Waldenström's macroglobulinemia harbors a unique proteome where Ku70 is severely underexpressed as compared with other B-lymphoproliferative disorders

Abstract

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70--a component of the non homologous end-joining pathway--might be relevant in disease pathophysiology.

Citation: Blood Cancer Journal (2012) 2, e88; doi:10.1038/bcj.2012.35& 2012 Macmillan Publishers Limited All rights reserved 2044-5385/12

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ORIGINAL ARTICLE

Waldenstrms macroglobulinemia harbors a unique proteome where Ku70 is severely underexpressed as compared with other B-lymphoproliferative disorders

A Perrot1,2,3, C Pionneau4,5, N Azar6, C Baillou5,7, FM Lemoine5,7, V Leblond5,6, H Merle-Bral5,6, M-C B n3, R Herbrecht8, S Bahram1,2,9 and L Vallat1,2,9

Waldenstrms macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic proles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); conrmed at both the protein and RNA levels in an independent cohort of patients. Hence, we dene a distinctive proteomic prole for WM where the downregulation of Ku70a component of the non homologous end-joining pathwaymight be relevant in disease pathophysiology.

Blood Cancer Journal (2012) 2, e88; doi:10.1038/bcj.2012.35; published online 7 September 2012

Keywords: Waldenstrm macroglobulinemia; proteomics; 2D-electrophoresis; XRCC6

INTRODUCTIONWaldenstrms macroglobulinemia (WM)alternatively designated as lymphoplasmacytic lymphomais an uncommon B-cell lymphoproliferative disorder (LPD) characterized primarily by a lymphoplasmacytic inltration of the bone marrow and by an immunoglobulin M monoclonal gammapathy.1,2

Although the precise molecular path(s) leading to WM remain largely unknown, there is clear evidence for the post-germinal center nature of the WM clonal cells, as the IGH (immunoglobulin heavy chain gene) is almost always subjected to somatic hyper-mutation;3,4 although without the presence of any intraclonal variation.5,6 Moreover and in contrast to immunoglobulin M multiple myeloma (MM) and chronic lymphocytic leukemia (CLL), post-switch clonotypic immunoglobulins are undetectable in WM B cells, suggesting therefore that absence of isotype switching;5,6

although the latter could be achieved in vitro in response to appropriate stimuli for example, CD40-ligand and IL-4.7 Immunoglobulin class switching requires a functional activation-induced cytidine deaminase8 and uses the robust non homologous end-joining (NHEJ) pathway.9 The Ku (Ku70/Ku80) heterodimer is a key factor in this pathway, acting as a scaffold for the recruitment of NHEJ core or such processing factors as the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the XRCC4/ligase IV complex.10,11

To progress in the understanding of molecular pathway(s) underlying the advent of the disease, gene-expression proling of WM cells has been previously performed; revealing a homogeneous expression prole, more similar to that of CLL than that of MM.12 A small set of genes was thereafter identied to be distinctly expressed in WM. They include interleukin-6 (IL6) and genes of the mitogen-activated protein kinase pathway. Upregulation of IL6 in WM was conrmed by an independent study.13 Aiming to compare WM cells with B-cell morphology and those with plasma cell morphology, this work concluded that B cells and plasma cells from WM patients exhibit distinct patterns of gene expression as compared with B cells and plasma cells from patients with CLL and MM.13

Few proteomic studies have been performed in WM. These include a proteomic analysis of signaling pathways performed in WM and MM samples, before and after treatment with a proteasome inhibitor.14 Clustering analysis allowed to identify proteins that were expressed by either of these disorders but not both, indicating differences in cellular responses to proteasome inhibition.14 Hatjiharissi et al.15on the other hand compared using an antibody-based protein microarray methodthe patterns of protein expression between untreated WM cells and normal bone marrow controls. These analyses identied upregulation of proteins of the Ras and Rho family, as well as of

1Laboratoire dImmunogntique Molculaire Humaine, Centre de Recherche dImmunologie et dHmatologie. Facult de Mdecine, Universit de Strasbourg, Strasbourg Cedex, France; 2Laboratoire Central dImmunologie, Plateau Technique de Biologie, Nouvel Hpital Civil, Hpitaux Universitaires de Strasbourg, Strasbourg Cedex, France;

