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. 2016 Aug 11;35(32):4269-81.
doi: 10.1038/onc.2015.493. Epub 2016 Jan 18.

Oncogenic CARMA1 couples NF-κB and β-catenin signaling in diffuse large B-cell lymphomas

M K Bognar  1 , M Vincendeau  1 , T Erdmann  2   3 , T Seeholzer  1 , M Grau  2   4 , J R Linnemann  5 , J Ruland  6   7 , C H Scheel  5 , P Lenz  4 , G Ott  8 , G Lenz  2   3 , S M Hauck  9 , D Krappmann  1
Affiliations

Oncogenic CARMA1 couples NF-κB and β-catenin signaling in diffuse large B-cell lymphomas

M K Bognar et al. Oncogene. .

Abstract

Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis.

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Figures

Figure 1
Figure 1
Proteomic analysis defines a functional interaction of oncogenic CARMA1 with the β-catenin destruction complex. (a) Schematic depiction of CARMA1 domains and generated ABC (blue) or GCB (orange) DLBCL-derived mutations of the coiled-coil (CC) domain. CARD: caspase recruitment domain; SH3: SRC homology 3; GUK: guanylate kinase; FS: Flag-Strep-Tag. (b) Oncogenic CARMA1 promotes constitutive NF-κB activity. NF-κB DNA binding was assessed in EMSA of unstimulated or 60 min P/I stimulated transduced BJAB cells. Oct1 EMSA was performed for control. (c) Oncogenic CARMA1 is constitutively recruited to BCL10-MALT1. CBM complex formation was assessed in stimulated (15 min P/I) or unstimulated transduced BJAB cells and analyzed in western blot after BCL10 IP. (d) LC-MS/MS identifies CARMA1 interaction partners consisting of proteins surrounding the β-catenin destruction complex. Three independent Strep-precipitation experiments were conducted in CARMA1 WT, WT stimulated (30 min P/I) and four different oncogenic CARMA1 mutant-transduced BJAB. The ratio of mean peptide abundances of each sample related to the mock precipitation control was calculated and depicted with +=2–10, ++=10–50 and +++=>50. Infinite stands for incalculable values, because no peptides were detected in mock control. † marks proteins that were identified by one single peptide. (e) Interaction database analysis of identified CARMA1 interaction partners emerges a network of proteins surrounding the β-catenin destruction complex. Known and predicted protein interactions using string database (string-db.org) was calculated based on experimental data with a medium confidence score. Only significant interactions (P<0.05) were used for string analyses. Published CK1α-CARMA1 interaction was integrated manually with dashed line. (f) Oncogenic CARMA1 interacts with proteins of the β-catenin destruction complex. CARMA1 StrepTactin pull-down in transduced BJAB cells and associated proteins were analyzed in western blot. (g) Proteins of the β-catenin destruction complex are associated to the CBM complex. The interaction of CK1α and β-catenin to the CBM complex was assessed in transduced BJAB cells and analyzed in western blot after BCL10 IP.
Figure 2
Figure 2
Oncogenic CARMA1 expression enhances β-catenin protein stability in BJAB cells. (a) β-catenin protein levels are enhanced in oncogenic CARMA1-expressing BJAB cells. Cell lysates were analyzed in western blot. Numbers indicate fold change of β-catenin normalized to CK1α in relation to mock. (b) β-catenin mRNA is not regulated in BJAB transduced with oncogenic CARMA1. Relative β-catenin mRNA level of WT and L225LI-transduced BJAB were determined in qRT–PCR (mean±s.e.m.; n=5). (c) Active β-catenin levels are enhanced in CARMA1 L225LI-transduced BJAB. Applied total β-catenin (βCat) amounts were adjusted after β-catenin IP and stained for unphosphorylated β-catenin in western blot. (d) β-catenin is stabilized in BJAB transduced with oncogenic CARMA1. CARMA1 WT and L225LI-transduced BJAB were treated with cycloheximid (CHX25 μg/ml) for indicated time points. β-catenin protein levels were assessed in western blot and normalized to β-Actin. Graph shows relative β-catenin levels normalized to β-Actin after CHX-treatment compared with untreated (0 min; n=2).
Figure 3
Figure 3
