NIK Controls Classical and Alternative NF-Kb Activation and Is Necessary for the Survival of Human T-Cell Lymphoma Cells

Published OnlineFirst March 27, 2013; DOI: 10.1158/1078-0432.CCR-12-3151

Clinical Cancer Human Cancer Biology Research

NIK Controls Classical and Alternative NF-kB Activation and Is Necessary for the Survival of Human T-cell Lymphoma Cells

Lina Odqvist1, Margarita Sanchez-Beato 1,3, Santiago Montes-Moreno1,10, Esperanza Martín-Sanchez 1, Raquel Pajares2, Lydia Sanchez-Verde 2, Pablo L. Ortiz-Romero4, Jose Rodriguez5, Socorro M. Rodríguez-Pinilla1,6, Francisca Iniesta-Martínez9, Juan Carlos Solera-Arroyo11, Rafael Ramos-Asensio12, Teresa Flores13, Javier Menarguez Palanca7, Federico García Bragado14, Puriﬁcacion Domínguez Franjo8, and Miguel A. Piris1,10

Abstract Purpose: Peripheral T-cell lymphomas (PTCL) are a heterogeneous entity of neoplasms with poor prognosis, a lack of effective therapies, and a largely unknown molecular pathology. Deregulated NF-kB activity has been associated with several lymphoproliferative diseases, but its importance in T-cell lymphomagenesis is poorly understood. We investigated the function of the NF-kB–inducing kinase (NIK), in this pathway and its role as a potential molecular target in T-cell lymphomas. Experimental Design: We used immunohistochemistry to analyze the expression of different NF-kB members in primary human PTCL samples and to study its clinical impact. With the aim of inhibiting the pathway, we used genetic silencing of NIK in several T-cell lymphoma cell lines and observed its effect on downstream targets and cell viability. Results: We showed that the NF-kB pathway was activated in a subset of PTCLs associated with poor overall survival. NIK was overexpressed in a number of PTCL cell lines and primary samples, and a pivotal role for NIK in the survival of these tumor cells was unveiled. NIK depletion led to a dramatic induction of apoptosis in NIK-overexpressing cell lines and also showed a more pronounced effect on cell survival than inhibitor of kappa B kinase (IKK) knockdown. NIK silencing induced a blockage of both classical and alternative NF-kB activation and reduced expression of several prosurvival and antiapoptotic factors. Conclusions: The results of the present study indicate that NIK could be a promising therapeutic target in these aggressive malignancies. Clin Cancer Res; 19(9); 2319–30. 2013 AACR.

Introduction an aggressive clinical course and poor outcome. First-line Peripheral T-cell lymphomas (PTCL) are a heterogeneous standard therapy is based on the combination chemother- family of non-Hodgkin lymphomas often associated with apy regimens usually used in B-cell lymphomas or solid tumors. As these regimens generally have poor response or high rates of recurrence, there is a need to develop targeted Authors' Afﬁliations: 1Molecular Pathology Programme and 2Biotechnol- therapies based on the signaling pathways that are aber- ogy Programme, Spanish National Cancer Research Centre (CNIO); 3Onco-hemathology Area, Instituto de Investigacion Sanitaria, HU Puerta rantly expressed in these T-cell malignancies (1, 2). de Hierro-Majadahonda, Madrid; 4Department of Dermatology, Institute Deregulated NF-kB activity plays a key role in the iþ12, Hospital 12 de Octubre Medical School University Complutense; development of multiple malignancies. Thus, constitutive 5Department of Internal Medicine, Hospital Universitario Severo Ochoa; 6DepartmentofPathology,Fundacion JimenezDíaz; 7Pathology Department, activation of NF-kB signaling has been observed in var- Hospital Gregorio Maranon;~ 8Pathology Department, Hospital Infanta Sofía, ious tumor types, including lymphomas, leukemias, and 9 10 Madrid; Hospital Virgen de la Arrixaca, Murcia; Pathology Department, solid tumors (3, 4). The NF-kB signaling pathway reg- Hospital Universitario Marques de Valdecilla, Universidad de Cantabria, IFIMAV, Santander; 11Pathology Department, Hospital Virgen de la Concha, ulates the transcription of many genes involved in cancer Zamora; 12Pathology Department, Hospital Son Espases, Palma de Mallorca; initiation and progression events, such as apoptosis, 13Pathology Department, University Hospital of Salamanca, Salamanca; and 14Pathology Department, Hospital Virgen del Camino, Pamplona, Spain proliferation, angiogenesis, and metastasis, and hence, several lymphoma types rely on NF-kB activity for their Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). proliferation and survival (4, 5). Genetic changes leading to constitutive activation of the pathway have been Corresponding Author: Margarita Sanchez-Beato, Instituto de Investigacion Sanitaria, Hospital Universitario Puerta de Hierro-Majada- detected in several hematologic tumors, emphasizing its honda, Calle Joaquín Rodrígo 2, E-28222 Majadahonda-Madrid, Spain. importance in the pathogenesis of these malignancies (6– Phone: 34-911916095; Fax: 34-911917863; E-mail: [email protected] 9). NF-kB can be activated either through the classical doi: 10.1158/1078-0432.CCR-12-3151 (canonical) pathway or the alternative (noncanonical) 2013 American Association for Cancer Research. pathway. Brieﬂy, activation of the classical pathway