3EA RHEM 4369, Laboratoire dImmunologie, Facult de mdecine, Nancy-Universit, Vanduvre-ls-Nancy, France; 4INSERM, Plateforme post-gnomique de la Piti-Salptrire (P3S), Paris, France; 5Universit Pierre et Marie Curie, Paris, France; 6Service dHmatologie, Hpital de la Piti-Salptrire, Paris, France; 7UMR CNRS 7211, Hpital de la Piti-Salptrire, Paris, France; 8Service dOncologie et dHmatologie, Hpital de Hautepierre, Hpitaux Universitaires de Strasbourg, Strasbourg, France and 9Laboratoire dExcellence (LabEx) TRANSPLANTEX, Centre de Recherche dImmunologie et dHmatologie. Facult de Mdecine, Universit de Strasbourg, Strasbourg Cedex, France. Correspondence: Professor S Bahram, Laboratoire dImmunogntique Molculaire Humaine, Centre de Recherche dImmunologie et dHmatologie, 4 rue Kirschleger, 67085 Strasbourg Cedex, France.

E-mail: [email protected] 28 March 2012; revised 16 July 2012; accepted 7 August 2012

Proteomics of WM vs other LPDs A Perrot et al

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cyclin-dependent kinases, apoptosis regulators and histone deacetylases.15 Moreover, high expression of the heat shock protein HSP90 was observed in cells from symptomatic WM patients.15

Here, we aimed to perform a comprehensive proteomic analysis of WM versus closely linked diseases with likely overlapping pathophysiologies. Similarly to our recent work in CLL16 and using

a two-dimensional gel electrophoresis (2D-E) approach,17 we

compared WM with marginal zone lymphoma (MZL) and CLL. Three notable lessons were learnt in this study: (1) in contrast to observations using gene-expression proling and limited proteomic analyses where WM is quasi indistinguishable from CLL, MM or normal B cells, we demonstrate astatistically signicantdistinct proteomic content for WM versus other LPDs;(2) we establish the existence for WM, of 17 majorly differentially expressed proteins in the same comparative analyses; (3) among the latter, we identify Ku70, part of the Ku heterodimer, and directly involved in class switch recombination, as one of the most differentially underexpressed proteins in WM cells as compared with other B-cell LPDs.

PATIENTS AND METHODSPatients and B-cell selectionTwenty six untreated patients with LPD (10 WM, 4 MZL, 10 CLL and 2 mantle cell lymphoma; MCL), were enrolled in this study. Peripheral blood and/or bone marrow were obtained for each patient after informed consent in accordance with the Helsinki Declaration. Clinical and biological features of all patients are provided in Table 1. Cells corresponding to3 WM, 3 MZL and 4 CLL samples were used for 2D-E analysis, whereas those from, respectively, 9 (4 WM, 3 MZL and 2 CLL) and 14 (5 WM, 2 MZL,5 CLL and 2 MCL) untreated patients were used for western blot and quantitative RT-PCR experiments. B cellsfor all LPDs with the exception

of WM and MZL used in 2D-Ewere puried by negative selection using the Rosettesep B-cell enrichment cocktail (StemCell Technologies, Vancouver, Canada) followed by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare, Saclay, France). B cells from whole blood or bone marrow of WM and MZL patients analyzed in 2D-E were sorted by ow cytometry (FACSAria II, Becton Dickinson, Le Pont-De-Claix, France) using anti-CD19 monoclonal antibody (CD19-PerCP Cy5.5, BD Biosciences, Le Pont de Claix, France), kappa or lambda monotypy (kappa-FITC/lambda-PE, Dako) and the immunophenotypic characteristics of the malignant clone (CD79b for the MZL2 patient) (CD79b-APC, BD Biosciences). The purity of samples isolated by rosette and ow cytometry sorting cells was 495%; often 497%.

2D-ELECTROPHORESIS (2D-E)

Ten samples were used in 2D-E analysis. For each cell suspension, total proteins were extracted in iso-electric focusing-specic lysis buffer containing 7 M urea, 2 M thiourea, 1% CHAPS, 10% isopropanol, 10% isobutanol, 0.5% Triton X100, 0.5% SB3-10, 30 mM Tris, 65 mM DTT, 0.5% IPG buffer 310 and 30 mM spermine. After centrifugation at 16 000 g for 30 min at 4 1C, proteins were precipitated with the Perfect-Focus Kit from G-Biosciences (Maryland, Heights, MO, USA) and resuspended in a buffer containing 7 M urea, 2 M thiourea, 1% CHAPS, 10% isopropanol, 10% isobutanol, 0.5% Triton X100, 0.5% SB3-10 and 30 mM Tris. The total protein concentration of each sample was established using the Bradford assay (Protein Assay, Bio-rad, Ivry sur Seine, France) with bovine serum albumin as standard. All protein extracts (50 mg per sample) were labeled using uorescent

