Journal: Blood cancer journal

Article Title: Bruton's tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax.

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: Fig. 1 Gene expression impact of BCL10 mutations. A Heatmap of differentially expressed genes in RIVA cells containing vector or BCL10 mutants, (FC ≥1.5x up or down). B Venn diagram of the number of differentially expressed genes in BCL10 mutants compared to vector. C Top 10 upregulated (adjusted p value ≤0.001) Jensen compartment complexes using genes upregulated with a FC ≥2. D The top overlapping Hallmark gene set pathways upregulated (FDR q-value ≤0.25) in BCL10 mutants. E Western blot of RIVA and HBL1 cells induced with doxycycline for 24 h and probed as indicated. F TNFα ELISA assay on cell supernatant from RIVA, HBL1, and U2932 cells containing vector or BCL10 mutants. G Boxplot of gene expression levels of TNFRSF13B and USP2 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Gene Expression, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Blood cancer journal

Article Title: Bruton's tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax.

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: Fig. 3 BCL10 mutants mediate lymphomagenesis through activation of key transcription factors. A Genes that were upregulated (FC ≥2) in both mutants were analyzed for enrichment in regulation by transcription factors from the ENCODE project through the online transcription factor analysis tool ChIP-X Enrichment Analysis Version 3 (ChEA3). Sixteen transcription factors were significantly upregulated in BCL10 mutants (FDR ≤0.05). B Schematic of transcription factors identified to be upregulated in (A). C Boxplot of gene expression levels of BATF and IRF4 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases. D Western blot of RIVA and HBL1 cells induced with doxycycline for 24 h and probed as indicated. E Western blot of RIVA and U2932 cells induced with doxycycline for 24 h and probed as indicated. F ELISA assays in RIVA AND U2932 cell supernatant probing for CXCL10 (RIVA - S136X: p = 0.0046, R58Q: p = 0.0003, U2932- S136X: p = 0.0003, R58Q: p = 0.0028).

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Activation Assay, Gene Expression, Mutagenesis, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Blood cancer journal

Article Title: Bruton's tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax.

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: Fig. 4 BCL10 mutants drive resistance to multiple drug classes. A Dose response viability assays of RIVA cells treated with pirtobrutinib (S136X: p = 0.033, R58Q: p = 0.019), acalabrutinib (S136X: p = 0.07, R58Q: p = 0.07), and ibrutinib (S136X: p = 0.023, R58Q: p = < 0.0001). B Epigenetic Library (TargetMol) on doxycycline induced RIVA cells. Hits are compounds that significantly inhibited cell viability and compounds that had an increase or decrease in viability two standard deviations from the mean of the difference between BCL10 S136X and vector after 72 h of 10 μM drug treatment. Targets in red highlight mutant-driven resistance, mutant-sensitive targets are in blue, and compounds that sensitized both vector and BCL10 S136X are in purple. C Schematic of hits from 4B. D Dose response viability assays of RIVA cells treated with selcidemstat (p=ns), tovorafenib (p = ns), ulixertinib (S136X: p = 0.0125, R58Q: p = 0.0103), JNKi (p = ns), duvelisib (S136X: p = 0.126, R58Q: p = 0.0264), idelalisib (S136X: p = ns, R58Q: p = 0.004), capivasertib (S136X: p = 0.0002, R58Q: p = 0.005), venetoclax (S136X: p = 0.008, R58Q: p = 0. 0.046), and AZD1208 (PIMi) (S136X: p = 0.019, R58Q: p = 0.040).

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Plasmid Preparation, Mutagenesis

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet: CARD and LATCH domain mutants disrupt the Opening Step in the CARD11 signaling cycle (A) NF-κB 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 IKKα and FLAG. The CYLD and HOIL-1 C-terminal cleavage products (CYLD Cter and HOIL-1 Cter ) 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.

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Luciferase, Activity Assay, Stable Transfection, Expressing, Mutagenesis, Retroviral, Transduction, Activation Assay, Two Tailed Test, Co-Immunoprecipitation Assay, Western Blot, Ubiquitin Proteomics, Construct, Transfection, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet: Effect of CARD and LATCH LOF mutations on HOIP and Bcl10 binding under inducible and constitutive conditions

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Binding Assay

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet: 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-1 Cter and CYLD Cter ) are indicated by arrows. The quantitated relative amounts of HOIL-1 Cter 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 CYLD Cter 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.

