TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma.
ABSTRACT: Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor kappaB (NF-kappaB). NF-kappaB activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus-positive (EBV(+)) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV(-) cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-kappaB target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-kappaB activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-kappaB activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV(-) than EBV(+) cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.
Project description:Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-kappaB transcription complex. However, except for a small fraction of cases, it remains unclear whether NF-kappaB activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A (RANK)) regulators of NF-kappaB. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-kappaB responses, is most commonly affected, with approximately 30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-kappaB. Thus, our results demonstrate that NF-kappaB activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-kappaB responses.
Project description:Mediastinal gray zone lymphoma (MGZL) has immunopathologic features between classical Hodgkin lymphoma (cHL) and primary mediastinal thymic B-cell lymphoma (PMBL), leading to uncertainty regarding its biological relationship to these entities. We performed gene expression profiling from patients with MGZL (20), cHL (18), and PMBL (17) and show MGZL clusters between cHL and PMBL. Expression signatures reveal germinal B-cell and IFN regulatory factor 4 (IRF4) signatures were relatively low in MGZL and cHL compared with PMBL, indicating downregulation of the B-cell program in MGZL, a hallmark of cHL. T-cell and macrophage signatures were higher in MGZL and cHL compared with PMBL, consistent with infiltrating immune cells, which are found in cHL. The NF?B signature was higher in MGZL than PMBL, and like cHL, MGZL and PMBL express NF?B inducing kinase (NIK), indicating noncanonical signaling. These findings indicate that while MGZL has distinctive clustering, it is biologically closer to cHL.
Project description:A20 is a ubiquitin modifying enzyme that restricts NF-kappaB signals and protects cells against tumor necrosis factor (TNF)-induced programmed cell death. Given recent data linking A20 (TNFAIP3) with human B cell lymphomas and systemic lupus erythematosus (SLE), we have generated mice bearing a floxed allele of Tnfaip3 to interrogate A20's roles in regulating B cell functions. A20-deficient B cells are hyperresponsive to multiple stimuli and display exaggerated NF-kappaB responses to CD40-induced signals. Mice expressing absent or hypomorphic amounts of A20 in B cells possess elevated numbers of germinal center B cells, autoantibodies, and glomerular immunoglobulin deposits. A20-deficient B cells are resistant to Fas-mediated cell death, probably due to increased expression of NF-kappaB-dependent antiapoptotic proteins such as Bcl-x. These findings show that A20 can restrict B cell survival, whereas A20 protects other cells from TNF-induced cell death. Our studies demonstrate how reduced A20 expression predisposes to autoimmunity.
Project description:A20, a negative regulator of NF-?B, has been implicated as a tumor suppressor gene in multiple types of B-cell lymphoma. AIDS-related lymphomas (ARLs) are high-grade B-cell lymphomas that are frequently associated with EBV infection. We examined a panel of ARLs for A20 alterations. FISH showed A20 deletion in 6 of 33 cases (18%). A20 mutations were found in 3 of 19 cases (16%), including 2 cases with deletions of the comple-mentary allele. Immunohistochemistry showed the absence of A20 protein in 7 of 55 samples (13%). In contrast to reports in Hodgkin lymphoma in which EBV infection and A20 alteration are mutually exclusive, A20 inactivation was observed in both EBV(+) and EBV(-) cases. The EBV latent membrane protein 1, which activates NF-?B, was not expressed in 12 of 13 cases with A20 loss. In ARLs loss of A20 may be an alternative mechanism of NF-?B activation in the absence of latent membrane protein 1 expression.
Project description:Unique and shared cytogenetic abnormalities have been documented for marginal zone lymphomas (MZLs) arising at different sites. Recently, homozygous deletions of the chromosomal band 6q23, involving the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) gene, a negative regulator of NF-kappaB, were described in ocular adnexal MZL, suggesting a role for A20 as a tumor suppressor in this disease. Here, we investigated inactivation of A20 by DNA mutations or deletions in a panel of extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Inactivating mutations encoding truncated A20 proteins were identified in 6 (19%) of 32 MZLs, including 2 (18%) of 11 EMZLs, 3 (33%) of 9 NMZLs, and 1 (8%) of 12 SMZLs. Two additional unmutated nonsplenic MZLs also showed monoallelic or biallelic A20 deletions by fluorescent in situ hybridization (FISH) and/or SNP-arrays. Thus, A20 inactivation by either somatic mutation and/or deletion represents a common genetic aberration across all MZL subtypes, which may contribute to lymphomagenesis by inducing constitutive NF-kappaB activation.