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NF-kB inhibition could be used as a therapeutic strategy. Translational Relevance However, most studies have only addressed the classical Peripheral T-cell lymphomas (PTCL) are aggressive pathway, leaving the alternative pathway poorly malignancies presenting poor clinical outcome. There described. There are no specific NF-kB inhibitor drugs in are currently no effective treatments or targeted therapies clinical use for PTCL today, indicating a need for more available for these patients. Thus, studies aimed to detailed studies, the identification of new targets, and the elucidate the mechanisms contributing to these tumors development of novel specific inhibitors in the pathway. and to identify new therapeutic targets are required to In the present study, we show that both the alternative improve their dismal prognosis. Here, we show for the and classical NF-kB pathways are activated in a subset of first time that primary PTCLs expressing nuclear NF-kB primary PTCL samples associated with poor clinical out- are characterized by a significantly worse clinical outcome. To block the NF-kB signaling pathway in PTCL, we come compared with NF-kB–negative tumors, support- showed a key role for NIK as a regulator of both pathways ing a rationale for the exploration of NF-kB–interfering and showed that NIK-depleted PTCL cells present strongly strategies. We further identify NF-kB–inducing kinase reduced cell viability. These results highlight NIK as an (NIK) as a novel potential therapeutic target in T-cell attractive molecular target in T-cell lymphomas. lymphomas and show that targeting NIK might be more effective than previously suggested inhibitor of kappa B Materials and Methods kinase (IKK) inhibition. This study opens up opportu- Patient samples and cell lines nities for further translational studies and will hopefully Use of patient samples in this study was approved by the contribute to the future development of new targeted Clinical Research Ethics Committee of Hospital Universi- drugs useful in PTCL. tario Marques de Valdecilla (HUMV; Santander, Spain). Tumor biopsies before treatment were obtained from the CNIO Tumour Bank Unit. T cells from peripheral blood of healthy donors or patients with Sezary syndrome were results in nuclear translocation of mainly p50/p65 het- isolated through negative selection using the RosetteSep erodimers through the phosphorylation of IkB and p105 Kit (StemCell Technologies). The proportion of T cells þ by the inhibitor of kappa B kinase (IKK) complex. Acti- (CD3 ) in the samples was checked by flow cytometry and vation of the alternative pathway results in nuclear accu- ensured to be more than 90%. For the gene expression data mulation of p52/RelB heterodimers and depends on the in primary samples, 37 frozen PTCL cases were used, activation of the NF-kB–inducing kinase (NIK) and IKKa including 19 PTCL-not otherwise specified (PTCL-NOS), phosphorylation (reviewed in ref. 10). NIK (MAP3K14)is 15 angioimmunoblastic T-cell lymphomas (AITL), and 3 a serine/threonine kinase described as critical for the anaplastic large cell lymphomas (ALCL). The human T-cell activation of the alternativepathwaybyinducingphos- lymphoma cell lines DERL-7 (hepatosplenic g-d T-cell lym- phorylation of IKKa and p100, leading to p100 proces- phoma) and SR-786 (ALCL) were obtained from the Ger- sing with subsequent p52 generation and nuclear trans- man Collection of Microorganism and Cell Cultures location (11, 12). Nevertheless, the involvement of NIK (DSMZ). HuT 78 (Sezary syndrome), HH (cutaneous T- in the activation of the classical pathway has also been cell lymphoma), and MJ (PTCL, HTLV-positive) were noticed (13, 14). Signals from CD40, B-cell activating obtained from the American Type Culture Collection, factor receptor (BAFF-receptor), and lymphotoxin b and the cell line My-La (Mycosis fungoides) was obtained receptor have been shown to induce NIK-dependent from the European Collection of Cell Cultures. All cell NF-kB activation (15–18). In multiple myeloma, several lines were previously authenticated (year 2010–2011) by genetic abnormalities, such as deletions and inactivating DSMZ. mutations of the TNF receptor–associated factor 3 (TRAF3), have been shown to lead to the stabilization Immunohistochemistry ofNIK(19).Similarly,inB-celllymphomas,activating Tissue microarrays of paraffin-embedded tumor biopsies mutations in the BAFF-receptor are known to activate NF- or cell lines were used for immunohistochemistry and kB in a NIK-dependent manner (20). Knockdown of NIK evaluated by the pathologists participating in the project in several types of malignancies, such as multiple mye- (M.A. Piris and S. Montes-Moreno). Cases presenting nucle- loma, diffuse large B-cell lymphoma (DLBCL), adult T- ar staining in more than 20% of tumor cells were considered cell leukemia, and melanoma, has been associated with positive, grade 1 (pos þ), whereas cases with strong nuclear antitumor effects (14, 16, 21, 22), suggesting that NIK staining in more than 50% of cells were considered positive, could be a therapeutic target in some cancers. However, grade 2 (pos þþ). Antibodies and conditions are summa- whether NIK is involved in NF-kB activation and tumor- rized in Supplementary Table S1. igenesis or not is dependent on the cell type and cellular context and has not been studied in PTCL. Cutaneous T- Survival and correlation analyses cell lymphoma cell lines and primary samples undergo The Kaplan–Meier method applying the log-rank test was apoptosis upon treatment with the proteasome inhibitor used to estimate the differences in overall survival (OS) bortezomib or an IKKb inhibitor (23–25), suggesting that between NF-kB–positive and -negative cases in a series of 77

