OPEN Leukemia (2016), 1–12 www.nature.com/leu ORIGINAL ARTICLE Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma KD Wurster1,2, F Hummel1,2, J Richter3, M Giefing3,4, S Hartmann5, M-L Hansmann5, S Kreher2, K Köchert1,2, D Krappmann6, W Klapper7, M Hummel8, S-S Wenzel2, G Lenz9, M Janz1,2, B Dörken1,2,10, R Siebert3,11 and S Mathas1,2,10 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. Leukemia advance online publication, 14 October 2016; doi:10.1038/leu.2016.238 INTRODUCTION of negative regulators of more downstream located signaling Classical Hodgkin lymphoma (cHL) is characterized by a unique components (for example, inhibitor of NF-κB(IκB)α mutations in 10,11 12,13 histopathological appearance in the affected lymph nodes the NF-κB pathway), genomic alterations of respective TFs 14 demonstrating only a minority of tumor cells, the so-called and the constitutive activation of upstream kinases. The strength Hodgkin and Reed–Sternberg (HRS) cells, that are embedded in an and plethora of pathway activation in HRS cells is exceptional abundant inflammatory microenvironment.1 It is assumed that among lymphoid malignancies. Interestingly, despite their com- HRS cells attract these inflammatory bystander cells by high-level plex transcriptional alterations including long-terminal repeat activation,15,16 HRS cells demonstrate a surprisingly constant production of cytokines and chemokines, and that the interaction phenotype. Our recent data on the pivotal role of IRF5 together between HRS cells and their surrounding cells supports their 1–3 with NF-κB regarding the orchestration of the HL-specific gene growth, survival and immune escape. The abundant production 7 fl fl expression program suggest that only a limited number of of in ammatory mediators by HRS cells re ects their highly pathogenic events coordinate the cHL phenotype. activated phenotype. In line with such a phenotype, HRS cells Given the number of high-level activated TFs in HRS cells show constitutive activation of a plethora of transcription factors involved in immediate-early gene regulation, we postulated that (TFs) that are physiologically only transiently activated, and that alterations of the ubiquitin system contribute to their simulta- are implicated in cellular activation and immediate-early gene neous activation. Based on a literature search in combination with induction. Among these TFs, nuclear factor-κB (NF-κB), signal gene expression data of HRS and non-Hodgkin (NH) B cell-derived transducer and activator of transcription (STAT) 3, 5 and 6, cell lines,17 we focused on PDLIM2 (also known as mystique or activator protein-1 (AP-1) and interferon regulatory factor-5 (IRF5) SLIM).18–20 PDLIM2 has been shown to act as ubiquitin E3-ligase – play key roles in HL pathogenesis.4 8 inducing inactivation and degradation of TFs.19,21 In addition, The mechanisms leading to activation of these TFs in HRS cells ubiquitin-independent mechanisms of PDLIM2 have been are complex and affect various layers of control of respective described.19 By these mechanisms, PDLIM2 controls the activity pathway components.1 These include deleterious mutations of of TFs, such as NF-κB-p65, STAT1 and STAT3,19,21–23 that are highly negative regulators of receptor-proximal activation modules,9 activated in HRS cells. Furthermore, alterations of PDLIM2 gene 1Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; 2Hematology, Oncology, and Tumor Immunology, Charité–Universitätsmedizin Berlin, Berlin, Germany; 3Institute of Human Genetics, Christian-Albrechts University Kiel, Kiel, Germany; 4Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; 5Dr Senckenberg Institute of Pathology, University of Frankfurt, Medical School, Frankfurt, Germany; 6Research Unit Cellular Signal Integration, Helmholtz Zentrum München für Gesundheit und Umwelt, Neuherberg, Germany; 7Department of Pathology, Haematopathology Section and Lymph Node Registry, Christian-Albrechts