Cyanine (Cy) dyes, as per the manufacturers instructions for minimal labeling (GE Healthcare). Cy3 and Cy5 were alternatively used to label protein extracts according to the dye switch method. For each gel, two labeled protein extractsexpected to

Table 1. Patients characteristics

Samples Origin Experiments Patients age Patients sex Diagnosis Cytogenetics

WM1_BM Bone marrow 2D-E and qPCR 79 M WM (IgM kappa) Trisomy 4WM1_B Blood 2D-EWM2_B Blood 2D-E 66 M WM (IgM kappa) Undetermined WM3_BM Bone marrow WB 72 M WM (IgM kappa) Undetermined WM4_B Blood WB 58 M WM (IgM kappa) Undetermined WM5_BM Bone marrow WB 59 F WM (IgM kappa) NormalWM6_BM Bone marrow WB 59 M WM (IgM kappa) Undetermined WM7_BM Bone marrow qPCR 63 F WM (IgM lambda) Undetermined WM8_BM Bone marrow qPCR 64 M WM (IgM kappa) Undetermined WM9_BM Bone marrow qPCR 51 M WM (IgM lambda) NormalWM10_BM Bone marrow qPCR 32 M WM (IgM kappa) Undetermined MZL1_B Blood 2D-E 61 F MZL (IgM kappa) Undetermined MZL2_B Blood 2D-E 57 F MZL (IgM lambda) Trisomy 18 and 3 MZL2_BM Bone marrow 2D-E and WBMZL3_BM Bone marrow WB and qPCR 59 F MZL (IgM kappa) Undetermined MZL4_B Blood WB and qPCR 64 F MZL (IgM kappa) NormalCLL1 Blood 2D-E 89 M CLL (IGH unmutated) Deletion 11qCLL1 Blood 2D-ECLL2 Blood 2D-E 67 F CLL (IGH unmutated) Deletions 13q and 17p CLL3 Blood 2D-E 64 M CLL (IGH mutated) NormalCLL4 Blood WB 77 M CLL (IGH undetermined) Deletion 13qCLL5 Blood WB 78 M CLL (IGH mutated) Trisomy 12CLL6 Blood qPCR 54 M CLL (IGH unmutated) Deletions 16q and 17p CLL7 Blood qPCR 52 M CLL (IGH mutated) NormalCLL8 Blood qPCR 78 M CLL (IGH undetermined) UndeterminedCLL9 Blood qPCR 79 F CLL (IGH undetermined) Deletion 13qCLL10 Blood qPCR 72 M CLL (IGH undetermined) Undetermined MCL1_BM Bone marrow qPCR 65 M MCL t(11;14)

MCL2_B Blood qPCR 57 M MCL t(11;14)

Abbreviations: CLL, chronic lymphocytic leukemia; IGH, immunoglobulin heavy chain gene; IgM, immunoglobulin M; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; qPCR, quantitative polymerase chain reaction; WB: western-blot; WM, Waldenstrms macroglobulinemia; 2D-E, two-dimensional electrophoresis.

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co-migratewere mixed to a strips rehydration buffer containing7 M urea, 2 M thiourea, 1% CHAPS, 10% isopropanol, 10% isobutanol, 0.5% Triton X100, 0.5% SB3-10, 40 mM DTT and 0.5% IPG buffer 47 for a total volume of 460 ml. Rehydration of a 24 cm

Immobiline pH 47 DryStrip (GE Healthcare) was achieved in the dark during 16 h. Iso-electric focusing was then performed at 20 1C for a total of 85 000 Vh using the Ettan II IPGphor system (GE Healthcare). After migration, the strips were equilibrated in SDS containing buffer (reduction and alkylation) before being loaded onto SDS polyacrylamide gels for separation according to molecular weight using an Ettan DALT Six Electrophoresis System (GE Healthcare). After migration, 2D-E gels were scanned using an Ettan DIGE Imager (GE Healthcare) according to the manufacturers instructions. Image analysis and statistical calculations were performed using the Progenesis SameSpots software (NonLinear Dynamics, Newcastle, UK) and the Multiple stains per gel without internal standards comparison method. All sample gel images were rst aligned. Spots were then automatically detected and ltered to eliminate non-protein spots. Statistical analyses (analysis of variance and principal component analyses) were performed on normalized spots data. For multigroup analysis of variance test, a q-value (a FDR corrected P-value) of o0.05 was considered signicant. The threshold chosen to appreciate variations was a 2.5-fold change when comparing WM versus CLL groups.