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Table 1. Characteristics of patients included in conducted following standard protocols. For NIK detection, cell lines were treated for 3 hours with 20 mmol/L protea- the survival analysis some inhibitor MG132 (Sigma). MG132 was exclusively added to cells for NIK detection, and separate untreated cells Characteristics Patients, n Percentage were collected to detect other proteins. Western blot anal- Gender yses were quantified using the ImageJ program (National Male 46 59.7 Institutes of Health, Bethesda, MD). Female 31 40.3 Age, y Gene expression microarray analyses 60 32 41.6 All gene expression experiments used 4 44 K Whole >60 45 58.4 Human Genome Oligo Microarrays (Agilent Technologies, Ann Arbor stage Inc.). For gene expression analysis of primary PTCL samples, I–II 17 22.1 37 frozen biopsies were used, and the Gene Set Enrichment III–IV 60 77.9 Analysis (GSEA) tool (http://www.broad.mit.edu/gsea/) IPI was applied to classify the expression profiles into func- MAP3K14 0–2 34 44.2 tional pathways. The gene sets correlated with 3–5 41 53.2 (NIK) expression in primary samples were identified using — 2 2.6 Pearson correlation. Gene sets with a false discovery rate ECOG (FDR) < 0.10 were considered significant. The microarray 0–1 53 68.8 data are available at the Gene Expression Omnibus under 2–4 24 31.2 accession number GSE36172. After NIK knockdown in cell LDH level lines, pellets were collected 48 hours after siRNA transfec- Normal 34 44.2 tion and RNA was extracted. Three independent experi- High 42 54.5 ments were carried out in My-La and SR-786 cell lines, and — 1 1.3 each sample was hybridized onto a separate microarray. PTCL subtype The gene sets up- or downregulated in NIK knockdown AITL 30 39.0 cells were identified with the GSEA tool by applying a PTCL-NOS 47 61.0 limma t test (FDR < 0.10). Genes differentially expressed Total 77 100.0 between control cells and NIK knockdown cells were identified using a paired t test (http://pomelo2.bioinfo.cnio.es) Abbreviations: ECOG, Eastern Cooperative Oncology and visualized using Gene Cluster and Treeview Group; IPI, International Prognostic Index; LDH, lactate (http://rana.lbl.gov/EisenSoftware.htm). dehydrogenase. RNA interference siRNAs against MAP3K14 (NIK), IKBKB (IKKb), and patients with PTCL (Table 1). Samples presenting nuclear CHUK (IKKa), or a nontemplate control (NTC; 100 staining of NF-kB (p52, p50, RelB, p65, or c-Rel) in more nmol/L stealth siRNA; Invitrogen) were used for genetic than 50% (pos þþ) of tumor cells were considered silencing. siRNAs were introduced into the cells by micro- positive for NF-kB. Multivariate survival analysis includ- poration (Microporator MP-100; Digital Bio). ing relevant clinical parameters (see Table 1) was conducted using a Cox regression analysis. To determine the NF-kB–binding activity independence of variables, the Pearson x2 test was used. Nuclear cell fractions were isolated using the BioVision SPSS version 15.0 (SPSS Inc.) was used to carry out these Nuclear/Cytosol Fractionation Kit (BioVision), and 5 mgof tests. Values of P < 0.05 were considered statistically nuclear extract was used to quantify the NF-kB transcription significant. activation using the ELISA-based TransAM NF-kB Family Transcription Factor Assay Kit (Active Motif), following the Quantitative RT-PCR manufacturer’s instructions. Nuclear extracts were plated in RNA was isolated using the RNeasy Mini Kit (Qiagen). triplicate. cDNA was synthesized from 500-mg DNaseI-treated RNA with SuperScript II Reverse Transcriptase (Invitrogen) Cell viability and cell-cycle analysis and random primers. Human GUSB and 18S expression Cell viability and cell cycle were assessed in a FACS were used as endogenous controls in samples and cell Canto II flow cytometer (BD Biosciences), and FlowJo lines. software (FlowJo version 7.6.1; TreeStar Inc.) was used for data quantification. Cells staining negative for Annexin Immunoblotting V–allophycocyanin conjugate (APC) and 40,6-diamidino- Protein was extracted using radioimmunoprecipitation 2-phenylindole (DAPI) were considered viable. To estimate assay (RIPA) lysis buffer supplemented with protease and the number of cells in each stage of the cell cycle, cells were phosphatase inhibitors, and Western blot analysis was fixed with ethanol and stained with propidium iodide.

