Margot Thome

Epstein-Barr virus (EBV) is a human herpesvirus that causes a variety of hematological malignancies, in particular B-, T- and NK-cell lymphomas. Latency proteins of the virus contribute to lymphomagenesis by activating the transcription factor NF-κB, however, the molecular mechanism underlying EBV-driven NF-κB activation remains incompletely understood. Here we show that EBV latent membrane proteins LMP1 and LMP2A or LMP2B synergize to activate the protease MALT1, a key driver of NF-κB-mediated lymphocyte proliferation. EBV-positive B-cell lines co-expressing LMP1 and LMP2 proteins exhibited constitutive MALT1 activity, and treatment with a MALT1 inhibitor or silencing of latent membrane protein-1 (LMP1) or LMP2 impaired MALT1-dependent substrate cleavage. LMP1 physically interacted with the MALT1-binding partner BCL10, while LMP2A and LMP2B interacted with MALT1. LMP1 also colocalized with LMP2 in dotted structures, suggesting that LMP1 and LMP2 synergize via the coordinated formation of LMP-BCL10-MALT1 (LBM) complexes. Finally, MALT1 protease inhibition led to reduced expression of a set of NF-κB-driven genes, and reduced tumor growth in an EBV-positive lymphoma xenograft model. These findings establish a key role for the protease MALT1 in LMP1/2-mediated, NF-κB-driven cellular transformation and provide a rationale for inhibiting MALT1 to treat EBV-positive B-cell lymphomas that co-express LMP1 and LMP2.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease, with the activated B-cell-like (ABC) subtype showing inferior outcomes. The ETS transcription factors ETS1 and FLI1 are recurrently gained and functionally relevant in DLBCL, yet their pathogenic role remains to be fully elucidated. Here, we describe their cooperation with the RNA regulatory machinery, demonstrating that the RNA helicase DDX21 is a central effector of the ETS1/FLI1 transcriptional network in ABC-DLBCL. Our proteomic analyses revealed that ETS1 physically interacted with DDX21 and other RNA processing factors. As ETS1, DDX21 was preferentially expressed in ABC-DLBCL, particularly in the MCD/C5 genetic subtype, and it was associated with adverse clinical outcomes in this lymphoma subtype. Genetic and pharmacological studies demonstrated that DDX21 was essential for ABC-DLBCL cell proliferation. DDX21 also coordinated various transcriptional programs, which were revealed by integrated RNA-Seq, small RNA-Seq, ChIP-Seq, and Capture Hi-C analyses. DDX21-dependent transcription was relevant for ribosome biogenesis, MYC signaling, cell cycle progression, and immune evasion, but also regulated non-coding RNA networks, including microRNAs and small nucleolar RNAs, in particular SNORA37. Collectively, our data establish DDX21 as a nucleolar hub linking ETS transcription factors to coding and non-coding RNA programs that sustain aggressive lymphoma biology. These findings suggest DDX21 and ETS-RNA helicase complexes as promising therapeutic vulnerabilities in ABC-DLBCL.

The RHOA G17V mutation is highly recurrent in T follicular helper (TFH) cell lymphoma of the angioimmunoblastic type (AITL; 60%-70% of cases) and frequently associated with mutations in other T-cell receptor signaling genes, including CD28. Here, we sought to elucidate how RHOA and CD28 variants may work in concert to sustain T-cell activation by generating stable Jurkat T-cell lines expressing wild-type (wt) RHOA or RHOA G17V with wt CD28 or CD28 T195P. Concomitant expression of RHOA G17V and CD28 T195P induced significantly higher levels of interleukin-2 (IL-2) production and NFAT nuclear factor of activated T cells (NFAT) and activator protein 1 (AP1) transcriptional activities than either variant alone upon T-cell activation with agonistic anti-CD3 and anti-CD28 antibodies. We identified the histone acetyltransferase p300 as a major interacting partner of RHOA G17V in our model and human primary T cells. p300 inhibition abolished the increased IL-2 secretion induced by CD3/CD28 stimulation in cells expressing RHOA G17V and/or CD28 T195P. Chromatin immunoprecipitations and immunofluorescence staining revealed an increase of p300-specific H3K18ac and H3K27ac marks at the IL-2 promoter and across whole genome, respectively, in cells expressing RHOA G17V. Finally, immunofluorescence staining of tumor samples from 4 patients with AITL carrying RHOA G17V variant and 4 carrying wt RHOA showed that neoplastic TFH cells with RHOA G17V have increased H3K18ac and H3K27ac levels compared with non-neoplastic T cells. Collectively, these findings uncover a new mechanism of action by which RHOA G17V potentiates CD28 T195P-induced NFAT and AP1 transcriptional activities by enhancing p300 histone acetyltransferase activity and expand the notion that epigenetic deregulation contributes to the pathogenesis of TFH lymphomas.