Protein identicationAnalytical gels were stained with SYPRO Ruby (Invitrogen

Corporation, Carlsbad, CA, USA) and used for robotized spot picking (EXQuest spot cutter, Bio-Rad, Marnes-la-Coquette, France). Gel plugs were washed twice with 25 mM ammonium bicarbonate in 50% acetonitrile then dehydrated in 100% acetonitrile, before being subjected to overnight in-gel trypsic digestion. Briey, each spot was incubated in a solution containing 50 mM ammonium bicarbonate, 10% acetonitrile and 20 mg/ml trypsin (G-Biosciences) on ice during 1 h, then overnight at 37 1C.

The supernatants were collected and gel plugs were incubated in0.1% TFA 50% acetonitrile in an ultrasonic bath during 10 min to extract residual peptides. The new supernatants were pooled with the former. Peptides were dried completely in a vacuum centrifuge then resuspended in 0.1% TFA 50% acetonitrile, to be analyzed by mass spectrometry (MS or MS/MS). Peptides were spotted onto a MALDI plate with matrix solution (50% acetonitrile,0.1% TFA, HCCA at saturation) diluted four-fold in 50% acetonitrile,0.1% TFA and analyzed by MS using an Autoex MALDI-TOF (Bruker Daltonics, Bremen, Germany). This instrument was operated in positive ion mode and externally calibrated in the peptide mass range of 7003200 m/z. MS/MS analysis was performed using the Ultimate 3000 nanoLC system (Dionex, Voisin le Bretonneux, France) coupled to an Esquire HCTultra nESI-IT-MS (Bruker Daltonics). A search for protein identity was carried out with MASCOT (http//www.matrixscience.com). Condent matches were dened by the MASCOT score and statistical signicance (Po0.05), the number of matching peptides and the percentage of total amino acid sequences covered by matching peptides.

Western-blot analysisCell lysates were prepared by incubating puried B-cell pellets on ice for 30 min in 200 ml of a lysis buffer containing 150 mM NaCl, 50 mM Tris pH8, 1% NP40, 10% glycerol, 0.5 mM EDTA and protease inhibitor Complete 1X (Roche Diagnostics, Mannheim, Germany). The samples were then centrifuged at 4 1C for 30 min at 16 000 g.

Samples containing 50 mg of protein were mixed with Laemmli buffer 1X and boiled for 5 min. Protein extracts were separated on 10% SDSPAGE, then transferred to nitrocellulose membranes (Hybond-C Extra, Amersham Biosciences, Buckinghamshire, UK). Nonspecic binding sites were blocked for 1 h in 5% milk in T-TBS

(50 mM Tris, 150 mM NaCl, 0.5% Tween-20). The membranes were then probed with anti-Ku (p70) (clone N3H10, Kamiya Biomedical, Seattle, WA, USA) during 3 h at room temperature. After thorough washing in T-TBS, incubation was carried out for 1 h in the presence of horseradish peroxidase-conjugated anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA, USA). All immunoblots were revealed by enhanced chemiluminescence using the Super Signal West Pico Substrate (Pierce Biotechnology, Inc., Rockford, IL, USA). Data were acquired by scanning-densitometry using a Chemi-Start (Vilber-Lourmat, Marne-La-Valle, France).

Quantitative real-time RT-PCRA quantitative real-time RT-PCR amplication of XRCC6 (encoding

Ku70) was performed as a second validation test. This was achieved for 14 subjects, respectively, 5 WM and 9 others B LPD including MZL (n 2), MCL (n 2) and CLL (n 5). Briey, total

RNA was extracted using the RNeasy mini kit (Qiagen, Courtaboeuf, France) and treated with DNase (Qiagen). cDNA was synthesized using Improm II reverse transcriptase (Promega, Lyon, France) and random hexadeoxynucleotide primers (Promega). The relative gene expression of XRCC6 was determined by concomitant amplication of GUSB (beta-D glucuronidase) as a reference gene using a LightCycler 480 (Roche). Assays were performed in duplicate using 5 ml of cDNA, 1X Taqman Universal Master Mix (Applied BioSystems, Warrington, UK) and 1X TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) for XRCC6 and GUSB in a total volume of 25 ml. LightCycler 480

Software (Roche) was used to calculate the relative gene expression of XRCC6 (2 DDCT method).