Direct interaction of CK1α to oncogenic CARMA1 results in the stabilization of β-catenin. (a) Reciprocal IP confirms the interaction of oncogenic CARMA1 L225LI to the β-catenin destruction complex. Endogenous IPs against CK1α, β-catenin and GSK3β in BJAB transduced with mock, CARMA1 WT or L225LI were analyzed in comparison with StrepTactin pull-down in western blot. (b) CK1α co-localizes with oncogenic CARMA1 F123I/K208M in the cytoplasm as shown by indirect immunofluorescence of endogenous CK1α (red, arrow) and overexpressed StrepII-CARMA1 (green, arrow). Cell nuclei were stained with Hoechst33342. CARMA1-CK1α co-aggregates are marked by arrows. (c) CK1α acts as a bridging factor of oncogenic CARMA1 and the β-catenin destruction complex. BJAB CARMA1 L244P cells were transduced with a DOX-inducible shRNA system containing non silencing (ns) or shRNA against β-catenin (shβCat) or CK1α (shCK1α). Following CARMA1 StrepTactin pull-down, association of the β-catenin destruction complex was monitored in western blot. Protein depletion was analyzed from cell lysates.
Figure 4
Figure 4
CK1α-GSK3β-β-catenin recruitment to CARMA1 is independent of BCL10 and NF-κB. (a) Schematic depiction of CARMA1 domains and generated mutants. CARMA1 R35A represents a BCL10-binding mutant, whereas CARMA1 Δlinker (Δ441-668), as well as GCB DLBCL-derived mutation L225LI in the CC domain activate NF-κB constitutively. (b) CARMA1 L225LI and Δlinker activate NF-κB constitutively. CARMA1 mutant-transduced BJAB cells were stimulated for indicated time points. NF-κB DNA binding was monitored in EMSA. Oct1 EMSA was performed for control. Numbers indicate fold change of β-catenin normalized to β-Actin in relation to mock. (c) CARMA1 Δlinker does not interact with the β-catenin destruction complex and does not induce β-catenin stabilization. Complex formation was monitored in western blot after StrepTactin pull-down of CARMA1 mutants in transduced BJAB cells. (d) CARMA1 R35A impairs inducible and constitutive BCL10 recruitment. Transduced BJAB cells were stimulated with P/I (30 min) as indicated. CARMA1 recruitment to BCL10 was analyzed in western blot after BCL10 IP. (e) CARMA1 R35A impairs inducible and constitutive NF-κB activation. Transduced BJAB cells were stimulated for indicated time points and NF-κB DNA binding was assessed in EMSA. Oct1 EMSA was performed for control. (f) Oncogenic CARMA1 associates with the β-catenin destruction complex independently of BCL10 binding. BJAB transduced with different CARMA1 constructs were stimulated with P/I (30 min) as indicated. Following CARMA1 StrepTactin pull-down, a subsequent analysis of binding partners was monitored in western blot. Numbers indicate fold change of β-catenin normalized to GSK3β in relation to mock.
Figure 5
Figure 5
High expression of β-catenin in ABC DLBCL is not connected to tumor cell viability. (a, b) High expression of β-catenin is often found in ABC DLBCL cell lines. β-catenin protein expression was analyzed in western blot (a) or in indirect β-catenin immunofluorescence staining (b). Staining was controlled by omission of primary β-catenin antibody (left). (c, d) CARMA1 interacts with the β-catenin destruction complex in ABC DLBCL cell lines. Endogenous IPs against CK1α (c) or β-catenin, GSK3β and BCL10 (d) in DLBCL cell lines were analyzed by western blot. (e) β-catenin acts as a general survival factor in BJAB. BJAB cells were transduced with a DOX-inducible shRNA system containing ns or shβCat. β-catenin knockdown was verified in western blot (right). Number of viable cells by trypan blue exclusion was determined 4 days after seeding the cells at 1 × 105 cells/ml in the presence of DOX (mean±s.e.m.; n=3). (f) β-catenin is not driving survival in HBL1 cells. BJAB and HBL1 were transduced with a DOX-inducible shβCat or shCARMA1. Knockdown was confirmed by western blot. Cell viability was determined in cell counts with trypan blue exclusion after 3 and 6 days of culture (mean±s.e.m.; n=3). (g) IHC of β-catenin in DLBCL as exemplified in two non-GCB DLBCL sections. (h) DLBCL were grouped into GCB and non-GCB subtypes using immunophenotyping according to the Hans algorithm. Positive stainings for β-catenin was over-represented in non-GCB DLBCL (one-tailed Fishers' exact test P=0.042).
