Fig 2: Model illustrating two steps impacted by dominant-negative CARD11 mutations in TCR signaling(A) CARD11 exists as an oligomer of unknown stoichiometry of at least 2 subunits in a closed conformation in the absence of antigen receptor signaling. Upon TCR signaling all wild-type (WT) CARD11 oligomers become active and proceed through the Opening, Cofactor Association, and Enzyme Activation Steps leading to the transient activation of NF-?B.(B) Mixed wild-type:mutant oligomers containing a strong dominant-negative mutant cannot proceed through the Opening Step, causing the oligomer to stay in the closed conformation following TCR signaling.(C) Oligomers consisting of the constitutively open reQM variant with all four REs disabled constitutively proceed through the Cofactor Association and Enzyme Activation Steps leading to constitutive activation of NF-?B.(D) Mixed wild-type:mutant reQM oligomers containing E57D or R72Q mutations fail to recruit MALT1 and optimally recruit HOIP and Bcl10 at the Cofactor Association Step.

Fig 3: CARD and LATCH domain mutants disrupt the Opening Step in the CARD11 signaling cycle(A) NF-?B luciferase reporter activity in CARD11-KO Jurkat T cells stably expressing myc-WT-FLAGx2 or myc-mutant-FLAGx2 CARD11 variants via retroviral transduction after stimulation with anti-CD3/anti-CD28 for 4 h. Mean fold activation with SD from one of two replicate experiments is represented. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the values of the CARD11 mutants obtained under stimulated conditions as compared with that observed for wild-type CARD11 under stimulated conditions: N25Y, p = 0.0252; E27K, p = 0.0405; C28F, p = 0.0399; R30W, p = 0.0388; K41M, p = 0.0399; Q55R, p = 0.0396; E57D, p = 0.0322 R72Q, p = 0.0406; K83M, p = 0.0407; and H129D, p = 0.0245.(B) CARD11-KO Jurkat T cells reconstituted with myc-WT-FLAGx2 or myc-mutant-FLAGx2 were stimulated with PMA/ionomycin for 30 min, followed by the denatured co-immunoprecipitation assay with rabbit anti-Bcl10 and Western blotting for linear ubiquitin and Bcl10.(C) Reconstituted cells were stimulated with anti-CD3/anti-CD28 cross-linking for 30 min followed by Western blotting for CYLD, HOIL-1, and the loading controls IKKa and FLAG. The CYLD and HOIL-1 C-terminal cleavage products (CYLDCter and HOIL-1Cter) are indicated by arrows.(D) Reconstituted cells were stimulated with PMA/ionomycin for 30 min, followed by the endogenous co-immunoprecipitation assay with rabbit anti-FLAG and Western blotting for HOIP, MALT1, Bcl10, and FLAG.(E and F) Constructs expressing CARD11 mutants in the constitutively open and active myc-tagged RE Quadruple Mutant (myc-reQM) context were transiently transfected in HEK293T cells with (E) 200 ng FLAG-Bcl10 or (F) 100 ng FLAG-HOIP for co-immunoprecipitation assays. At 40–41 h, cells were lysed and co-immunoprecipitated with rabbit anti-FLAG followed by Western blotting with anti-myc and anti-FLAG. The following concentrations of DNA were used to achieve roughly equivalent protein expression for the FLAG-Bcl10 co-immunoprecipitation: 400 ng reQM, 575 ng N25Y-reQM, 600 ng E27K-reQM, 600 ng C28F-reQM, 675 ng R30W-reQM, 575 ng K41M-reQM, 650 ng Q55R-reQM, 400 ng E57D-reQM, 700 ng R72Q-reQM, 400 ng K83M-reQM, and 450 ng H129D-reQM. The following concentrations of DNA were adjusted to achieve roughly equivalent protein levels for the FLAG-HOIP co-immunoprecipitation: 500 ng reQM in the left panel and 400 ng reQM in the right panel, 500 ng of each reQM mutant.(G) CARD11-KO Jurkat T cells stably reconstituted with myc-WT-FLAGx2 or myc-mutant-FLAGx2 were stimulated with anti-CD3/anti-CD28 for 30 min followed by Western blotting to detect p-JNK and JNK2.(H) Reconstituted CARD11-KO Jurkat T cells were stimulated with anti-CD3/anti-CD28 for 24 h and IL-2 production was measured by ELISA. Data are represented as mean pg/mL of IL-2 with SD for one representative experiment performed with three technical replicates. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the values of the CARD11 mutants obtained under stimulated conditions as compared with that observed for wild-type CARD11 under stimulated conditions: N25Y, p = 0.0027; E27K, p = 0.0024; C28F, p = 0.0024; R30W, p = 0.0024; K41M, p = 0.0024; Q55R, p = 0.0024; E57D, p = 0.0024 R72Q, p = 0.0024; K83M, p = 0.0024; and H129D, p = 0.0016.