University Kiel, Kiel, Germany; 8Institute of Pathology, Charité–Universitätsmedzin Berlin, Berlin, Germany; 9Division of Translational Oncology, Department of Medicine A, University Hospital Münster, and Cluster of Excellence EXC 1003, Cells in Motion, Münster, Germany; 10German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany and 11Institute of Human Genetics, University Hospital Ulm, Ulm, Germany. Correspondence: Dr S Mathas, Max-Delbrück-Center for Molecular Medicine, and Charité - Universitätsmedizin Berlin, Hematology, Oncology and Tumor Immunology, Robert-Rössle-Straße 10, D-13125 Berlin, Germany. E-mail: [email protected] Received 19 January 2016; revised 2 August 2016; accepted 9 August 2016; accepted article preview online 19 August 2016 Loss of PDLIM2 in human lymphoma KD Wurster et al 2 expression have been observed in malignancies,23–25 and the RNA preparation and reverse transcriptase-PCR analyses PDLIM2 gene locus in 8p21.3 is in the minimal commonly deleted Total RNA preparation, complementary DNA synthesis and semiquantita- region in 8p21.3 in several B-cell non-Hodgkin lymphomas.26,27 tive PCR and real-time quantitative PCR analyses were performed as Here, we describe an inactivation of the putative ubiquitin previously described.17,28 Primers are listed in Supplementary Table 1. E3-ligase PDLIM2 as unifying defect of HRS cells. PDLIM2 loss of All PCR products were verified by sequencing. expression is associated with various mechanisms, including genomic alterations and promoter DNA methylation. Functionally, Analysis of apoptosis and proliferation loss of PDLIM2 promotes growth of HRS cells, facilitates the The percentage of viable and apoptotic cells was determined by Annexin activation of inflammatory TFs and results in deregulation of V-fluorescein isothiocyanate/propidium iodide double staining (Bender fl 7 differentially expressed cHL-associated genes, suggesting a MedSystems, Vienna, Austria) and ow cytometry as previously described. Proliferation of cells was determined by measurement of [3H]-thymidine pathogenic role in cHL. Furthermore, we demonstrate loss of incorporation using standard protocols. PDLIM2 expression in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. Western blot analyses Whole-cell and nuclear extract preparation and western blotting were performed as described5 with 30 μg whole-cell or 20 μg nuclear extracts. MATERIALS AND METHODS Primary antibodies were: PDLIM2 (sc-79988), IκBα (sc-371; both from Santa Cell lines and culture conditions Cruz, Heidelberg, Germany), PDLIM2 (ab68220; Abcam, Cambridge, UK) β HRS (L428, L1236, KM-H2, L591 (EBV+), U-HO1, SUP-HD1, HDLM-2, L540, and -actin (A5316, Sigma-Aldrich). L540Cy), pro-B lymphoblastic leukemia (Reh), Burkitt lymphoma (Namalwa, BL-60, BJAB), diffuse large B-cell lymphoma (SU-DHL-4), ALCL (K299, SU- Immunohistochemistry DHL-1, DEL, JB6, all anaplastic lymphoma kinase (ALK) positive; Mac-1, Mac- Immunohistochemical stainings of 20 primary cHL, 10 follicular lymphoma, 2A, FE-PD, DL40, all ALK negative), T-cell leukemia-derived (Jurkat, KE-37, 15 diffuse large B-cell lymphoma and 10 Burkitt lymphoma cases Molt-14, H9) and HEK293 cells were cultured as previously described.28 were performed as previously described.33 Primary antibodies (PDLIM2, Cells were electroporated in OPTI-MEM I using Gene-Pulser Xcell (Bio-Rad, PA5-31484, Thermo Scientific, Darmstadt, Germany; PDLIM2, ab68220, Munich, Germany) with 950 μF and 0.18 kV (L540Cy, HEK293) or 500 μF Abcam; IκBα, sc-371, Santa Cruz) were applied in a dilution of 1:100 and 0.3 kV (Reh, L591). Transfection efficiency was determined by pEGFP- (PDLIM2 antibodies) or 1:500 (IκBα) overnight. N3 (Clontech Laboratories, Mountain View, CA, USA) co-transfection. L591 and L540Cy cells were transfected with 40 μg of a pcDNA3-PDLIM2 Interphase cytogenetics transcript variant 2 (tv 2) construct along with 10 μg pEGFP-N3. GFP+ cells Fluorescence