Figure 6
Figure 6
β-catenin stabilization by oncogenic CARMA1 augments TCF/LEF activation. (a) WNT gene signatures are not significantly regulated in transduced BJAB cells. WNT gene signatures by microarray expression profiling were compared in R35A, R35A/L225LI, WT and mock transduced BJAB cells in reference to L225LI-transduced cells. P-values are based on Student's t-tests against untransduced BJAB and error bars depict s.e.m.s. In gene set enrichment analyses, enrichment score (ES) was calculated to 0.33, P=0.627 via permutation test. (b) Induced β-catenin nuclear translocation is enhanced in BJAB CARMA1 L225LI. Transduced BJAB cells were stimulated with LiCl overnight. β-catenin localization was monitored in confocal immunofluorescence microscopy and the percentage of cells with nuclear β-catenin was quantified (mean±s.e.m.; n=3). (c) Induced TCF/LEF reporter activity is enhanced in BJAB-expressing oncogenic CARMA1. BJAB cells expressing different CARMA1 mutants were transduced with a TCF/LEF luciferase reporter. Cells were stimulated with LiCl overnight as indicated and relative TCF/LEF-regulated luciferase activity was determined (mean±s.e.m.; n=5). Protein levels were analyzed by western blot. (d) TCF/LEF reporter activity depends on β-catenin. CARMA1 WT or L225LI-expressing BJAB cells were transduced with a DOX-inducible shRNA system targeting β-catenin. Cells were treated with LiCl overnight. Relative TCF/LEF-regulated luciferase activity was determined (mean±s.e.m.; n=3).
Figure 7
Figure 7
β-catenin influences the expression of distinct NF-κB target genes. (a) β-catenin interacts with p65 in BJAB-expressing oncogenic CARMA1. β-catenin–p65 association in transduced BJAB cells was assessed after p65 IP and western blot analyses. (b, c) Expression of distinct NF-κB target genes is regulated by β-catenin. Left: mRNA levels of NF-κB target genes in untransduced BJAB, BJAB-expressing CARMA1 WT, L225LI or R35A/L225LI were measured in qRT–PCR. Values were normalized to CARMA1 WT. Right: BJAB cells expressing CARMA1 WT or L225LI were transduced with a DOX-inducible shRNA system containing ns or shβCat. All values were related to RPII (mean±s.e.m.; n=4). (d) IL-10 and CCL3 secretion is partially influenced by β-catenin. Left: IL-10 and CCL3 secretion of untransduced BJAB, BJAB-expressing CARMA1 WT, L225LI or R35A/L225LI was determined in ELISA. Right: BJAB-expressing CARMA1 WT or L225LI were transduced with a DOX-inducible shRNA system containing ns or shβCat. Secreted IL-10 and CCL3 was measured in supernatants using ELISA (mean±s.e.m.; n=3). (e) CCL3 expression is inhibited by β-catenin in CARMA1 L244P, S243P or F123I/K208M mutant BJAB. BJAB cells were transduced with a DOX-inducible shRNA system containing ns or shβCat. Left: CCL3 mRNA expression was determined in qRT–PCR (normalized to RPII) and values were related to CARMA1 WT (mean±s.e.m.; n=3). Right: secreted CCL3 was measured in supernatants using ELISA (mean±s.e.m.; n=3). (f) Model for regulation of NF-κB and β-catenin cross-talk through CARMA1. Oncogenic mutations (*) or chronic BCR signaling induce parallel recruitment of BCL10 and CK1α to CARMA1. Whereas the classical CARMA1-BCL10-MALT1 (CBM) complex activates IKK/NF-κB signaling, CK1α recruits main components of the β-catenin destruction complex to CARMA1, which leads to β-catenin stabilization. Even though enhanced β-catenin expression alone is not sufficient to induce WNT signature genes, β-catenin influences expression of distinct NF-κB target genes. Whereas most genes are not influenced by β-catenin, full induction of some target genes (CD40, IL-10) requires β-catenin, whereas the expression of other genes is decreased by β-catenin (CCL3).
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