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Reagent information, primer sequences, and additional NIK is overexpressed in PTCL cell lines and primary protocols are described in the online Supplementary samples, and its expression is correlated with NF-kB Data. activation Because NIK is involved in the activation of NF-kBin some cells and settings, we examined whether NIK was Results involved in NF-kBactivationinT-celllymphomas.We Nuclear NF-kB is present in primary PTCL samples and observed a remarkable overexpression of NIK mRNA in is associated with worse clinical outcome several PTCL cell lines and primary Sezary syndrome Even though NF-kB activation has been reported in samples compared with T lymphocytes from healthy PTCL, there are few studies of patient material, and the donors (Fig. 2A). The cell lines expressing high levels of expression pattern of the various NF-kBmembershasnot NIK mRNA also expressed elevated protein levels of NIK been described in detail. We conducted immunohis- (Fig. 2B). Although DERL-7 lacked nuclear expression of tochemistry on paraffin-embedded tissues from patients alternative NF-kB factors (p52 and RelB), the other cell with the most common types of PTCL: AITL, ALCL, and line with low NIK levels, HuT 78, expressed both nuclear PTCL-NOS, to examine the nuclear expression of NF-kB. p52 and RelB (Supplementary Table S3). However, HuT Nuclear expression of components defining both the 78 expresses a truncated form of p100 (Fig. 2B), which classical (p50 and c-Rel) and alternative (p52 and RelB) has previously been described to activate the alternative pathways (Fig. 1A and B) was detected in the majority of pathway in a NIK-independent manner (30). To detect PTCLs. To represent different levels of NF-kB expression, NIK protein levels in primary PTCL samples, we tested a 2 positivity thresholds were used: 20% (pos þ)and50% handful of different NIK antibodies in paraffin-embedded (pos þþ) positive nuclei. Surprisingly, the commonly used tissues, but none of these provided consistent results. For marker for NF-kB activation, p65, showed only cytoplasmic that reason, we used gene expression microarray data to expression in most cases, suggesting that other factors of the compare the expression of NIK (MAP3K14)andNF-kB family are more frequently involved in NF-kBsignalingin target genes (6) in a series of 37 PTCL samples. Using PTCL. A subset of cases expressed highly elevated levels of GSEA, we found a significant positive correlation between NF-kB(posþþ), being most frequent in the CD30-positive the expression of NIK and NF-kB target genes (Fig. 2C), ALCLs. Moreover, a significant positive correlation (P < suggesting that NIK may be involved in NF-kB signaling 0.05) was established between nuclear p50 and p52 expres- in these tumors. Other gene sets significantly correlated sion (Fig. 1C), indicating frequent activation of both path- with the expression of NIK are listed in Supplementary ways in the same sample. Table S4. Contradictory results have been reported for the clinical correlation of NF-kB in different tumors (5, 26–28). NIK is involved in both classical and alternative NF-kB To estimate the impact of nuclear NF-kB expression on signaling in T-cell lymphomas the clinical outcome of patients with PTCL, we did a Whether NIK participates in classical NF-kB pathway Kaplan–Meier analysis to compare the OS between NF- activation as well as in alternative pathway regulation is kB–positive and –negative tumors in a series of 77 PTCL currently unclear. To study the role of NIK in NF-kB sig- cases (Fig. 1D; see patient characteristics in Table 1). naling in T-cell lymphoma cells, we knocked down NIK Tumors with more than 50% positive nuclei for any using 2 different siRNAs in 2 PTCL cell lines (My-La and SR- NF-kB factor were considered positive. Similar thresholds 786). The 2 siRNA sequences induced different levels of have previously been used for immunohistochemistry of knockdown efficiencies (Fig. 3A), allowing us to study the NF-kB in other studies in the lymphoma field (6, 28, 29). effect of a dose-dependent decrease in NIK. As expected, the Only PTCL-NOS and AITL subtypes were included in the levels of p52 were reduced and the levels of p100 were survival analysis, as these patients did not present any initially increased after NIK knockdown, indicating an significant difference in basal OS due to PTCL subtype attenuation of p100 processing. We also observed a similar (Supplementary Fig. S1). When NF-kBwastakeninto but somewhat delayed decrease of p50 levels and an account, however, patients with NF-kB–positive tumors increase in the levels of p105, linking NIK to the regulation had a significantly worse OS compared with those in the of classical NF-kB activation as well (Fig. 3A and C and NF–kB-negative group (log-rank test; P ¼ 0.003) with a 2- Supplementary Fig. S2). Figure 3B confirms that the knock- year OS of 41.7% compared with 67.9%, respectively. NF- down of NIK gave rise to decreased nuclear levels of both kB activation in PTCL was still significantly associated p52 and p50. The NF-kB DNA-binding activity of all 5 with inferior OS when the impact of other clinical cov- proteins, measured by TransAM ELISA assay, was decreased, ariates was taken into account, although the International supporting a role for NIK in both classical and alternative Prognostic Index (IPI) was still a more accurate estimate NF-kB regulation in PTCL (Fig. 3D). of prognosis in our series (Supplementary Table S2). The observation that NF-kB signaling is activated in a subset NIK is necessary for the survival of PTCL cell lines with of T-cell lymphomas with very poor clinical outcome high NIK levels suggests the merit of evaluating NF-kB inhibition strate- According to our data, My-La and SR-786 are character- gies in these tumors. ized by NIK overexpression and an active NF-kB pathway

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A p52 p50 p65 RelB c-Rel

B 100% 90% 80% 70% pos ++ 60% pos + 50%

% Cases % neg 40% 30% 20% 10% 0% p52 p50 RelB c-Rel p65 p52 p50 RelB c-Rel p65 p52 p50 RelB c-Rel p65

PTCL-NOS AITL ALCL CD 1.0 100 P = 0.05 90 0.8 P = 0.003 80 70 60 0.6 NF-κB negative

50 OS NF-κB positive 40 0.4 30 % p50-positive cases p50-positive % 20 0.2 10 0 p52-negative p52-positive 0.0

0 20 40 60 80 100 120 Months

Figure 1. Expression of NF-kB in human PTCL samples. A, immunohistochemical staining of p100/p52, p105/p50, p65, RelB, and c-Rel in primary paraffin- embedded PTCL samples shows nuclear staining of NF-kB in most cases (B). The category pos þ indicates nuclear staining of more than 20% of the tumor, whereas pos þþ indicates nuclear staining in more than 50% of the tumor. The percentage of the total number of cases (57 PTCL-NOS, 42 AITLs, and 28 ALCLs) in each PTCL subtype is shown. Comparison of the numbers of tumors with high levels of nuclear expression (pos þþ) of p50 or p52 reveals a significant positive correlation between their expression, in which the majority of cases positive for p52 also were positive for p50 (C). D, Kaplan–Meier curve representing the OS times of patients with NF-kB–positive and –negative PTCL. The NF-kB–positive group (red line) has a significantly worse OS (P < 0.05; log-rank test) than the NF-kB–negative group (blue line).