RESULTSHere we present a rst comprehensive 2D-E analysis of WM versus other LPDs.

WM is a unique proteomic entityQuantitative proteomic analysis by 2D-E was performed for 10 protein extracts obtained from B cells isolated from peripheral blood and/or bone marrow of 2 WM (3 samples), 2 MZL (3 samples) and 3 CLL (4 samples) patients. The inclusion of blood and bone marrow cells from the same patients (WM1 and MZL2) was aimed to foresee the extent to which cell origin/ developmental stage might have any inuence in proteomic prole in a same individual.

Multivariate analysis of protein expression was rst performed using a principal component analysis. Used as an explorative tool to investigate the clustering of all WM, MZL and CLL samples, principal component analysis analysis demonstrated that WM clustered distinctly from MZL and CLL (Figure 1). Hence, the protein content of WM cells is signicantly different from those of MZL or CLL. Of note, one WM and one MZL patient segregated quite differently in principal component analysis, although without overlapping with other LPDs, and the stray WM patient still was nearest to the other two (Figure 1).

Several proteins are differentially expressed in WM as compared with MZL and CLL

Among the 1051 polypeptide spots analyzed within different samples, 356 showed a differential expression. Most differentially expressed polypeptide spots (with a 2.5-fold change) between WM and CLL samples, and between WM and MZL samples, as identied by mass spectrometry, are presented in Table 2. The rst highlight from this analysis is that the detected differentially expressed proteins are notfor the vast majorityamong the candidate genes/proteins, which would have expected to be critical in WMthat is, in direct connection with leukomogenesis,

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Figure 1. Principal component analysis (PCA) distinguishes WM, MZL and CLL cells. PCA as a score plot of a spot map of the 10 protein extracts (3 WM, 3 MZL and 4 CLL) color-coded according to the legend, projected onto the rst two principal components. The PC1 and PC2 axes segregate WM samples (pink spots) from both MZL (purple spots) and CLL (blue spots) groups.

immune-related loci and so onbut are rather conned to ubiquitously expressed structural/housekeeping proteins for example, cytoskeleton, metabolism. Remarkably the vast majority of these proteins show a similar expression pattern that is, up/ downregulation in WM vs CLL or MZL hinting again to the fact that WM harbors a distinct proteome entity versus these two other closely considered LPDs. A few notable distinctly expressed proteins are the following: phosphoglucomutase-2 and annexin A6, which were strongly overexpressed, especially when comparing WM and CLL; conversely, peroxiredoxin and Ku70 (X-ray repair cross-complementing protein 6), showed the strongest downregulation in WM compared with CLL. The same trend, although less pronounced, was observed between WM and MZL. Several components of the cytoskeleton were also differentially expressed in WM cells, namely moesin, gelsolin and lamin-B1. Ku70 proteomic prole was examined more thoroughly: 2D and 3D representations together with statistical expression data are shown in Figure 2. No variation of the proteomic content between blood and bone marrow cells, neither for WM nor MZL was noted for this protein.

Lower expression of Ku70 (XRCC6) protein and gene expression in WM cells by comparison with other LPDs

Ku70 wasfor obvious reasons of its direct implication in immunoglobulin class switchingthe candidate protein retained for validation on a larger cohort of patients. First, western blot experiments performed on 9 untreated patients (whose characteristics are summarized in Table 1) clearly conrmed a much lower expression that is, quasi absence of Ku70 in WM as compared with MZL and CLL (Figure 3). Second, in order to investigate whether the quantitative defect of Ku70 protein was related to a lack of XRCC6 RNA transcript or to a post-translational process, gene expression of XRCC6 was assessed using quantitative RT-PCR (using GUSB as reference gene) in 14 patients with WM or other lymphoid malignancies. XRCC6 expression was found to be signicantly lower in MW cells as compared with other LPDs including MZL, CLL and MCL (Figure 4); thus suggesting that the observed protein down-regulation was due to a transcriptional shut down.

DISCUSSIONWM, a rare, distinct, B LPD, is dened by lymphoplasmacytic inltration of the bone marrow and a monoclonal immunoglobulin

M paraproteinemia, respectively, ascertained by bone marrow biopsy and serum protein immunoelectrophoresis/immunoxation. However, before performing the latter tests in order to reach diagnostic certitude, there is/are at present no unique non invasive diagnostic biomarker(s) available helping to guide the clinician in an often diversity of nonspecic early symptoms for example, fatigue, anemia, thrombocytopenia, hepatosplenomegaly and lymphadenopathy.

