Lorena Fontan

MALT1 Small Molecule Inhibitors Specifically Suppress ABC-DLBCL In Vitro and In Vivo

MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.

Structural Architecture of the CARMA1/Bcl10/MALT1 Signalosome: Nucleation-Induced Filamentous Assembly

The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains (CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.

Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1+Lin− hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.

Lymphoma stem cells: enough evidence to support their existence?

While leukemia-originating stem cells are critical in the initiation and maintenance of leukemias, the existence of similar cell populations that may generate B-cell lymphoma upon mutation remains uncertain. Here we propose that committed lymphoid progenitor/precursor cells with an active V-D-J recombination program are the initiating cells of follicular lymphoma and mantle cell lymphoma when targeted by immunoglobulin (IG)- gene translocations in the bone marrow. However, these pre-malignant lymphoma-initiating cells cannot drive complete malignant transformation, requiring additional cooperating mutations in specific stem-cell programs to be converted into the lymphoma-originating cells able to generate and sustain lymphoma development. Conversely, diffuse large B-cell lymphoma and sporadic Burkitt’s lymphoma derive from B lymphocytes that acquire translocations through IG-hyper-mutation or class-switching errors within the germinal center. Although secondary reprogramming mutations are generally required, some cells such as centroblasts or memory B cells that have certain stem cell-like features, or lymphocytes with MYC rearrangements that deregulate self-renewal pathways, may bypass this need and directly function as the lymphoma-originating cells. An alternative model supports an aberrant epigenetic modification of gene sets as the first occurring hit, which either leads to retaining stem-cell features in hematopoietic stem or progenitor cells, or reprograms stemness into more committed lymphocytes, followed by secondary chromosomal translocations that eventually drive lymphoma development. Isolation and characterization of the cells that are at the origin of the different B-cell non-Hodgkin’s lymphomas will provide critical insights into the disease pathogenesis and will represent a step towards the development of more effective therapies.

Pathogenic role of B-cell receptor signaling and canonical NF-κB activation in mantle cell lymphoma.

To interrogate signaling pathways activated in mantle cell lymphoma (MCL) in vivo, we contrasted gene expression profiles of 55 tumor samples isolated from blood and lymph nodes from 43 previously untreated patients with active disease. In addition to lymph nodes, MCL often involves blood, bone marrow, and spleen and is incurable for most patients. Recently, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrated important clinical activity in MCL. However, the role of specific signaling pathways in the lymphomagenesis of MCL and the biologic basis for ibrutinib sensitivity of these tumors are unknown. Here, we demonstrate activation of B-cell receptor (BCR) and canonical NF-κB signaling specifically in MCL cells in the lymph node. Quantification of BCR signaling strength, reflected in the expression of BCR regulated genes, identified a subset of patients with inferior survival after cytotoxic therapy. Tumor proliferation was highest in the lymph node and correlated with the degree of BCR activation. A subset of leukemic tumors showed active BCR and NF-κB signaling apparently independent of microenvironmental support. In one of these samples, we identified a novel somatic mutation in RELA (E39Q). This sample was resistant to ibrutinib-mediated inhibition of NF-κB and apoptosis. In addition, we identified germ line variants in genes encoding regulators of the BCR and NF-κB pathway previously implicated in lymphomagenesis. In conclusion, BCR signaling, activated in the lymph node microenvironment in vivo, appears to promote tumor proliferation and survival and may explain the sensitivity of this lymphoma to BTK inhibitors.

Downregulation of FOXP1 is required during germinal center B-cell function.

B-cell maturation and germinal center (GC) formation are dependent on the interplay between BCL6 and other transcriptional regulators. FOXP1 is a transcription factor that regulates early B-cell development, but whether it plays a role in mature B cells is unknown. Analysis of human tonsillar B-cell subpopulations revealed that FOXP1 shows the opposite expression pattern to BCL6, suggesting that FOXP1 regulates the transition from resting follicular B cell to activated GC B cell. Chromatin immunoprecipitation-on-chip and gene expression assays on B cells indicated that FOXP1 acts as a transcriptional activator and repressor of genes involved in the GC reaction, half of which are also BCL6 targets. To study FOXP1 function in vivo, we developed transgenic mice expressing human FOXP1 in lymphoid cells. These mice exhibited irregular formation of splenic GCs, showing a modest increase in naïve and marginal-zone B cells and a significant decrease in GC B cells. Furthermore, aberrant expression of FOXP1 impaired transcription of noncoding γ1 germline transcripts and inhibited efficient class switching to the immunoglobulin G1 isotype. These studies show that FOXP1 is physiologically downregulated in GC B cells and that aberrant expression of FOXP1 impairs mechanisms triggered by B-cell activation, potentially contributing to B-cell lymphomagenesis.

Germinal centre protein HGAL promotes lymphoid hyperplasia and amyloidosis via BCR-mediated Syk activation

The human germinal centre associated lymphoma (HGAL) gene is specifically expressed in germinal centre B-lymphocytes and germinal centre-derived B-cell lymphomas, but its function is largely unknown. Here we demonstrate that HGAL directly binds Syk in B-cells, increases its kinase activity upon B-cell receptor stimulation and leads to enhanced activation of Syk downstream effectors. To further investigate these findings in vivo, HGAL transgenic mice were generated. Starting from 12 months of age these mice developed polyclonal B-cell lymphoid hyperplasia, hypergammaglobulinemia and systemic reactive AA amyloidosis, leading to shortened survival. The lymphoid hyperplasia in the HGAL transgenic mice are likely attributable to enhanced B-cell receptor signalling as shown by increased Syk phosphorylation, ex vivo B-cell proliferation and increased RhoA activation. Overall, our study shows for the first time that the germinal centre protein HGAL regulates B-cell receptor signalling in B-lymphocytes which, without appropriate control, may lead to B-cell lymphoproliferation.

Molecular Pathways: Targeting MALT1 Paracaspase Activity in Lymphoma

MALT1 mediates the activation of NF-κB in response to antigen receptor signaling. MALT1, in association with BCL10 and CARD11, functions as a scaffolding protein to activate the inhibitor of IκB kinase (IKK) complex. In addition, MALT1 is a paracaspase that targets key proteins in a feedback loop mediating termination of the NF-κB response, thus promoting activation of NF-κB signaling. Activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL), which tend to be more resistant to chemotherapy, are often biologically dependent on MALT1 activity. Newly developed MALT1 small-molecule inhibitors suppress the growth of ABC-DLBCLs in vitro and in vivo. This review highlights the recent advances in the normal and disease-related functions of MALT1. Furthermore, recent progress targeting MALT1 proteolytic activity raises the possibility of deploying MALT1 inhibitors for the treatment of B-cell lymphomas and perhaps autoimmune diseases that involve increased B- or T-cell receptor signaling. Clin Cancer Res; 19(24); 6662–8. ©2013 AACR.

MALT1 Inhibition Is Efficacious in Both Naïve and Ibrutinib-Resistant Chronic Lymphocytic Leukemia

The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Brutons tyrosine kinase (BTK), is essential for B-cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2, a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease. Cancer Res; 77(24); 7038-48. ©2017 AACR.

Discovering what makes STAT signaling TYK in T-ALL.

SUMMARY RNA interference screening establishes TYK2 dependence in T-cell acute lymphoblastic leukemia (T-ALL), leading to identification of TYK2-activating mutations and increased IL-10 receptor signaling in T-ALL cell lines. Cancer Discov; 3(5); 494-6. ©2013 AACR.