Phosphorylation of serine-893 in CARD11 suppresses the formation and activity of the CARD11-BCL10-MALT1 complex in T and B cells.

CARD11 acts as a gatekeeper for adaptive immune responses after T cell or B cell antigen receptor (TCR/BCR) ligation on lymphocytes. PKCθ/ß-catalyzed phosphorylation of CARD11 promotes the assembly of the CARD11-BCL10-MALT1 (CBM) complex and lymphocyte activation. Here, we demonstrated that PKCθ/ß-dependent CARD11 phosphorylation also suppressed CARD11 functions in T or B cells. Through mass spectrometry-based proteomics analysis, we identified multiple constitutive and inducible CARD11 phosphorylation sites in T cells. We demonstrated that a single TCR- or BCR-inducible phosphorylation on Ser893 in the carboxyl terminus of CARD11 prevented the activation of the transcription factor NF-κB, the kinase JNK, and the protease MALT1. Moreover, CARD11 Ser893 phosphorylation sensitized BCR-addicted lymphoma cells to toxicity induced by Bruton's tyrosine kinase (BTK) inhibitors. Phosphorylation of Ser893 in CARD11 by PKCθ controlled the strength of CARD11 scaffolding by impairing the formation of the CBM complex. Thus, PKCθ simultaneously catalyzes both stimulatory and inhibitory CARD11 phosphorylation events, which shape the strength of CARD11 signaling in lymphocytes.

MALT1 Phosphorylation Controls Activation of T Lymphocytes and Survival of ABC-DLBCL Tumor Cells.

The CARMA1/CARD11-BCL10-MALT1 (CBM) complex bridges T and B cell antigen receptor (TCR/BCR) ligation to MALT1 protease activation and canonical nuclear factor κB (NF-κB) signaling. Using unbiased mass spectrometry, we discover multiple serine phosphorylation sites in the MALT1 C terminus after T cell activation. Phospho-specific antibodies reveal that CBM-associated MALT1 is transiently hyper-phosphorylated upon TCR/CD28 co-stimulation. We identify a dual role for CK1α as a kinase that is essential for CBM signalosome assembly as well as MALT1 phosphorylation. Although MALT1 phosphorylation is largely dispensable for protease activity, it fosters canonical NF-κB signaling in Jurkat and murine CD4 T cells. Moreover, constitutive MALT1 phosphorylation promotes survival of activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) cells addicted to chronic BCR signaling. Thus, MALT1 phosphorylation triggers optimal NF-κB activation in lymphocytes and survival of lymphoma cells.

BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation.

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.

MALT1 activation by TRAF6 needs neither BCL10 nor CARD11.

The MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) paracaspase couples antigen receptors on lymphocytes to downstream signaling events. Activation of MALT1 is known to involve stimulus-dependent CBM complex formation, that is, the recruitment of BCL10-bound MALT1 to a CARD-Coiled Coil protein. Beyond this canonical, CBM-dependent mechanism of MALT1 activation, recent studies suggest that MALT1 protease activity may be triggered by alternative mechanisms. For instance, the E3-ligase TRAF6 can activate MALT1 proteolytic function and induce MALT1 auto-cleavage. However, the interplay between CBM and TRAF6 with regard to MALT1 activation has remained incompletely elucidated. Here, by generating CRISPR/Cas9-derived knock-out Jurkat T-cells, we show that TRAF6 was dispensable for CARD11/BCL10-dependent MALT1 activation upon T-cell stimulation. However, ectopically-expressed TRAF6 could induce MALT1 activity in Jurkat T-cells devoid of either CARD11 or BCL10. These data provide unequivocal evidence that TRAF6-mediated MALT1 activation does not require the upstream scaffold CARD11 or the interaction between MALT1 and BCL10. Thus, TRAF6 may be part of a previously unidentified non-canonical pathway that triggers MALT1 protease activity independently of canonical CBM signalosomes.

S6 Kinase is essential for MYC-dependent rDNA transcription in Drosophila.

Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, we performed a transient expression screen for known oncogenic signalling pathways in Drosophila. Strikingly, from the broad range of pathways tested, we found that ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, we demonstrated that one of the mechanism(s) by which MYC and S6K cooperate is through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

The nucleolus as a fundamental regulator of the p53 response and a new target for cancer therapy.

BACKGROUND: Recent studies have highlighted the fundamental role that key oncogenes such as MYC, RAS and PI3K occupy in driving RNA Polymerase I transcription in the nucleolus. In addition to maintaining essential levels of protein synthesis, hyperactivated ribosome biogenesis and nucleolar function plays a central role in suppressing p53 activation in response to oncogenic stress. Consequently, disruption of ribosome biogenesis by agents such as the small molecule inhibitor of RNA Polymerase I transcription, CX-5461, has shown unexpected, potent, and selective effects in killing tumour cells via disruption of nucleolar function leading to activation of p53, independent of DNA damage. SCOPE OF REVIEW: This review will explore the mechanism of DNA damage-independent activation of p53 via the nucleolar surveillance pathway and how this can be utilised to design novel cancer therapies. MAJOR CONCLUSION AND GENERAL SIGNIFICANCE: Non-genotoxic targeting of nucleolar function may provide a new paradigm for treatment of a broad range of oncogene-driven malignancies with improved therapeutic windows. This article is part of a Special Issue entitled: Translation and Cancer.

E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis.

Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.