Fig 4: Dominant-negative variants perturb the ability of the mixed wild-type:mutant CARD11 oligomer to proceed through the Opening Step of the CARD11 signaling cycle(A and B) WT Jurkat T cells that were stably reconstituted with myc-WT-FLAGx2 or myc-mutant-FLAGx2 were stimulated with PMA/ionomycin for 30 min, followed by the endogenous co-immunoprecipitation with (A) anti-Bcl10 and Western blotting for FLAG and Bcl10 or (B) anti-FLAG and Western blotting for HOIP and FLAG. The quantitated relative amounts of FLAG-tagged CARD11 variants in the IP samples from stimulated cells normalized to that in the input lysate for the endogenous Bcl10 IP are the following: WT 1.00, N25Y 0.14, R30W ND, R72Q ND, K83M 0.11, H129D 0.18, and E27K ND, where ND is not detected. The quantitated relative amounts of HOIP proteins in the IP samples from stimulated cells normalized to that in the input lysate for the endogenous FLAG IP are the following: WT 1.00, R30W 0.05, R72Q ND, K83M 0.25, H129D 0.83, and E27K ND, where ND is not detected.(C and D) MALT1 protease activation at 30 min was assessed by Western blot analysis of input samples from the endogenous anti-Bcl10 co-immunoprecipitation for CARD11 and (C) HOIL-1 or (D) CYLD. The C-terminal cleavage products (HOIL-1Cter and CYLDCter) are indicated by arrows. The quantitated relative amounts of HOIL-1Cter in stimulated cells normalized to the CARD11 control are the following: WT 1.00, N25Y 0.61, R30W 0.21, R72Q 0.16, K83M 0.45, H129D 0.81, and E27K 0.17. The quantitated relative amounts of CYLDCter in stimulated cells normalized to the CARD11 control are the following: WT 1.00, R30W 0.30, R72Q 0.42, K83M 0.86, H129D 1.63. E27K 0.65, and N25Y 1.31.(E) Stably reconstituted WT Jurkat T cells were stimulated with anti-CD3/anti-CD28 for 24 h and IL-2 production was measured by ELISA. Results are the average of replicate experiments and are represented as mean pg/mL IL-2 with SD. A two-tailed unpaired Student's t test with Welch's correction resulted in p values of p < 0.0001 for the values of all CARD11 mutants obtained under stimulated conditions as compared with that observed for WT CARD11 under stimulated conditions.(F) Stably reconstituted WT Jurkat T cells were stimulated with anti-CD3/anti-CD28 for 30 min followed by Western blotting for p-JNK, JNK2, and CARD11. The quantitated relative amounts of p-JNK in stimulated cells normalized to the JNK2 control are the following: WT 1.00, R30W 0.47, R72Q 0.05, K83M 0.40, H129D 1.07, E27K 1.24, and N25Y 0.62.

Fig 5: RBF197 and RBF208 decreases overall translation and eIF4A1 dependent pathway proteins in double-hit lymphoma. A SUnSET assay was performed by exposing cells to puromycin (1 µg/mL), post compound treatments for 30 min, and subsequently lysed. Representative immunoblots were probed with anti-puromycin, anti-eIF4A, and anti-eIF4E. GAPDH was probed as an internal loading control. B Relative fold change in expression levels with on treatment of RBF197 or RBF208 at 1 and 3 µM concentrations. C Representative immunoblots of cMYC, MCL1, PARP1, BCL2, CDK7, NRF2, Cyclin E and CARD11 on treatment with RBF197 and RBF208 at 1 and 3 µM concentrations respectively. Vinculin was used as a loading control. D Relative fold change in protein levels of the above-mentioned proteins with on treatment of RBF197 and RBF208, respectively. E Heat map of translation efficiency values for RBF197 and RBF208 in RC cell line. F Effect of RBF197 and RBF208 on DLBCL colony formation. Representative image of the colony formation in RC (malignant) and GMO13604 (non-malignant) cells. G The total number of colonies grown in RC, and GMO13604 cells upon treatment with 1 and 3 µM of RBF197 and RBF 208, respectively. All treated groups were normalized with DMSO controls and expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s correction analysis. ap < 0.05; cp < 0.001, dp < 0.0001 vs DMSO control groups, ap < 0.05, ßp < 0.001, ¥p < 0.0001 vs 3 µM treatment groups