(Fig. 2A and B and Supplementary Table S3). We measured longer period, we repeated the siRNA transfection 4 days cell viability after NIK silencing in these cells by flow after the first microporation. Strikingly, after 1 week of NIK cytometry using Annexin V and DAPI staining. NIK knock- depletion, nearly all cells had undergone apoptosis, show- down led to a dramatic increase in cell death compared with ing the essential role of NIK in the viability of these tumor NTC-transfected cells, suggesting that NIK is necessary for cells (Fig. 4B). The appearance of cleaved caspase-3 after the survival of these cells. The cell death observed after NIK NIK knockdown in both My-La and SR-786 indicates an knockdown increased as knockdown became more efficient induction of a caspase-dependent apoptotic pathway (Fig. (Fig. 4A). To study the effect of NIK knockdown over a 4C). Interestingly, NIK silencing in DERL-7 or HuT 78 cell

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A NIK expression in PTCL cell lines NIK expression in Sézary syndrome T cells 35 18 30 16 14 25 12 20 10 15 8 6 10 4 5 Relative NIK expression NIK Relative expression NIK Relative 2 0 0 Control Control Control My-La SR-786HuT 78 DERL-7 HH MJ Control Control Control SS 1 SS 2 SS 3 SS 4 SS 5 1 2 3 1 2 3 BC Enrichment plot of NF-κB target genes My-La SR-786 HuT 78 DERL-7 HH MJ

NIK

α-Tubulin

p100

p52

p105

p50 Rank in ordered dataset

α-Tubulin FDR value: 0.093

Figure 2. NIK expression in PTCL cell lines and samples. A, reverse-transcription quantitative PCR analysis of NIK expression in PTCL cell lines and isolated T cells from patients with Sezary syndrome (SS1–SS5) showed strongly elevated levels of NIK compared with T cells from healthy donors (control 1–3). B, NIK protein levels were detected after MG-132 treatment, and a comparable expression pattern was seen between NIK mRNA and protein levels among the cell lines. C, GSEA of gene expression microarray data from 37 PTCL cases shows a signiﬁcant positive correlation (FDR < 0.10) between the expression of NIK (MAP3K14) and NF-kB target genes.

lines, presenting low NIK levels, had no effect on cell NIK knockdown leads to decreased expression of NF- survival (Fig. 4D), suggesting that NIK targeting has a kB target genes and downregulation of prosurvival selective effect only on the cells presenting elevated NIK genes levels. No obvious alterations of the cell-cycle distribution To gain a better insight into the mechanisms involved in were observed after NIK knockdown, apart from there being NIK-dependent survival, we analyzed the gene expression more cells in sub-G1 (Supplementary Fig. S3). profile induced 48 hours after NIK knockdown in My-La To rule out whether the toxicity of NIK knockdown was and SR-786 cells. Knockdown efficiencies are shown in due to a blockade of the classical or alternative NF-kB Supplementary Fig. S5. GSEA applying a limma t test com- pathway, we knocked down either IKKa,IKKb,orboth,in paring siNIK1 and siNIK2 with control cells was conducted. these cell lines (Fig. 4E and Supplementary Fig. S4). The gene sets significantly lost (FDR < 0.10) in the siNIK Surprisingly, IKKa and IKKb knockdown (separately or cells included the NF-kB target genes, the JAK–STAT in combination) only led to a slight increase in apoptosis pathway, and targets of XBP1, and are described in Supple- compared with NIK knockdown. Moreover, only the mentary Table S5. NF-kB target genes were significantly knockdown of NIK, but not the knockdown of IKK, was underrepresented after NIK knockdown, indicating again able to strongly reduce the levels of p52 and p50 (Fig. 4E a pivotal role for NIK in NF-kB activation (Fig. 5A). A paired and F). These results suggest that NIK might have, at least t test revealed 395 genes in My-La and 94 genes in SR-786, in part, IKK-independent roles that confer survival on which were significantly and differentially expressed (FDR these cells, and that targeting NIK could be a more < 0.05 and log2 fold change > 0.6 in either direction) effective therapeutic approach than IKK inhibition in between NIK knockdown and control cells (Fig. 5B; see these tumors. Supplementary Table S6 for the complete list of genes).

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A My-La SR-786 B 24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h Nucleus siNTC – + – – + – – + – – + – – siNTC – + – – + – – + – – + – – siNIK – – 1 2 – 1 2 – 1 2 – 1 2 siNIK – – 1 2 – 1 2 – 1 2 – 1 2 NTC siNIK1 siNIK2 NIK NIK p52 α -Tubulin α-Tubulin 1.00 0.42 0.10

p100 p100 p50 1.00 0.38 0.11 p52 p52 Lamin B1 α-Tubulin α-Tubulin

p105 p105

p50 p50

α-Tubulin α-Tubulin

C p52 and p50 levels in My-La after NIK knockdown D 1.2

1.0 NIK knockdown in My-La 0.8 p52 2 p50 0.6 1.8 Ratio vs NTC Ratio 0.4 1.6 0.2 1.4 siNTC 1.2 0.0 siNIK NTC siNIK1siNIK2 NTC siNIK1siNIK2 NTC siNIK1siNIK2 NTC siNIK1siNIK2 1 24 h 48 h 72 h 96 h 0.8 0.6

p52 and p50 levels in SR-786 after NIK knockdown 0.4 1.4 0.2 DNA-binding activity (OD 450 nm)DNA-binding activity (OD 450 1.2 0 1.0 p52 RelB p50 p65 c-Rel 0.8 p52 p50 0.6 Ratio vs NTC Ratio 0.4 0.2 0.0 NTC siNIK1siNIK2 NTC siNIK1siNIK2 NTC siNIK1siNIK2 NTC siNIK1siNIK2 24 h 48 h 72 h 96 h

Figure 3. Knockdown of NIK in T-cell lymphoma cell lines. A, NIK was knocked down using 2 siRNAs (siNIK1 and siNIK2) or a NTC (siNTC) in My-La and SR-786. After NIK knockdown, the expression of NIK, p100/p52, and p105/p50 was analyzed by Western blot analysis. B, nuclear expression of p50 and p52 after NIK knockdown in My-La. The quantification of the signal is represented under each blot as a ratio against the siNTC-transfected cells. C, quantification of the immunoblots confirms a decrease in the levels of both p50 and p52 after NIK knockdown. The data are represented as the mean SD of 3 independent experiments. D, DNA-binding activity of p52, RelB, p50, p65, and c-Rel 48 hours after NIK knockdown was measured using the ELISA-based TransAM assay and reveals a reduction of classical and alternative NF-kB activation.