Project description:Primary mediastinal large B-cell lymphomas (PMBLs) are aggressive tumors that typically present as large mediastinal masses in young women. PMBLs share clinical, transcriptional, and molecular features with classical Hodgkin lymphoma (cHL), including constitutive activation of nuclear factor ?B (NF-?B), JAK/STAT signaling, and programmed cell death protein 1 (PD-1)-mediated immune evasion. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBLs led to recent approval by the US Food and Drug Administration and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. Here, we report a comprehensive analysis of recurrent genetic alterations -somatic mutations, somatic copy number alterations, and structural variants-in a cohort of 37 newly diagnosed PMBLs. We identified a median of 9 genetic drivers per PMBL, including known and newly identified components of the JAK/STAT and NF-?B signaling pathways and frequent B2M alterations that limit major histocompatibility complex class I expression, as in cHL. PMBL also exhibited frequent, newly identified driver mutations in ZNF217 and an additional epigenetic modifier, EZH2. The majority of these alterations were clonal, which supports their role as early drivers. In PMBL, we identified several previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high tumor mutational burden, microsatellite instability, and an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutational signature. The shared genetic features between PMBL and cHL provide a framework for analyzing the mechanism of action of PD-1 blockade in these related lymphoid malignancies.
Project description:Classical Hodgkin lymphoma (CHL) is a neoplasm characterized by robust inflammatory infiltrates and heightened expression of the immunosuppressive PD-1/PD-L1 pathway. Although anti-PD-1 therapy can be effective in >60% of patients with refractory CHL, improved treatment options are needed for CHLs which are resistant to anti-PD-1 or relapse after this form of immunotherapy. A deeper understanding of immunologic factors in the CHL microenvironment might support the design of more effective treatment combinations based on anti-PD-1. In addition, because the Epstein-Barr virus (EBV) residing in some CHL tumors is strongly immunogenic, we hypothesized that characteristics of the tumor immune microenvironment in EBV+ CHL would be distinct from EBV- CHL, with specific implications for designing combination treatment regimens. Employing immunohistochemistry for immune cell subsets and checkpoint molecules, as well as gene expression profiling, we characterized 32 CHLs from the Johns Hopkins archives, including 12 EBV+ and 20 EBV- tumors. Our results revealed a dichotomous cellular and cytokine immune milieu in EBV+ vs EBV- CHL. EBV+ tumors displayed a T helper 1 (Th1) profile typical of effective antitumor immunity, with increased infiltration of CD8+ T cells and coordinate expression of the canonical Th1 transcription factor Tbet (TBX21), interferon-? (IFNG), and the IFN-?-inducible immunosuppressive enzyme indoleamine 2,3-dioxygenase. In contrast, EBV- tumors manifested a pathogenic Th17 profile and ongoing engagement of the interleukin-23 (IL-23)/IL-17 axis, with heightened phosphorylated signal transducer and activator of transcription 3 expression in infiltrating lymphocytes. These findings suggest that drugs blocking the IL-23/IL-17 axis, which are already in the clinic for treating certain autoimmune disorders, may enhance the therapeutic impact of anti-PD-1 therapy in EBV- CHL.
Project description:MYD88 mutations are one of the most recurrent mutations in hematologic malignancies. However, recent mouse models suggest that MYD88<sub>L265P</sub> alone may not be sufficient to induce tumor formation. Interplay between MYD88<sub>L265P</sub> and other genetic events is further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88<sub>L265P</sub>. However, we are still lacking information about the consequence of MYD88<sub>L265P</sub> in combination with TNFAIP3 loss in human B cell lymphoma. Review of our genetic data on diffuse large B cell lymphoma (DLBCL) and Waldenstrom macroglobulinemia (WM), found that a large percentage of DLBCL and WM cases that have a MYD88 mutation also harbor a TNFAIP3 loss, 55% DLBCL and 28% of WM, respectively. To mimic this combination of genetic events, we used genomic editing technology to knock out TNFAIP3 in MYD88<sub>L265P</sub> non-Hodgkin's lymphoma (NHL) cell lines. Loss of A20 expression resulted in increased NF-?B and p38 activity leading to upregulation of the NF-?B target genes BCL2 and MYC. Furthermore, we detected the increased production of IL-6 and CXCL10 which led to an upregulation of the JAK/STAT pathway. Overall, these results suggest that MYD88<sub>L265P</sub> signaling can be enhanced by a second genetic alteration in TNFAIP3 and highlights a potential opportunity for therapeutic targeting.
Project description:The TNFAIP3 (tumor necrosis factor alpha-induced protein 3) gene encodes a ubiquitin editing enzyme, A20, that restricts NF-kappaB-dependent signaling and prevents inflammation. We show that three independent SNPs in the TNFAIP3 region (rs13192841, rs2230926 and rs6922466) are associated with systemic lupus erythematosus (SLE) among individuals of European ancestry. These findings provide critical links between A20 and the etiology of SLE.
Project description:EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.