Profiling the activity of the para-caspase MALT1 in B-cell acute lymphoblastic leukemia for potential targeted therapeutic application.

B cell acute lymphoblastic leukemia (B-ALL) remains a hard-to-treat disease with a poor prognosis in adults. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for B-cell receptor (BCR)-mediated NF-κB activation. Inhibition of MALT1 in preclinical models has proven efficacious in many B-cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and diffuse large B-cell lymphoma. We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of its inhibition. Targeting MALT1 with both Z-VRPR-fmk and MI-2 efficiently kills B-ALL cells independent of the cell-of-origin (pro, pre, mature) or the presence of the Philadelphia chromosome, and spares normal B-cells. The mechanism of cell death was through apoptotic induction, mostly in cycling cells. The proteolytic activity of MALT1 can be studied by measuring its ability to cleave its substrates. Surprisingly, with the exception of mature B-ALL, we did not detect cleavage of MALT1 substrates at baseline, nor after proteasomal inhibition or following activation of pre-BCR. To explore the possibility of a distinct role for MALT1 in B-ALL, independent of signaling through BCR, we studied the changes in gene expression profiling following a 24-hour treatment with MI-2 in 12 B-ALL cell lines. Our transcriptome analysis revealed a strong inhibitory effect on MYC-regulated gene signatures, further confirmed by Myc protein downregulation, concomitant with an increase in the Myc degrader FBXW7. In conclusion, our evidence suggests a novel role for MALT1 in B-ALL through Myc regulation and provides support for clinical testing of MALT1 inhibitors in B-ALL.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. SIGNIFICANCE: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

Identification of MALT1 feedback mechanisms enables rational design of potent antilymphoma regimens for ABC-DLBCL.

MALT1 inhibitors are promising therapeutic agents for B-cell lymphomas that are dependent on constitutive or aberrant signaling pathways. However, a potential limitation for signal transduction-targeted therapies is the occurrence of feedback mechanisms that enable escape from the full impact of such drugs. Here, we used a functional genomics screen in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cells treated with a small molecule irreversible inhibitor of MALT1 to identify genes that might confer resistance or enhance the activity of MALT1 inhibition (MALT1i). We find that loss of B-cell receptor (BCR)- and phosphatidylinositol 3-kinase (PI3K)-activating proteins enhanced sensitivity, whereas loss of negative regulators of these pathways (eg, TRAF2, TNFAIP3) promoted resistance. These findings were validated by knockdown of individual genes and a combinatorial drug screen focused on BCR and PI3K pathway-targeting drugs. Among these, the most potent combinatorial effect was observed with PI3Kδ inhibitors against ABC-DLBCLs in vitro and in vivo, but that led to an adaptive increase in phosphorylated S6 and eventual disease progression. Along these lines, MALT1i promoted increased MTORC1 activity and phosphorylation of S6K1-T389 and S6-S235/6, an effect that was only partially blocked by PI3Kδ inhibition in vitro and in vivo. In contrast, simultaneous inhibition of MALT1 and MTORC1 prevented S6 phosphorylation, yielded potent activity against DLBCL cell lines and primary patient specimens, and resulted in more profound tumor regression and significantly improved survival of ABC-DLBCLs in vivo compared with PI3K inhibitors. These findings provide a basis for maximal therapeutic impact of MALT1 inhibitors in the clinic, by disrupting feedback mechanisms that might otherwise limit their efficacy.

Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and Activates Immune Surveillance in Lymphoma.

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6-HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse CREBBP-mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating lymphocytes to kill DLBCL cells in an MHC class I and II-dependent manner, and synergized with PD-L1 blockade in a syngeneic model in vivo. Hence, HDAC3 inhibition represents a novel mechanism-based immune epigenetic therapy for CREBBP-mutant lymphomas. SIGNIFICANCE: We have leveraged the molecular characterization of different types of CREBBP mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in CREBBP-mutant cells in tandem with promoting antitumor immunity.This article is highlighted in the In This Issue feature, p. 327.

Quinoline and thiazolopyridine allosteric inhibitors of MALT1.

Quinolines and thiazolopyridines were developed as allosteric inhibitors of MALT1, with good cellular potency and exquisite selectivity. Mouse pharmacokinetic (PK) profiling showed these to have low in vivo clearance, and moderate oral exposure. The thiazolopyridines were less lipophilic than the quinolines, and one thiazolopyridine example was active in our hIL10 mouse pharmacodynamic (PD) model upon oral dosing.

Peptide-based covalent inhibitors of MALT1 paracaspase.

Potent and selective substrate-based covalent inhibitors of MALT1 protease were developed from the tetrapeptide tool compound Z-VRPR-fmk. To improve cell permeability, we replaced one arginine residue. We further optimized a series of tripeptides and identified compounds that were potent in both a GloSensor reporter assay measuring cellular MALT1 protease activity, and an OCI-Ly3 cell proliferation assay. Example compounds showed good overall selectivity towards cysteine proteases, and one compound was selected for further profiling in ABL-DLBCL cells and xenograft efficacy models.

Germinal center-derived lymphomas: The darkest side of humoral immunity.