Reverse transcription quantitative PCR (RT-qPCR) was con- gram in NIK-expressing versus NIK-nonexpressing cells. ducted on selected genes to validate the gene expression Apart from known NF-kB target genes, NIK depletion also data (Fig. 5C and D). Several NF-kB target genes involved in modulated the expression of other genes involved in tumor- cancer cell survival were downregulated upon NIK silenc- igenesis, such as Yes-associated protein 1 (YAP1), paraox- ing, such as the antiapoptotic BCL2L1 [Bcl-x(L)] and CFLAR onase 2 (PON2), and Kruppel-like factor 2 (KLF2). In (c-FLIP) as well as several interleukins. Interleukin (IL)-6 summary, NIK knockdown leads to a decrease in the and IL-21 are cytokines with known functions in cell pro- expression of NF-kB target genes and modulates the expres- liferation and survival of cancer cells and were strongly sion of many genes involved in tumor growth and survival. downregulated after NIK depletion in both cell lines. Curi- ously, despite an activated NF-kB pathway in DERL-7 and Discussion HuT 78 as well, only the NIK-expressing My-La and SR-786 The molecular pathology of PTCLs has been poorly express high levels of these interleukins (Supplementary characterized, and the dismal prognosis of these neoplasms Fig. S6), indicating a different NF-kB transcriptional pro- and the lack of efﬁcient therapies demand further studies

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ABNIK knockdown in My-La 80 ** 70 ** NTC siNIK 60 * * MOCK 50 siNTC

40 siNIK1 * ** 30 siNIK2

% Nonviable cells % Nonviable 20 10 0

48 h72 h96 h DAPI

NIK knockdown in SR-786 50 45 * 40 35 * 30 25 * Annexin-V-APC 20 * 15 % Nonviable cells % Nonviable 10 D 12 5 My La 0 SR-786 48 h 72 h 96 h 10 HuT 78 C 24 h 48 h 72 h 96 h 8 DERL-7 siNTC – + – – + – – + – – + – – siNIK – – 1 2 – 1 2 – 1 2 – 1 2 6 Cleaved caspase-3 4

My-La death cell in Increase

α-Tubulin (fold change siNIK/siNTC) 2 Cleaved caspase-3 0 SR-786 0 h 48 h 72 h 96 h α-Tubulin

F 45 SR-786 ** 40 35 E 72 h 96 h 30 25 siNTC +––––––+–––––– 20 * siIKKα –12 ––2 ––12 ––2 – * 15 siIKKβ –––122––––122–

% Nonviable cells 10 siNIK ––––––2––––––2 5 IKKβ 0 IKKα siNTC siIKKα 1siIKKα 2siIKKβ 1siIKKβ 2siIKKα + siNIK2 siIKKβ p100 90 My-La 80 * p52 70 60 50 p105 40 ** * 30 * * * p50 % Nonviable cells 20 10 α-Tubulin 0 siNTC siIKKα 1siIKKα 2siIKKβ 1siIKKβ 2siIKKα + siNIK2 siIKKβ

Figure 4. The effect of NIK knockdown on PTCL cell survival. A, NIK knockdown (siNIK) induces cell death in My-La and SR-786 cells compared with untransfected (MOCK) or nontemplate-transfected (siNTC) cells, as measured by ﬂow cytometry using DAPI and Annexin V staining. The data are represented as the mean SD of 3 independent knockdown experiments. B, only 1.26% of cells remain viable 7 days after NIK knockdown in My-La (DAPI/Annexin V–negative). C, the cleavage of caspase-3 was detected after NIK depletion in both MyLa and SR-786 cells. D, increase in cell death in cell lines with high (My-La and SR-786) or low (HuT 78 and DERL-7) NIK expression, represented as the ratio between the values of cell death in NIK knockdown (siNIK2) and control (siNTC) cells. E, knockdown of IKKa,IKKb, or NIK in My-La and evaluation of the expression of p100/p52 and p105/p50. F, percentage of nonviable SR-786 cells after IKKa,IKKb, or NIK silencing show a more potent induction of apoptosis after NIK knockdown compared with IKK knockdown. *, P < 0.05; **, P < 0.01.

to clarify their molecular background and to identify (21, 23). NF-kB activation has previously been reported in new therapeutic targets. Previous data suggest that NF-kB a subset of human PTCLs using gene expression data activation is a key step in T-cell lymphoma pathogenesis and immunohistochemistry for classical NF-kB subunits

2326 Clin Cancer Res; 19(9) May 1, 2013 Clinical Cancer Research

NIK in T-cell Lymphoma

A B My-La SR-786 Enrichment plot of NF-κB target genes siNIK: 1 1 2 2 2 siNIK: 1 1 2 2 2 PON2 IL6 YAP1 IL21 TNFSF4 ADAM8 IL6 IL15 XBP1 NFKBIA IL21 IL22 IL9 ADAM8

YAP1 LTA

CCL5 IL26 IL1R2 CCL17 IL1A FAS CD44 TNFAIP6

Rank in ordered dataset IL1A C My-La 1.4 BIRC3 TNFSF4 NFKBIE 1.2 LTB 1 NTC 0.8 siNIK1 IL9 siNIK2 0.6 CFLAR LTA 0.4 Relative expression Relative 0.2 NFKBIA GADD45A 0 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h IL15 IL6 IL21 BCL2L1 CFLAR NFKBIA BIRC3 BCL2L1 SR-786 1.6 1.4 1.2 NTC 1 siNIK1 0.8 siNIK2 0.6 0.4 Relative expression Relative 0.2 0 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h IL6 IL21 BCL2L1 CFLAR NFKBIA

