 nm).

Fig. 2.

Differential interaction of CBM signaling proteins with mCADS. (A–D) Confocal IF microscopy analysis of WT and GOF mutants of CARD11-FLAG expressed ectopically in JPM50.6 T cells. Cells were co-stained with Abs against (A) BCL10 and (B) MALT1 as well as anti-FLAG-AlexaFluor 647, 24 hrs post-transfection. JPM50.6 T cells were transfected with (C) WT or (D) E134G CARD11-FLAG then stained with Abs against caspase 8, TAK1, or TRAF6 and FLAG. GFP reporter expression indicates NF-κB activity, although variability of GFP signal was noted after cell permeabilization. Data are representative of >3 independent experiments. Scale bar = 5 μm.

Fig. 3.

mCADS are sites of active IKK signaling. (A) Confocal IF microscopy of BJAB B cells transfected with WT or mutant CARD11-FLAG constructs and stained with anti-FLAG, anti-MALT1, and anti-phospho-IKKα/β Abs. Merge images include DAPI staining. Scale bar = 5 μm. (B) Transfected BJAB cell lysates were separated by SDS-PAGE and immunoblotted for CARD11-FLAG expression. β-actin served as a loading control. (C) Transfected BJAB cells were analyzed by flow cytometry 24 hrs post-transfection using CD83 upregulation as a marker NF-κB activation. (D) Confocal IF microscopy of JPM50.6 T cells transfected with WT or mutant CARD11-FLAG constructs and stained with as in (A). (E) Transfected JPM50.6 cell lysates were separated by SDS-PAGE and immunoblotted for CARD11-FLAG expression. β-actin served as a loading control. (F) NF-κB-driven GFP reporter activity was measured in JPM50.6 transfectants by flow cytometry 24 hrs post-transfection. All data are representative of >3 independent experiments.

Fig. 4.

WT CARD11 does not form mCADS after AgR stimulation. (A) JPM50.6 T cells were transfected with empty vector (EV), WT, or GOF CARD11-FLAG mutants. After 24 hrs, cells were left unstimulated (left panel) or stimulated with anti-CD3/CD28 agonistic Abs for 4 hrs. NF-κB-driven GFP reporter activity was measured by flow cytometry. (B) Confocal IF microscopy of JPM50.6 T cells transfected and stimulated as in (A). At 1, 4, and 24 hr post-stimulation, cells were stained using anti-FLAG, anti-MALT1, and anti-ph-IKKαAbs. Merge images include DAPI staining. Scale bar = 5 μm. (C) The % of transfected cells (FLAG⁺) with visible aggregates was scored in multiple fields (>100 cells/construct/experiment). Average ± SD was calculated from 2 independent scorers. Data are representative of 3 independent experiments.

Fig. 5.

Specific LOF mutations disrupt mCADS formation and NF-κB signaling. (A) Confocal IF microscopy of JPM50.6 T cells transfected with E134G CARD11-FLAG constructs ± additional LOF mutations (L815P, ΔGUK, R35A) and stained 24 hrs later using anti-FLAG, anti-MALT1, and anti-ph-IKKα/β Abs. Merge images include DAPI staining. Scale bar = 5 μm. (B) Immunoblotting for CARD11-FLAG expression in lysates made from transfected JPM50.6 cells in (A). β-actin served as a loading control. (C) NF-κB-driven GFP reporter activity was measured in JPM50.6 transfectants in (A) by flow cytometry 24 hrs post-transfection. (D) Confocal IF microscopy of BJAB B cells transfected and stained as in (A). (E) Immunoblotting for CARD11-FLAG expression in lysates made from transfected BJAB cells in (A). β-actin served as a loading control. (F) NF-κB-dependent induction of CD83 expression was measured in BJAB transfectants in (A) by flow cytometry 24 hrs post-transfection. Data are representative of >3 independent experiments.

Fig. 6.

NEMO is dispensable for IKKα/β phosphorylation at mCADS, but required for downstream NF-κB induction. (A) Confocal IF microscopy of JPM50.6 T cells transfected with WT, E134G or G123D CARD11-FLAG constructs stained using anti-FLAG, anti-BCL-10, and anti-NEMO (IKKγ) Abs at 24 hrs post-transfection. (B) Confocal IF microscopy of NEMO-deficient JM4.5.2 T cells transfected with WT, E134G or G123D CARD11-FLAG constructs and stained using anti-FLAG, anti-MALT1, and anti-ph-IKKα/β Abs at 24 hrs post-transfection. Merge images include DAPI staining. Scale bars = 5 μm. (C) NF-κB activity was measured in cell JPM50.6 and JM4.5.2. transfectants from (A) and (B) using a dual luciferase assay. (D) Immunoblotting of lysates prepared from transfected cells for CARD11-FLAG and NEMO expression. β-actin served as a loading control. Data are representative of >3 independent experiments.

Fig. 7.

Endogenous mCADS formation in DLBCL lines carrying GOF CARD11 mutations. (A) Confocal IF microscopy of DLBCL cell lines stained with anti-CARD11 and anti-MALT1 Abs. Lines marked in red carry GOF CARD11 mutations. Merge images include DAPI staining. Scale bar = 5 μm. (B) The % of cells with mCADS (gray bars) and/or visible aggregates (black bars) was scored in multiple fields (>100 cells/DLBCL line). Average ± SD was calculated from 2 independent scorers. (C) Immunoblot analysis for relative CARD11 protein expression in lysates prepared from DLBCL lines. All data are representative of >3 independent experiments.