One of the unusual features of germinal center (GC) B cells is that they manifest many hallmarks of cancer cells. Accordingly, most B-cell neoplasms originate from the GC reaction, and characteristically display abundant point mutations, structural genomic lesions, and clonal diversity from the genetic and epigenetic standpoints. The dominant biological theme of GC-derived lymphomas is mutation of genes involved in epigenetic regulation and immune receptor signaling, which come into play at critical transitional stages of the GC reaction. Hence, mechanistic studies of these mutations reveal fundamental insight into the biology of the normal and malignant GC B cell. The BCL6 transcription factor plays a central role in establishing the GC phenotype in B cells, and most lymphomas are dependent on BCL6 to maintain survival, proliferation, and perhaps immune evasion. Many lymphoma mutations have the commonality of enhancing the oncogenic functions of BCL6, or overcoming some of its tumor suppressive effects. Herein, we discuss how unique features of the GC reaction create vulnerabilities that select for particular lymphoma mutations. We examine the interplay between epigenetic programming, metabolism, signaling, and immune regulatory mechanisms in lymphoma, and discuss how these are leading to novel precision therapy strategies to treat lymphoma patients.

TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors.See related commentary by Shingleton and Dave, p. 1515.This article is highlighted in the In This Issue feature, p. 1494.

PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival.

The germinal center (GC) reaction plays an important role in generating humoral immunity and is believed to give rise to most B-cell lymphomas. GC entry and exit are tightly regulated processes, controlled by the actions of transcription factors such as BCL6. Herein, we demonstrate that protein arginine methyltransferase 5 (PRMT5), a symmetric dimethyl arginine methyltransferase, is also necessary for GC formation and affinity maturation. PRMT5 contributes to GC formation and affinity maturation at least in part through its direct interaction with and methylation of BCL6 at arginine 305 (R305), a modification necessary for the full transcriptional repressive effects of BCL6. Inhibition of PRMT5 in B-cell lymphoma lines led to significant upregulation of BCL6 target genes, and the concomitant inhibition of both BCL6 and PRMT5 exhibited synergistic killing of BCL6-expressing lymphoma cells. Our studies identify PRMT5 as a novel regulator of the GC reaction and highlight the mechanistic rationale of cotargeting PRMT5 and BCL6 in lymphoma.

Specific covalent inhibition of MALT1 paracaspase suppresses B cell lymphoma growth.

The paracaspase MALT1 plays an essential role in activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) downstream of B cell and TLR pathway genes mutated in these tumors. Although MALT1 is considered a compelling therapeutic target, the development of tractable and specific MALT1 protease inhibitors has thus far been elusive. Here, we developed a target engagement assay that provides a quantitative readout for specific MALT1-inhibitory effects in living cells. This enabled a structure-guided medicinal chemistry effort culminating in the discovery of pharmacologically tractable, irreversible substrate-mimetic compounds that bind the MALT1 active site. We confirmed that MALT1 targeting with compound 3 is effective at suppressing ABC DLBCL cells in vitro and in vivo. We show that a reduction in serum IL-10 levels exquisitely correlates with the drug pharmacokinetics and degree of MALT1 inhibition in vitro and in vivo and could constitute a useful pharmacodynamic biomarker to evaluate these compounds in clinical trials. Compound 3 revealed insights into the biology of MALT1 in ABC DLBCL, such as the role of MALT1 in driving JAK/STAT signaling and suppressing the type I IFN response and MHC class II expression, suggesting that MALT1 inhibition could prime lymphomas for immune recognition by cytotoxic immune cells.

How Biophysical Forces Regulate Human B Cell Lymphomas.

The role of microenvironment-mediated biophysical forces in human lymphomas remains elusive. Diffuse large B cell lymphomas (DLBCLs) are heterogeneous tumors, which originate from highly proliferative germinal center B cells. These tumors, their associated neo-vessels, and lymphatics presumably expose cells to particular fluid flow and survival signals. Here, we show that fluid flow enhances proliferation and modulates response of DLBCLs to specific therapeutic agents. Fluid flow upregulates surface expression of B cell receptors (BCRs) and integrin receptors in subsets of ABC-DLBCLs with either CD79A/B mutations or WT BCRs, similar to what is observed with xenografted human tumors in mice. Fluid flow differentially upregulates signaling targets, such as SYK and p70S6K, in ABC-DLBCLs. By selective knockdown of CD79B and inhibition of signaling targets, we provide mechanistic insights into how fluid flow mechanomodulates BCRs and integrins in ABC-DLBCLs. These findings redefine microenvironment factors that regulate lymphoma-drug interactions and will be critical for testing targeted therapies.

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, Bruton's 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.

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.

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.

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.

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.

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 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 human germinal centre-associated lymphoma directly binds to Syk in B cells, increases its kinase activity on B-cell receptor stimulation and leads to enhanced activation of Syk downstream effectors. To further investigate these findings in vivo, human germinal centre-associated lymphoma transgenic mice were generated. Starting from 12 months of age these mice developed polyclonal B-cell lymphoid hyperplasia, hypergammaglobulinemia and systemic reactive amyloid A (AA) amyloidosis, leading to shortened survival. The lymphoid hyperplasia in the human germinal centre-associated lymphoma 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 human germinal centre-associated lymphoma regulates B-cell receptor signalling in B-lymphocytes which, without appropriate control, may lead to B-cell lymphoproliferation.

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.