D KLF-2 expression KLF2 60 50 NTC 40 siNIK1 -2 0 +3 siNIK2 CASP7 30 20

Relative expression 10 0 KLF2 My-La SR-786 -2 0 +3

Figure 5. Gene expression proﬁle after NIK knockdown. Whole genome microarray analysis was conducted 48 hours after NIK knockdown in My-La and SR- 786. A, GSEA enrichment plot of the NF-kB target genes. The NF-kB target genes were signiﬁcantly underrepresented in the NIK knockdown cells. B, heatmaps of differentially expressed genes (FDR < 0.05 and log2 fold change > 0.6) between control and NIK knockdown cells. Negative log2 fold changes (ratio siNIK/control) are represented in green (downregulation in siNIK cells) and positive fold changes are represented in red (upregulation in siNIK cells). C and D, relative gene expression in siNIK cells compared with the NTC measured by RT-qPCR.

(24, 27, 29, 31). However, only a few studies have described samples, characterized by the nuclear expression of p50, the alternative pathway status or the clinical impact of this p52, RelB, and c-Rel, whereas nuclear expression of p65 was activation. Here, we found markers of activation of the usually absent. Nuclear expression of classical or alternative classical and alternative pathways in a subset of PTCL components was signiﬁcantly associated with worse OS in

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Odqvist et al.

patients with PTCL, suggesting that aberrant NF-kB activa- ylation of p100 in an IKKa-independent manner has been tion may confer enhanced survival or treatment resistance reported (11). It is not clear whether the effect on p105 on these tumors, and these data support the exploration of processing is a direct effect of NIK or an indirect effect of therapies that interfere with NF-kB activation. These find- reduced alternative activity. ings mimic those already described in DLBCL, in which the To investigate the possible downstream effectors of NIK- more aggressive activated B cell–like (ABC) subtype is regulated tumorigenesis, we conducted gene expression distinguished by increased NF-kB activation (5, 26). In analysis of NIK-silenced T-cell lymphoma cells. As expected, contrast with our results, Martinez-Delgado and colleagues many of the genes differentially expressed between NIK- (27) associated the expression of NF-kB–related genes, silenced and control cells were NF-kB target genes known to using gene expression microarrays, with a favorable clinical be important in regulating tumor growth and survival. The outcome in PTCL. These differences might be explained by expression of antiapoptotic proteins is one of the mechan- the genes included in the NF-kB signature (not restricted to isms by which tumor cells manage to survive in the envi- NF-kB target genes) and the signals from the tumor stroma ronment and to resist chemotherapy (39). After NIK knock- reported by Martinez-Delgado and colleagues versus the down, we observed a downregulation of antiapoptotic nuclear expression of NF-kB subunits as assessed by IHC in genes such as CFLAR (Bcl-xl), BIRC3 (cIAP2), and BCL2L1 our study. (c-FLIP), which might explain the strong induction of Even though there is evidence that NF-kB inhibition has an apoptosis observed in these cells after NIK inhibition. The antitumor effect in some circumstances (5, 32), the need interleukins IL6 and IL21, which were highly expressed in remains to identify therapeutic targets in the pathway and to cell lines that were sensitive to NIK knockdown but not in develop specific NF-kB inhibitors. NF-kB inhibitors targeting resistant cell lines, were strongly downregulated after NIK mutated or aberrantly expressed molecular targets in a par- depletion. These cytokines are important for B- and T-cell ticular tumor might be preferable to broad NF-kB inhibition development and a proper regulation of the immune that also abolishes normal NF-kB activation. We showed that response but have also been widely studied in several NIK was highly overexpressed in a subset of PTCL cell lines tumors owing to their protumorigenic activity and their and tumor samples and that its expression was significantly roles as targets for cancer therapy (40, 41). Both of these associated with NF-kB activation. NIK has previously been cytokines are NF-kB target genes but are also involved in found to be overexpressed at the RNA and/or protein level in JAK–STAT signaling, which also is consistent with our other cancers, such as melanoma, multiple myeloma, observation that NIK knockdown leads to downregulation DLBCL, mucosa-associated lymphoid tissue (MALT) lym- of the JAK–STAT pathway. The expression of other genes phoma, and adult T-cell leukemia (14, 16, 21, 22, 33). In involved in tumorigenesis, not described as NF-kB targets, some cases, genetic alterations such as gene amplifications, was also modulated after NIK knockdown. For example, the translocations, or mutations in NIK, or alterations in genes expression of the oncogene YAP1 (42) was reduced and an regulating the stability of NIK protein, are described, empha- upregulation of KLF2, a tumor suppressor with antiproli- sizing the role of NIK in tumorigenesis (14, 19, 34). From ferative effects known to be silenced in tumor cells (43), was previous reports, the role of NIK in the alternative NF-kB observed. This can indicate either NF-kB–independent pathwayis clear, but the involvement of NIK in the regulation functions of NIK or interaction between the NF-kB pathway of the classical pathway seems to be signal and cell type and other pathways (44). dependent (13, 35). For example, in melanoma and pancre- The upstream signaling events leading to NIK over- atic cancer, NIK only affects the alternative pathway (22, 36), expression and/or NF-kB activation in T-cell lymphomas whereas NIK regulates both pathways in DLBCL and multiple remain to be clarified. Several factors are known to trigger myeloma (14, 16). In PTCL, we showed that NIK is involved NF-kB activation in other tumors, including signals pro- in both classical and alternative pathway activation, as NIK vided by the tumor microenvironment or mutations that knockdown led to decreased expression and DNA-binding give rise to a constitutive activation of the pathway. In activity of both classical (p65, p50, and c-Rel) and alternative PTCL, apart from the previously reported p100 trunca- (p52 and Rel-B) NF-kB transcription factors. Moreover, NIK tions (7, 45), genetic lesions in NIK or other NF-kB knockdown also altered the expression of many genes com- pathway genes have still not been reported. Other pos- monly associated with classical NF-kB activation, such as IL6, sible contributions to NF-kBactivationinT-celllympho- IL10,andNFKBIA (37, 38). However, NIK does not seem to mas could be oncogenic viruses (46), signals from CD30 be strictly essential for NF-kB activation in all PTCL cells, as or the T-cell receptor (47, 48), or epigenetic mechanisms, we found that nuclear NF-kB was not exclusively expressed in such as the previously reported polycomb-mediated NIK-overexpressing cell lines. Thus, the pathway may be repression of mir31 (49). Even though we observed a activated by different mechanisms in these cells, and a proper lack of mir31 in our cell lines, this absence was indepen- identification of upstream mechanisms is important for dent of the levels of NIK (data not shown). It could also efficient NF-kB inhibition. The fact that NIK knockdown led be hypothesized that the activation of NIK and NF-kBcan to a more efficient reduction of p50 and p52, compared with be an effect of deregulated signaling of other pathways IKKa or IKKb knockdown, suggests that NIK can, at least in linked to T-cell lymphomagenesis, such as the phosphoi- part, regulate the NF-kB pathway by IKK-independent nositide 3-kinase/AKT, Notch, or JAK–STAT pathway, mechanisms. Consistent with these results, direct phosphor- whichcanallresultinNF-kB activation (44).

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The present study reveals a pivotal role for NIK in the Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Odqvist, S. Montes-Moreno, L. Sanchez-Verde, survival of T-cell lymphoma cells. NIK knockdown strongly P.L. Ortiz-Romero, J. Rodriguez, S.M. Rodrı´guez-Pinilla, F. Iniesta-Martıńez, reduces the cell viability of PTCL cell lines and is shown to R. Ramos-Asensio, T. Flores, J.M. Palanca, F.G. Bragado, P.D. Franjo, M.A. be more effective than IKK inhibition in these cells. Impor- Piris Analysis and interpretation of data (e.g., statistical analysis, biosta- tantly, the fact that cells with a low level of expression of NIK tistics, computational analysis): L. Odqvist, M. Sanchez-Beato, E. Martıń- were not affected by NIK knockdown suggests a selective Sanchez, J. Rodriguez, S.M. Rodrı´guez-Pinilla, T. Flores, M.A. Piris Writing, review, and/or revision of the manuscript: L. Odqvist, M. toxicity of NIK inhibition in NIK-overexpressing lymphoma Sanchez-Beato, P.L. Ortiz-Romero, J.M. Palanca, M.A. Piris cells. Moreover, although NIK-deficient mice exhibit defects Administrative, technical, or material support (i.e., reporting or orga- in lymphoid organogenesis, they do not present any gross nizing data, constructing databases): L. Odqvist, E. Martıń-Sanchez, R. Pajares, L. Sanchez-Verde, P.L. Ortiz-Romero, S.M. Rodrı´guez-Pinilla, J.C. phenotypic changes (50), suggesting that pharmacologic Solera-Arroyo inhibition of NIK might be safer than broad NF-kB inhi- Study supervision: M. Sanchez-Beato, M.A. Piris bition. To develop NF-kB–based cancer therapies efficiently and safely, it is necessary to identify molecular targets as well Acknowledgments as biomarkers with which to stratify patients who are likely The authors thank the Tumour Bank Unit of the CNIO (Marıá-Jesus Artiga, Laura Cereceda, and Maria Encarnacion Castillo) and all the members to benefit from the therapy. PTCL are highly aggressive of the laboratory at the CNIO and IFIMAV, especially Beatriz Herreros and malignancies that currently lack efficient therapies. Our Daniel Martin Perez, for their help and discussion, and Pierfrancesco Vargiu findings indicate that NIK is a promising molecular target for technical supervision. The authors also thank Francisco X. Real for excellent scientific discussion. in NIK-overexpressing PTCL, a conclusion that should be taken into account in further validation and development of Grant Support specific NIK inhibitors. This work was supported by grants from the Ministerio de Ciencia e Innovacion (SAF2008-03871), Fondo de Investigaciones Sanitarias (FIS 08/ Disclosure of Potential Conflicts of Interest 0856 and RTICC RD06/0020/0107), and Asociacion Espanola~ Contra el Pablo L. Ortiz-Romero has received honoraria from serving on the speak- Cancer. L. Odqvist is supported by a grant from the Comunidad Autonoma ers’ bureau of Bristol-Myers Squibb and Eisay and is a consultant/advisory de Madrid, Spain. M. Sanchez-Beato is supported by a Miguel Servet contract board member of AOP Orphan. No potential conflicts of interest were (CP11/00018) from the Fondo de Investigaciones Sanitarias. disclosed by the other authors. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Authors' Contributions this fact. Conception and design: L. Odqvist, M. Sanchez-Beato, P.L. Ortiz-Romero, M.A. Piris Development of methodology: L. Odqvist, E. Martıń-Sanchez, L. Sanchez- Received October 8, 2012; revised February 12, 2013; accepted March 10, Verde 2013; published March 27, 2013.

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2330 Clin Cancer Res; 19(9) May 1, 2013 Clinical Cancer Research

NIK Controls Classical and Alternative NF-κB Activation and Is Necessary for the Survival of Human T-cell Lymphoma Cells

Lina Odqvist, Margarita Sánchez-Beato, Santiago Montes-Moreno, et al.

Clin Cancer Res 2013;19:2319-2330. Published OnlineFirst March 27, 2013.

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