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Dominant Negative Mutation, Mutagenesis, Stable Transfection, Immunoprecipitation, Western Blot, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet: Dominant-negative CARD11 variants in the open, reQM conformation perturb the ability of the all-mutant reQM CARD11 oligomer to recruit MALT1 (A) NF-κB luciferase reporter activity in CARD11-KO Jurkat T cells stably transduced with myc-WT CARD11-FLAGx2 or myc-reQM CARD11-FLAGx2 with or without CARD11 LOF mutations. Mean fold activation with SD is depicted relative to Jurkat T cells reconstituted with WT CARD11. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the reQM CARD11 mutants as compared with that observed for reQM CARD11: E57D-reQM, p = 0.0016; and R72Q-reQM, p = 0.0013. (B) Reconstituted cells were harvested for denatured co-immunoprecipitation with rabbit anti-Bcl10 and Western blotting for linear ubiquitin, Bcl10, and FLAG. (C and D) MALT1 protease activation was assessed by Western blot analysis for HOIL-1, CYLD, and FLAG. The HOIL-1 and CYLD C-terminal cleavage products (HOIL-1 Cter and CYLD Cter ) are indicated by arrows. (E–G) Cells were harvested for the endogenous co-immunoprecipitation with mouse anti-Bcl10 (E) or rabbit anti-FLAG (F and G) and lysate inputs and immunoprepcipiates were probed by Western blotting with the indicated antibodies. (H) The myc-reQM CARD11 variants were transiently transfected in HEK293T cells with 500 ng FLAG-MALT1 and 300 ng untagged Bcl10 as indicated for co-immunoprecipitation assays. At 40–41 h, cells were lysed and co-immunoprecipitated with rabbit anti-FLAG followed by Western blotting for myc, FLAG, and Bcl10. The following concentrations of DNA were used to achieve roughly equivalent protein expression for the FLAG-MALT1 co-immunoprecipitation: 400 ng reQM, 375 ng E57D-reQM, and 700 ng R72Q-reQM.

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Dominant Negative Mutation, Mutagenesis, Luciferase, Activity Assay, Stable Transfection, Transduction, Activation Assay, Two Tailed Test, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Transfection, Expressing

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet: Dominant-negative variants in the open, reQM conformation perturb the ability of the mixed wild-type:reQM mutant CARD11 oligomers to inducibly recruit cofactors (A) NF-κB luciferase reporter activity in WT Jurkat T cells transiently transfected with 100 ng of myc-WT CARD11 or 100ng myc-reQM CARD11 with or without dominant-negative mutations. Mean fold activation with SD is depicted relative to unstimulated wild-type Jurkat T cells. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for stimulated cells expressing reQM variants as compared with that observed for WT CARD11: E57D-reQM, p = 0.0002; R72Q-reQM, p < 0.0001; and reQM, p = 0.0145. (B) NF-κB luciferase reporter activity in WT Jurkat T cells stably reconstituted with myc-WT CARD11-FLAGx2, myc-E57D-reQM-FLAGx2, or myc-R72Q-reQM CARD11-FLAGx2. Mean fold activation with SD is depicted relative to unstimulated Jurkat T cells reconstituted with an empty vector. A two-tailed unpaired Student's t test with Welch's correction resulted in the following p values for the values of cells reconstituted with CARD11 variants as compared with that observed for WT Jurkat cells without CARD11 reconstitution under stimulated conditions: WT, p = 0.0043, E57D-reQM, p = 0.0008; and R72Q-reQM, p = 0.0001. (C) Wild-type Jurkat T cells that were stably reconstituted with myc-WT-FLAGx2 or myc-reQM-FLAGx2 variants were harvested for the denatured co-immunoprecipitation with rabbit anti-Bcl10 and Western blotting for linear ubiquitin, Bcl10 FLAG, and CARD11. (D) MALT1 protease activation was assessed by Western blot analysis for HOIL-1, CYLD, FLAG, and CARD11. The HOIL-1 and CYLD C-terminal cleavage products (HOIL-1 Cter and CYLD Cter ) are indicated by the arrows. (E–G) Cells were harvested for the endogenous co-immunoprecipitation with rabbit anti-FLAG (E and F) or mouse anti-Bcl10 (G) and lysate inputs and immunoprecipitates were probed by Western blotting for the indicated antibodies. (H) WT Jurkat T cells stably reconstituted with WT or reQM mutants were stimulated with anti-CD3/anti-CD28 for 24 h and IL-2 production was measured by ELISA. Results are the representative of one replicate experiment 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 the following p values for cells reconstituted with CARD11 variants as compared with that observed for WT Jurkat cells without CARD11 reconstitution under stimulated conditions: WT, p = 0.0134, E57D-reQM, p = 0.0009; and R72Q-reQM, p = 0.0007.

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Dominant Negative Mutation, Mutagenesis, Luciferase, Activity Assay, Transfection, Activation Assay, Two Tailed Test, Expressing, Stable Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet: 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-κB. (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-κB. (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.

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Dominant Negative Mutation, Activation Assay, Mutagenesis, Variant Assay

Journal: iScience

Article Title: Mechanistic impact of oligomer poisoning by dominant-negative CARD11 variants

doi: 10.1016/j.isci.2022.103810

Figure Lengend Snippet:

Article Snippet: The remaining sample was further diluted to 0.1% SDS by adding 10 mL of DIPLB and 10 μg of rabbit anti-Bcl10 antibody (Bethyl Laboratories, A303–579A) and the samples incubated with rotation at 4 °C for 4 h. The samples were then incubated with nProtein A Sepharose at 30 μL bed volume overnight with rotation at 4°C, washed 5 times for 10 min with rotation at 4 °C, and harvested and analyzed as described above for the endogenous co-IP assay.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, Transfection, Enzyme-linked Immunosorbent Assay, Reporter Gene Assay, Luciferase, Plasmid Preparation, Control, Sequencing, Mutagenesis, Software, Immunoprecipitation, Lysis