The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody–based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain–containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

The protease MALT1 promotes lymphocyte activation and lymphomagenesis by cleaving a limited set of cellular substrates, most of which control gene expression. Here, we identified the integrin-binding scaffold protein Tensin-3 as a MALT1 substrate in activated human B cells. Activated B cells lacking Tensin-3 showed decreased integrin-dependent adhesion but exhibited comparable NF-κB1 and Jun N-terminal kinase transcriptional responses. Cells expressing a noncleavable form of Tensin-3, on the other hand, showed increased adhesion. To test the role of Tensin-3 cleavage in vivo, mice expressing a noncleavable version of Tensin-3 were generated, which showed a partial reduction in the T cell–dependent B cell response. Interestingly, human diffuse large B cell lymphomas and mantle cell lymphomas with constitutive MALT1 activity showed strong constitutive Tensin-3 cleavage and a decrease in uncleaved Tensin-3 levels. Moreover, silencing of Tensin-3 expression in MALT1-driven lymphoma promoted dissemination of xenografted lymphoma cells to the bone marrow and spleen. Thus, MALT1-dependent Tensin-3 cleavage reveals a unique aspect of the function of MALT1, which negatively regulates integrin-dependent B cell adhesion and facilitates metastatic spread of B cell lymphomas.

Mucosa-associated lymphoid tissue protein-1 (MALT-1, also known as paracaspase) is a caspase-like protease playing an essential role in mammalian immunity and oncogenesis. Humans and mice with muta...

The protease Malt1 controls the development and function of lymphocytes and promotes lymphomagenesis by cleaving a limited set of cellular substrates, many of which regulate gene transcription. Here, we report the identification of the integrin-binding scaffold protein Tensin-3 as a Malt1 substrate in activated B cells. B cells expressing a non-cleavable form of Tensin-3 (TNS3-nc) showed normal NF-κB and JNK transcriptional responses but increased and prolonged integrin-dependent adhesion upon activation. Moreover, mice expressing a non-cleavable form of Tensin-3 displayed reduced antibody production in response to immunization with a T-cell dependent antigen. We also explored the role of Tensin-3 in diffuse large B cell lymphomas and mantle cell lymphomas characterized by constitutive Malt1 activity, which showed strong constitutive Tensin-3 cleavage and a correlating reduction in total Tensin-3 levels. Silencing of Tensin-3 expression in Malt1-driven lymphoma models did not affect cellular proliferation but enhanced the dissemination of xenografted lymphoma cells. Thus, Malt1-dependent Tensin-3 cleavage limits integrin-dependent B-cell adhesion and promotes humoral immune responses and metastatic spreading of B cell lymphomas in a transcription-independent manner.

The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1-generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism. Copyright © 2019 the Author(s). Published by PNAS.

A major challenge in designing chimeric antigen receptor (CAR) T cells for cancer immunotherapy is to identify tumor antigens that are absolutely cancer-specific, in order to avoid killing of normal cells. This exciting paper proposes a smart approach to target surface receptors such as EGFRs that are overexpressed in certain cancers, but also expressed (at much lower levels) in healthy tissue. To achieve this, the authors designed therapeutic T cells that can distinguish between cells expressing high or low levels of the human EGFR2 (HER2), by expressing a combination of two receptors. The first one (synNotch) recognizes HER2 with low affinity and acts as a high-antigen density filter. When fully activated, this receptor induces expression of a high affinity CAR specific for HER2, which then confers ultrasensitive HER2-dependent cytotoxicity to the engineered T cells. So far, the authors have successfully tested the functionality of their approach in vitro and in vivo, using mice that were xenografted human cancer cells expressing different levels of HER2. Collectively, these findings raise hopes that similar approaches may be applicable to treat human tumors in the future.

Inactivating mutations of TNFAIP3 (A20), an anti-inflammatory ubiquitin-editing enzyme, are very frequent in Hodgkin lymphoma, but how A20 inactivation contributes to lymphomagenesis remains largely unknown. This study took a highly interesting approach to better understand the molecular contribution of A20. Using CRISPR library screens targeting ubiquitin regulators, the authors discovered that the survival of Hodgkin lymphoma cells with A20 loss-of-function mutations was indeed highly dependent on the activity of the linear ubiquitin chain assembly complex (LUBAC) and NF-kB activity. Introduction of wild-type A20 into A20 negative cells led to a decrease in NF-kB activity and cell survival, which depended on A20’s capacity to bind to linear ubiquitin chains. Furthermore, the authors identified the kinase activity of TAK1 as a target of the LUBAC-A20 signaling axis and showed that TAK1 kinase activity was essential for NF-kB activity and survival of A20-deficient HL cells in vitro and in xenograft mouse models. These findings are important because they reveal a novel A20-LUBAC-NF-kB signaling axis and identify LUBAC and TAK1 as attractive therapeutic targets for the treatment of A20 mutant cases of Hodgkin lymphoma.