Array technology, especially transcriptome analysis, has been widely used in clonal hematological malignancies.18,19 WM has not

escaped this trend, as mentioned earlier. Proteome analysis more cumbersome presentlyhas been less widely used and in case of WM has been performed in only two reports with self imposed limitations/biases. Here, we present a rst comprehensive 2D-E analysis of WM, as compared with other LPDs. This study yielded more signicant differences for 17 proteins (Table 2). Besides, the possibly critical downregulation of Ku70, a few other modications of housekeeping/structural proteins were observed. These include molecules involved in glucose metabolism, redox balance, cell communication, protein metabolism, regulation of translation or nucleic acid binding. Phosphoglucomutase-2 appears to be selectively overexpressed in WM compared with CLL and MZL.20,21 Inversely, glutaredoxin-3 (GLRX3), lactoylglutathione lyase (GLO1) and peroxiredoxin-6 (PRDX6) appeared to be underexpressed in WM cells as compared with CLL and MZL cells.22 GLO1 belongs to the glyoxalase complex, which catalyzes the conversion of methylglyoxal to D-lactate.23 Peroxiredoxins are a ubiquitous family of antioxidant enzymes that functions as tumor suppressor to certain haematopoietic cancers in mice.24

PRDX6, the most underexpressed protein in WM in our study, is a bifunctional enzyme having both peroxidase and phospholipase A2 activities, with roles in oxidative stress-induced and TNF-induced apoptosis.25

Cytoskeleton proteins also appeared to be differentially expressed in WM. Gelsolin is an actin-binding protein that is a key regulator of actin lament assembly and disassembly.26,27

Gelsolin has been reported increased in vincristine (an anti-microtubule agent)-resistant acute leukemia, whilst moesin was decreased in the same cells.28 Moesin, which expression was decreased in WM in our study, is a member of the ERM protein family, which appears to function as cross-linker between plasma membranes and actin-based cytoskeletons for cellcell recognition, signaling and cell movement phenomena. Finally, we observed that several isoforms of annexin A6, a calcium-dependent membrane-binding protein, were upregulated in WM cells.29

Again, perhaps the most notable alteration observed in our study was the silencing of Ku70 in WM cells. This was further conrmed by western blot and mRNA analysis in a second cohort of patients, strengthening the consistency of this anomaly. The Ku (Ku70/Ku86) heterodimer was rst discovered as an auto-antigen in patients with autoimmune disorders.30 The genes encoding these proteins are located on chromosomes 22q13 and 2q33-35.31

Ku is the DNA-targeting subunit of a DNA-dependent protein kinase (DNA-PK), which is a serine/threonine kinase consisting of a 465-kDa catalytic subunit (DNA-PKcs) and the heterodimeric Ku regulatory complex. The latter binds to DNA double-stranded ends and other discontinuities in the DNA32 and recruits the DNA-PKcs of the complex.33 Classical NHEJ involves the DNAPK complex, essential for lymphoid development, especially VDJ recombination and Ig switching. Indeed Ku70 / mice lack B-cell maturation and the absence of Ku70 confers hypersensitivity to ionizing radiation and deciency in DNA double-stranded breaks repair,34 translating in an extreme radiosensitivity and specic VDJ recombination defects,3537 as well as high levels of

chromosomal aberrations.38,39 In man, DNA-PK activity and

increased expression of both Ku70 and Ku86 have been shown to correlate with resistance to therapeutic molecules notably in

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Proteomics of WM vs other LPDs A Perrot et al

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nucleobase,nucleoside,nucleotideandnucleicacid

metabolism

463 3.5 1.6P13639Elongationfactor2EEF20.00357118%146.41/953386.75/96931Translationregulatoractivity,proteinmetabolism,

translation

685143.3Q96G03Phosphoglucomutase-2PGM20.00123510%56.28/682836.35/79175Catalyticactivity,metabolism,energypathways

653143P08133AnnexinA6ANXA60.000672236%235.42/758735.57/76803Calciumionbinding,cellcommunication,signal

transduction

7695.81.6P61978Heterogeneousnuclear

ribonucleoproteinK

HNRNPK0.00110041%155.39/509765.24/71014Ribonucleoprotein,regulationofnucleobase,

nucleoside,nucleotideandnucleicacidmetabolism

4765.21.2P06396GelsolinGSN0.0069422%125.9/806415.82/91105Structuralconstituentofcytoskeleton,cellgrowth

and/ormaintenance

7094.81.7P20700Lamin-B1LMNB10.00915232%215.11/664085.3/75379Structuralmoleculeactivity,cellgrowthand/or

maintenance

4784.41.6P06396GelsolinGSN0.0145026%165.9/806415.74/91105Structuralconstituentofcytoskeleton,cellgrowth

and/ormaintenance

8113.92P31150RabGDPdissociation

inhibitoralpha

GDI10.0066827%85.0/505835.05/68326GTPaseactivatoractivity,cellcommunication,signal

transduction

10173.43.4Q8TBC4NEDD8-activating

UBA30.017521%85.3/518525.38/55484Ubiquitin-specicproteaseactivity,protein

metabolism

6783.32.8P08133AnnexinA6ANXA60.00472236%245.42/758735.57/76803Calciumionbinding,cellcommunication,signal

transduction

4823.3 1P06396GelsolinGSN0.0213826%155.9/806415.91/90785Structuralconstituentofcytoskeleton,cellgrowth

and/ormaintenance

UBA50.00614919%54.79/452914.89/59448Proteinbinding,proteinmetabolism

HNRNPL0.00626812%58.46/641336.83/70435RNAbinding,regulationofnucleobase,nucleoside,

nucleotideandnucleicacidmetabolism

1673 2.61.6Q04760LactoylglutathionelyaseGLO10.0056626%75.12/207785.01/22387Lyaseactivity,metabolism,energypathways

1291 2.92.6O76003Glutaredoxin-3GLRX30.000225122%65.31/374325.53/41031Molecularfunctionunknown,biologicalprocess

unknown

DNAJC90.0246043%135.58/299105.85/31685Chaperoneactivity,proteinmetabolism

1046 3.11.1P13489RibonucleaseinhibitorRNH10.00232215%64.71/499734.71/53948Translationregulatoractivity,regulationof

462 4 2.1P13639Elongationfactor2EEF20.01993%26.41/953386.83/96704Translationregulatoractivity,proteinmetabolism,

translation

651 4.5 2.5P26038MoesinMSN0.0372023%136.09/678206.41/82098Structuralconstituentofcytoskeleton,cellgrowth

and/ormaintenance

XRCC60.041147%56.23/698436.47/77798DNAbinding,regulationofnucleobase,nucleoside,

nucleotideandnucleicacidmetabolism

XRCC60.0422541%236.23/698436.57/77344DNAbinding,regulationofnucleobase,nucleoside,

nucleotideandnucleicacidmetabolism

1 12 4.4P30041Peroxiredoxin-6PRDX60.0243846%106.0/250356.44/25730Peroxidaseactivity,metabolism,energypathways

Abbreviations:ANOVA,analysisofvariance;CLL,chroniclymphocyticleukemia;FC,foldchange;MW,molecularweight;MZL,marginalzonelymphoma;WM,Waldenstromsmacroglobulinemia.a Spotnumbers

refertoIDspots.b FCwascalculatedusingProgenesisSameSpotssoftware;negativeFCsignifyingthatpolypeptidespotisunderexpressedinWMsamplesascomparedwithCLLorMZLcells,andpositiveFC

signifyingthatspotisoverexpressedinWMcells.c AccessionnumberintheUniProtdatabase(http://uniprot.org).d FullproteinnamerecommendedbytheUniProtconsortium.e GenenamesaccordingtoHUGO

GeneNomenclature.f q-valuefrommultigroupANOVAwascalculatedusingProgenesisSameSpotssoftware.g MASCOTscoreindicatesthecondenceofproteinidenticationusingtheMascotsearchengine

(http://www.matrixscience.com),dependingonaminoacidsequencecoverage(in%)andonnumberofmatchedpeptidesusedforidenticationintheSwiss-Protdatabase(http://expasy.org/sprot/).

h TheoreticalpIandmolecularweightobtainedfromtheMascotdatabase.i ObservedmolecularweightandpIcalculatedbyDeCyderversion6.5software,accordingtolocationinthegel.j Proteinfunctionand

biologicalprocesswereassignedinaccordancewiththeHumanProteinReferenceDatabase(http://hprd.org).

Functionj

Observed

pI/MWi

Theoretical

pI/MWh

No.of

peptides

matchedg

Sequence

cover%g

MASCOT

scoreg

Table2.MostdifferentiallyexpressedproteinsbetweenWM,CLLandMZLcells

Spot

no.a

ANOVAf

Proteinnamed Genee q-value

enzymeE1catalytic

subunit

complementingprotein

6

complementingprotein

6

Access

numberc

9282.51.5Q9GZZ9Ubiquitinactivating

enzymeE1domain

containing1

798 2.5 1.6P14866Heterogeneousnuclear

ribonucleoproteinL

1465 3 2.3Q8WXX5DnaJhomologsubfamily

Cmember9

713 5.6 3.4P12956X-rayrepaircross-

712 7.4 3.3P12956X-rayrepaircross-

FCb

WMvs

MZL

FCb

WM

vs

CLL

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Figure 2. 2D-E analysis of Ku70 protein expression. Spots were analyzed with Progenesis SameSpots (NonLinear Dynamics). (a) Representative focus of the Ku70 protein (spot no. 712) on WM, MZL and CLL samples 2D-gel images. (b) 3D-representation of the Ku70 volume ratios difference between WM, MZL and CLL samples. (c) Statistical analysis of spot no. 712: signicant decrease of Ku70 expression in WM cells as compared with CLL cells (*Po0.05).

Figure 3. Western-blot validation of the Ku70 2D-E prole. Western blot results of Ku70 expression in samples from WM, MZL and CLL cells. Actin was used as loading control.

XRCC6

***

Relative quantification

5

4

3

2

1

0

the context of CLL.40 Minor defects in the NHEJ pathway moreover have been shown to confer predisposition to leukemia.41 Most

LPDs arise via transformation of post-germinal center B cells with chromosomal mutations.42 Immunodeciencies with both abnormal DNA repair43 and genetic polymorphisms of NHEJ components44,45 have been associated to an increased susceptibility to the development of lymphoid malignancies, suggesting that an aberrant NHEJ pathway could lead to lymphomagenesis. However, when investigated, no mutation or deletion of Ku86 has been observed in CLL or acute lymphoblastic leukemia cells.46 Acute lymphoblastic leukemia cells were found to express high levels of DNA-PKcs, Ku86 and Ku70 protein, whereas CLL cells displayed a lower expression of DNA-PKcs and

Ku86 but not Ku70.47 By quantitative RT-PCR in several types of LPD (not including WM), a lower expression of Ku70-encoding XRCC6 gene was observed compared with reactive lymph nodes.48

WM (n=5)

other B LPD (n=10)

Figure 4. Quantitative real-time PCR validation for XRCC6. Relative gene-expression quantication for XRCC6 (Ku70) and GUSB (beta-D

glucuronidase) as reference gene, in WM and other B LPD (MZL, MCL and CLL) samples (***Po0.001 by Students t-test).

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Lower expression of Ku70 transcripts by quantitative RT-PCR could be the result of at least two phenomenons: (a) genetic (point mutation etc.) or epigenetic (promoter methylation, microRNA etc.) mechanisms; recently described as key regulators in WM biology;49 (b) as part of a larger transcriptional downregulation that is, expression of BLIMP1a transcriptional repressor involved in terminal differentiation of B cells to plasma cells has been shown to shut down immunoglobulin class switching through inhibition of activation-induced cytidine deaminase, Ku70, Ku86, DNA-PKcs and STAT6.50

In conclusion, this is a comprehensive proteomic analysis of the WM cells in comparison with that of two other B LPDs. This study shows that WM harbors a unique proteome with regards to CLL and MZL cells. A rather limited set of proteins were found to be differentially expressed with no usual suspects being part of this list where the majority of proteins were rather housekeeping/ structural proteins. The conrmation of the downregulation of Ku70part of the Ku heterodimer, a critical factor in class switch recombination (lacking in WM)and its mechanisms need to be further investigated in model systems.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGEMENTS

We would like to thank Iozo Delic (CEA, Fontenay-aux-roses, France) and Ali Dalloul (EA RHEM, Nancy, France) for helpful advice and Manuel Chapelle (Plateforme Protomique/ Spectromtrie de masse, Institut Jacques Monod, Paris, France) for allowing us to use the EXQuest spot cutter. AP was supported by a fellowship from the Fondation pour la Recherche Mdicale. This work was supported by the Ligue rgionale Alsace contre le Cancer, the Association pour la Recherche contre le Cancer (ARC), Fondation pour la Recherche Mdicale (FRM) and Agence Nationale pour la Recherche (ANR) and the Laboratoire dExcellence TRANSPLANTEX; a member of the Strasbourg University Initiative in Excellence (IDEX).

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