B cell differentiation is controlled by a complex network of lineage-restricted transcription factors. How perturbations to this network alter B cell fate remains poorly understood. Here we show that classical Hodgkin lymphoma tumor cells, which originate from mature B cells, have lost the B cell phenotype as a result of aberrant expression of transcriptional regulators. The B cell-specific transcription factor program was disrupted by overexpression of the helix-loop-helix proteins ABF-1 and Id2. Both factors antagonized the function of the B cell-determining transcription factor E2A. As a result, expression of genes specific to B cells was lost and expression of genes not normally associated with the B lineage was upregulated. These data demonstrate the plasticity of mature human lymphoid cells and offer an explanation for the unique classical Hodgkin lymphoma phenotype.

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

Transcription factor AP-1 is constitutively activated and IRF4 drives growth and survival in ALK+ and ALK– anaplastic large cell lymphoma (ALCL). Here we demonstrate high-level BATF and BATF3 expression in ALCL. Both BATFs bind classical AP-1 motifs and interact with in ALCL deregulated AP-1 factors. Together with IRF4, they co-occupy AP-1-IRF composite elements, differentiating ALCL from non-ALCL. Gene-specific inactivation of BATFs, or global AP-1 inhibition results in ALCL growth retardation and/or cell death in vitro and in vivo. Furthermore, the AP-1-BATF module establishes TH17/group 3 innate lymphoid cells (ILC3)-associated gene expression in ALCL cells, including marker genes such as AHR, IL17F, IL22, IL26, IL23R and RORγt. Elevated IL-17A and IL-17F levels were detected in a subset of children and adolescents with ALK+ ALCL. Furthermore, a comprehensive analysis of primary lymphoma data confirms TH17–, and in particular ILC3-skewing in ALCL compared with PTCL. Finally, pharmacological inhibition of RORC as single treatment leads to cell death in ALCL cell lines and, in combination with the ALK inhibitor crizotinib, enforces death induction in ALK+ ALCL. Our data highlight the crucial role of AP-1/BATFs in ALCL and lead to the concept that some ALCL might originate from ILC3.

IntroductionTranscription factor nuclear factor kappa B (NF-B) plays a key role in the regulation of immune and inflammatory responses, functions as a potent inhibitor of apoptosis, and is involved in malignant transformation of different cell types (for recent reviews see Rayet and Gelinas, 1 Karin and Ben-Neriah, 2 and Bonizzi and Karin 3 ). Depending on the stimulus, the duration of stimulation, and the cellular context, the NF-B family members p50, p52, p65 (RelA), RelB, and c-Rel form different homo-or heterodimers. p50 and p52 are derived from precursor molecules (p105 and p100), which can act as inhibitors of NF-B (IB) proteins. The activation of the NF-B pathway is controlled by IB proteins, which retain NF-B in the cytoplasm. Following activation, they are degraded by the proteasome while NF-B translocates into the nucleus where it activates transcription. 2 The IB kinase (IKK) complex plays a central role in this pathway by mediating the initial phosphorylation of IB proteins. 4 The Bcl-3 protein shares structural features with I〉 proteins. 5,6 It was initially discovered by investigation of the t(14;19)(q32.3; q13.2) translocation in B-cell chronic lymphocytic leukemia (B-CLL), which is associated with poor prognosis. 7 In line with an oncogenic function, E-Bcl-3 transgenic mice develop lymphoproliferative disorders, 8 Bcl-3 directly transforms cells, 9 and Bcl-3 excerts an antiapoptotic effect in B and T lymphocytes. 10,11 Furthermore, Bcl-3 overactivity has been suggested to be involved in the pathogenesis of breast cancer, 12 and Bcl-3 overexpression has been reported in a subgroup of anaplastic large-cell lymphomas (ALCLs). 13 Consistent with a noninhibitory function, Bcl-3 is located mainly in the nucleus, and it can function as a regulator at NF-B DNA binding sites. 5,6,[14][15][16][17] Bcl-3-mediated regulation of NF-B activity might occur indirectly by dissociation of the inhibitory NF-B (p50) 2 and (p52) 2 complexes from DNA 5 or, alternatively, by a function as transcriptional coactivator. [16][17][18] ALCLs are unique lymphomas originating from cytotoxic T cells (for recent review see Stein et al 19 ). They are defined by the proliferation of predominantly large lymphoid cells and expression of the tumor necrosis factor (TNF)-receptor family member CD30 and the cytotoxic molecules perforin and granzyme B. ALCLs express the anaplastic lymphoma kinase (ALK) in 50% to 70% of the cases, 19 most commonly in the context of the chromosomal translocation t(2;5)(p23;q35). The resulting nucleophosmin (NPM)-ALK fusion protein promotes cellular transformation. ALCLs share a number of molecular aberrations with classical Hodgkin lymphoma (cHL), which in the majority of cases derives from B cells. 20 Constitutive AP-1 (c-Jun and JunB) 21 or Notch-1 overactivity 22 is found in both of these lymphoma entities. Interestingly, the B-cell-derived neoplastic Hodgkin/Reed-Sternberg (HRS) cells of cHL have lost the B-cell-specific gene expression program, 23 whereas the T-cell-derived ALCLs have lost most of the...

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TFbinding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.T ranscription factor (TF) activities have to be tightly controlled because their aberrant regulation alters tissue-specific gene expression programs and contributes to cancer pathogenesis. Therefore, the identification of altered TF activities in malignancies is of crucial importance to understand malignant transformation and to develop new treatment strategies. Deregulated TF activities are commonly observed in hematopoietic malignancies including human lymphomas and leukemias, and the link between structural or functional alterations in TFs and malignant transformation has been documented in various in vitro and in vivo studies (1-3). Apart from the direct modulation of cellular processes like cellular growth or cell death, alterations of the activity of even single TFs might enforce malignant transformation by switching differentiation programs and consequently altering the cellular fate of the respective cells, as exemplarily demonstrated for the B-lymphoid TF PAX5 (4, 5).Among lymphoid malignancies, one of the most prominent examples for complex patterns of deregulated TFs is classical Hodgkin lymphoma (HL), a common B cell-derived malignancy (6). Pathogenic hallmarks of the malignant Hodgkin/Reed-Sternberg (HRS) cells of HL include the constitutive activation of TFs that are only transiently activated in normal B cells, such as nuclear factor kappa B (NF-κB) or activator protein-1 (AP-1), and a profound deregulation of lineage-specific TFs such as E2A (6-8). Thus, although originating from B-lymphoid cells, HRS cells have lost their B cell-specific gene expression pattern and instead upregulate expression of genes characteristic for other hematopoietic lineages. However, the nature of the TFs initiating and maintaining HRS-specific gene expression remains poorly understood.As an unbiased approach for the identification of deregulated TF activities central to lymphoma biology, we identified HL-specific accessible chromatin regions...

Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed–Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis.