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Proteomics Profiling of CLL Versus Healthy B-cells Identifies Putative Therapeutic Targets and a Subtype-independent Signature of Spliceosome Dysregulation*

  • Harvey E. Johnston
    Affiliations
    From the Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, General Hospital, University of Southampton, Southampton, UK;

    Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton, UK;
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  • Matthew J. Carter
    Affiliations
    From the Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, General Hospital, University of Southampton, Southampton, UK;
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  • Marta Larrayoz
    Affiliations
    Cancer Genomics, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK.;
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  • James Clarke
    Affiliations
    Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK;
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  • Spiro D. Garbis
    Affiliations
    Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton, UK;

    Clinical and Experimental Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK;
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  • David Oscier
    Affiliations
    Department of Molecular Pathology, Royal Bournemouth Hospital, Bournemouth, UK;
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  • Jonathan C. Strefford
    Affiliations
    Cancer Genomics, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK.;
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  • Andrew J. Steele
    Affiliations
    Leukemia and Lymphoma Molecular Mechanisms and Therapy Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK;
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  • Renata Walewska
    Affiliations
    Department of Haematology, Royal Bournemouth Hospital, Bournemouth, UK
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  • Mark S. Cragg
    Correspondence
    To whom correspondence should be addressed: University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, UK. Fax:+44-(0)-23-80704061;.
    Affiliations
    From the Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, General Hospital, University of Southampton, Southampton, UK;
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  • Author Footnotes
    * This work was supported by an MRC iCASE studentship, a supplementary MRC fellowship award to H.J. and grants from Bloodwise (10012 and 12050; 11052, 12036), the Kay Kendall Leukaemia Fund (873), Cancer Research UK (C34999/A18087, ECMC C24563/A15581), and the Bournemouth Leukaemia Fund.
    This article contains supplemental material.
Open AccessPublished:January 24, 2018DOI:https://doi.org/10.1074/mcp.RA117.000539
      Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell cancer exhibiting a wide spectrum of disease courses and treatment responses. Molecular characterization of RNA and DNA from CLL cases has led to the identification of important driver mutations and disease subtypes, but the precise mechanisms of disease progression remain elusive. To further our understanding of CLL biology we performed isobaric labeling and mass spectrometry proteomics on 14 CLL samples, comparing them with B-cells from healthy donors (HDB). Of 8694 identified proteins, ∼6000 were relatively quantitated between all samples (q<0.01). A clear CLL signature, independent of subtype, of 544 significantly overexpressed proteins relative to HDB was identified, highlighting established hallmarks of CLL (e.g. CD5, BCL2, ROR1 and CD23 overexpression). Previously unrecognized surface markers demonstrated overexpression (e.g. CKAP4, PIGR, TMCC3 and CD75) and three of these (LAX1, CLEC17A and ATP2B4) were implicated in B-cell receptor signaling, which plays an important role in CLL pathogenesis. Several other proteins (e.g. Wee1, HMOX1/2, HDAC7 and INPP5F) were identified with significant overexpression that also represent potential targets. Western blotting confirmed overexpression of a selection of these proteins in an independent cohort. mRNA processing machinery were broadly upregulated across the CLL samples. Spliceosome components demonstrated consistent overexpression (p = 1.3 × 10−21) suggesting dysregulation in CLL, independent of SF3B1 mutations. This study highlights the potential of proteomics in the identification of putative CLL therapeutic targets and reveals a subtype-independent protein expression signature in CLL.
      Chronic lymphocytic leukemia (CLL)
      The abbreviations used are: CLL, Chronic lymphocytic leukaemia; HDB, Healthy donor B-cells; IGHV, Immunoglobulin heavy-chain variable-region gene; MBC, Memory B-cells; M-CLL, IGHV-mutated CLL; NBC, Naive B cells; NOTCH1, Neurogenic locus notch homolog protein 1; PBMCs, Peripheral blood mononuclear cells; Rs, Regulation score; SF3B1, Splicing factor 3B subunit 1; TMT, Tandem mass tags; Tri12, Trisomy 12; U-CLL, IGHV-unmutated CLL.
      1The abbreviations used are: CLL, Chronic lymphocytic leukaemia; HDB, Healthy donor B-cells; IGHV, Immunoglobulin heavy-chain variable-region gene; MBC, Memory B-cells; M-CLL, IGHV-mutated CLL; NBC, Naive B cells; NOTCH1, Neurogenic locus notch homolog protein 1; PBMCs, Peripheral blood mononuclear cells; Rs, Regulation score; SF3B1, Splicing factor 3B subunit 1; TMT, Tandem mass tags; Tri12, Trisomy 12; U-CLL, IGHV-unmutated CLL.
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      • Maitra A.
      • Leach S.D.
      • Drake C.G.
      • Halushka M.K.
      • Prasad T.S.
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      • Kerr C.L.
      • Bader G.D.
      • Iacobuzio-Donahue C.A.
      • Gowda H.
      • Pandey A.
      A draft map of the human proteome.
      ,
      • Uhlen M.
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      • Hallstrom B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson A.
      • Kampf C.
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      • Asplund A.
      • Olsson I.
      • Edlund K.
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      • Zwahlen M.
      • von Heijne G.
      • Nielsen J.
      • Ponten F.
      Proteomics. Tissue-based map of the human proteome.
      ).
      Our current discovery-stage study has applied isobaric labels and LC-MS proteomics to the characterization of isolated B-cell material from 3 healthy donors and 14 CLL patients. CLL samples were selected to include a range of clinically relevant CLL subtypes associated with poor prognosis versus healthy donor B-cells with the aim of assessing CLL-specific differential protein expression. The resulting quantitative proteomes identified a strong signature common to CLL, highlighting several potential therapeutic targets and suggesting mechanisms, such as spliceosome overexpression, contributing to pathogenesis.

      DISCUSSION

      CLL has been the subject of numerous investigations applying genomics and transcriptomics that have contributed greatly to the clinical and biological understanding of the disease (
      • Klein U.
      • Tu Y.
      • Stolovitzky G.A.
      • Mattioli M.
      • Cattoretti G.
      • Husson H.
      • Freedman A.
      • Inghirami G.
      • Cro L.
      • Baldini L.
      • Neri A.
      • Califano A.
      • Dalla-Favera R.
      Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.
      ,
      • Strefford J.C.
      The genomic landscape of chronic lymphocytic leukaemia: biological and clinical implications.
      ,
      • Ferreira P.G.
      • Jares P.
      • Rico D.
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      • Martinez-Trillos A.
      • Villamor N.
      • Ecker S.
      • Gonzalez-Perez A.
      • Knowles D.G.
      • Monlong J.
      • Johnson R.
      • Quesada V.
      • Djebali S.
      • Papasaikas P.
      • Lopez-Guerra M.
      • Colomer D.
      • Royo C.
      • Cazorla M.
      • Pinyol M.
      • Clot G.
      • Aymerich M.
      • Rozman M.
      • Kulis M.
      • Tamborero D.
      • Gouin A.
      • Blanc J.
      • Gut M.
      • Gut I.
      • Puente X.S.
      • Pisano D.G.
      • Martin-Subero J.I.
      • Lopez-Bigas N.
      • Lopez-Guillermo A.
      • Valencia A.
      • Lopez-Otin C.
      • Campo E.
      • Guigo R.
      Transcriptome characterization by RNA sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic leukemia.
      ,
      • Haslinger C.
      • Schweifer N.
      • Stilgenbauer S.
      • Dohner H.
      • Lichter P.
      • Kraut N.
      • Stratowa C.
      • Abseher R.
      Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.
      ). However, low correlations observed between mRNA and protein expression limits insight from these studies (
      • Gygi S.P.
      • Rochon Y.
      • Franza B.R.
      • Aebersold R.
      Correlation between protein and mRNA abundance in yeast.
      ,
      • Vogel C.
      • Marcotte E.M.
      Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.
      ). Indeed, a comparison between a previous transcriptomics analysis of CLL (
      • Haferlach T.
      • Kohlmann A.
      • Wieczorek L.
      • Basso G.
      • Kronnie G.T.
      • Bene M.C.
      • De Vos J.
      • Hernandez J.M.
      • Hofmann W.K.
      • Mills K.I.
      • Gilkes A.
      • Chiaretti S.
      • Shurtleff S.A.
      • Kipps T.J.
      • Rassenti L.Z.
      • Yeoh A.E.
      • Papenhausen P.R.
      • Liu W.M.
      • Williams P.M.
      • Foa R.
      Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group.
      ) and these proteomics results highlight minimally correlated differential expression (supplemental Fig. S2). Proteomics has been applied to CLL in several investigations providing insight into potential differences between subtypes and some CLL-specific signatures (
      • Eagle G.L.
      • Zhuang J.
      • Jenkins R.E.
      • Till K.J.
      • Jithesh P.V.
      • Lin K.
      • Johnson G.G.
      • Oates M.
      • Park K.
      • Kitteringham N.R.
      • Pettitt A.R.
      Total proteome analysis identifies migration defects as a major pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia.
      ,
      • Alsagaby S.A.
      • Khanna S.
      • Hart K.W.
      • Pratt G.
      • Fegan C.
      • Pepper C.
      • Brewis I.A.
      • Brennan P.
      Proteomics-based strategies to identify proteins relevant to chronic lymphocytic leukemia.
      ,
      • Barnidge D.R.
      • Jelinek D.F.
      • Muddiman D.C.
      • Kay N.E.
      Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.
      ,
      • Barnidge D.R.
      • Tschumper R.C.
      • Jelinek D.F.
      • Muddiman D.C.
      • Kay N.E.
      Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC-MS/MS.
      ,
      • Glibert P.
      • Vossaert L.
      • Van Steendam K.
      • Lambrecht S.
      • Van Nieuwerburgh F.
      • Offner F.
      • Kipps T.
      • Dhaenens M.
      • Deforce D.
      Quantitative proteomics to characterize specific histone H2A proteolysis in chronic lymphocytic leukemia and the myeloid THP-1 cell line.
      ,
      • Miguet L.
      • Bechade G.
      • Fornecker L.
      • Zink E.
      • Felden C.
      • Gervais C.
      • Herbrecht R.
      • Van Dorsselaer A.
      • Mauvieux L.
      • Sanglier-Cianferani S.
      Proteomic analysis of malignant B-cell derived microparticles reveals CD148 as a potentially useful antigenic biomarker for mantle cell lymphoma diagnosis.
      ). To date, however, CLL proteomics studies have lacked sufficient coverage to identify most expressed proteins and are yet to fully explore comparisons with healthy donor B-cell controls.
      This study aimed to implement advances in quantitative LC-MS proteomics to provide a detailed characterization of a broad spectrum of CLL samples and evaluate changes in protein expression relative to B-cells derived from healthy donors. Overall, this investigation provided a substantial, reproducible (supplemental Fig. S1) and representative (Fig. 3) description of the CLL proteome to a depth of almost 6000 proteins. Additionally, the accuracy of the results for individual samples was highlighted by the expression of key subtype markers (supplemental Fig. S1) suggesting the potential of the presented methods for the dissection of subtype-specific differences in CLL and other cancers in the future.
      The most striking finding was that of a consistent subtype-independent expression profile across the CLL samples (Fig. 2B). Given the heterogeneous clinical nature of CLL, some variation and clustering of subtypes was anticipated, although homogeneity among CLL subtypes has also been observed previously by transcriptomics (
      • Rosenwald A.
      • Alizadeh A.A.
      • Widhopf G.
      • Simon R.
      • Davis R.E.
      • Yu X.
      • Yang L.
      • Pickeral O.K.
      • Rassenti L.Z.
      • Powell J.
      • Botstein D.
      • Byrd J.C.
      • Grever M.R.
      • Cheson B.D.
      • Chiorazzi N.
      • Wilson W.H.
      • Kipps T.J.
      • Brown P.O.
      • Staudt L.M.
      Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.
      ,
      • Klein U.
      • Tu Y.
      • Stolovitzky G.A.
      • Mattioli M.
      • Cattoretti G.
      • Husson H.
      • Freedman A.
      • Inghirami G.
      • Cro L.
      • Baldini L.
      • Neri A.
      • Califano A.
      • Dalla-Favera R.
      Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.
      ). This suggests that the phenotypic differences between CLL subtypes may either be a product of post-translational modifications, microenvironment interactions or CLL niche-specific characteristics. Further studies with greater sample numbers will be required to better understand these potentially subtle differences in protein expression.
      Phenotypic differences exist between CLL cells in lymph nodes and peripheral blood (
      • Pasikowska M.
      • Walsby E.
      • Apollonio B.
      • Cuthill K.
      • Phillips E.
      • Coulter E.
      • Longhi M.S.
      • Ma Y.
      • Yallop D.
      • Barber L.D.
      • Patten P.
      • Fegan C.
      • Ramsay A.G.
      • Pepper C.
      • Devereux S.
      • Buggins A.G.
      Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration.
      ), suggesting that evaluation of CLL cells from additional niches may be required to understand the differences in disease behaviors observed between subtypes. Furthermore, evaluation of fractionated B-cell subsets from several niches would serve as more informative controls. Indeed, given recent insights from methylation studies relating to the likely cell of origin for different CLL subtypes, B-cell subsets relevant to each CLL sample (i.e. MBC for M-CLL and NBC for U-CLL) should be evaluated, guided by their CpG methylation signatures (
      • Oakes C.C.
      • Seifert M.
      • Assenov Y.
      • Gu L.
      • Przekopowitz M.
      • Ruppert A.S.
      • Wang Q.
      • Imbusch C.D.
      • Serva A.
      • Koser S.D.
      • Brocks D.
      • Lipka D.B.
      • Bogatyrova O.
      • Weichenhan D.
      • Brors B.
      • Rassenti L.
      • Kipps T.J.
      • Mertens D.
      • Zapatka M.
      • Lichter P.
      • Dohner H.
      • Kuppers R.
      • Zenz T.
      • Stilgenbauer S.
      • Byrd J.C.
      • Plass C.
      DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia.
      ,
      • Oakes C.C.
      • Claus R.
      • Gu L.
      • Assenov Y.
      • Hullein J.
      • Zucknick M.
      • Bieg M.
      • Brocks D.
      • Bogatyrova O.
      • Schmidt C.R.
      • Rassenti L.
      • Kipps T.J.
      • Mertens D.
      • Lichter P.
      • Dohner H.
      • Stilgenbauer S.
      • Byrd J.C.
      • Zenz T.
      • Plass C.
      Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia.
      ,
      • Queiros A.C.
      • Villamor N.
      • Clot G.
      • Martinez-Trillos A.
      • Kulis M.
      • Navarro A.
      • Penas E.M.
      • Jayne S.
      • Majid A.
      • Richter J.
      • Bergmann A.K.
      • Kolarova J.
      • Royo C.
      • Russinol N.
      • Castellano G.
      • Pinyol M.
      • Bea S.
      • Salaverria I.
      • Lopez-Guerra M.
      • Colomer D.
      • Aymerich M.
      • Rozman M.
      • Delgado J.
      • Gine E.
      • Gonzalez-Diaz M.
      • Puente X.S.
      • Siebert R.
      • Dyer M.J.
      • Lopez-Otin C.
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      • Campo E.
      • Lopez-Guillermo A.
      • Martin-Subero J.I.
      A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.
      ,
      • Kulis M.
      • Heath S.
      • Bibikova M.
      • Queiros A.C.
      • Navarro A.
      • Clot G.
      • Martinez-Trillos A.
      • Castellano G.
      • Brun-Heath I.
      • Pinyol M.
      • Barberan-Soler S.
      • Papasaikas P.
      • Jares P.
      • Bea S.
      • Rico D.
      • Ecker S.
      • Rubio M.
      • Royo R.
      • Ho V.
      • Klotzle B.
      • Hernandez L.
      • Conde L.
      • Lopez-Guerra M.
      • Colomer D.
      • Villamor N.
      • Aymerich M.
      • Rozman M.
      • Bayes M.
      • Gut M.
      • Gelpi J.L.
      • Orozco M.
      • Fan J.B.
      • Quesada V.
      • Puente X.S.
      • Pisano D.G.
      • Valencia A.
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      • Gut I.
      • Lopez-Otin C.
      • Campo E.
      • Martin-Subero J.I.
      Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.
      ). Currently, we are not aware of proteomics data describing these B-cell subsets.
      The findings presented here provide several potential novel hypotheses for further investigation. CKAP4, for instance (Fig. 2D, 3), was robustly identified as a highly abundant, overexpressed, putative surface protein in CLL and validated in a separate cohort by Western blotting. This offers potential mechanistic insight into CLL and presents a prospective clinical tool. In addition to roles in the endoplasmic reticulum and as a transcription factor (
      • Sandoz P.A.
      • van der Goot F.G.
      How many lives does CLIMP-63 have?.
      ), CKAP4, also known as CLIMP-63, can act as a cell surface receptor for tissue plasminogen activator (tPA) (
      • Razzaq T.M.
      • Bass R.
      • Vines D.J.
      • Werner F.
      • Whawell S.A.
      • Ellis V.
      Functional regulation of tissue plasminogen activator on the surface of vascular smooth muscle cells by the type-II transmembrane protein p63 (CKAP4).
      ), surfactant protein A (SP-A) (
      • Gupta N.
      • Manevich Y.
      • Kazi A.S.
      • Tao J.Q.
      • Fisher A.B.
      • Bates S.R.
      Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes.
      ) and anti-proliferative factor (APF) (
      • Conrads T.P.
      • Tocci G.M.
      • Hood B.L.
      • Zhang C.O.
      • Guo L.
      • Koch K.R.
      • Michejda C.J.
      • Veenstra T.D.
      • Keay S.K.
      CKAP4/p63 is a receptor for the frizzled-8 protein-related antiproliferative factor from interstitial cystitis patients.
      ). APF treatment of a bladder cancer cell line resulted in reduced proliferation, attributable to substantially reduced phosphorylation of AKT and GSK3β and an increased expression of p53 (
      • Shahjee H.M.
      • Koch K.R.
      • Guo L.
      • Zhang C.O.
      • Keay S.K.
      Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells.
      ). Interestingly, substantial CKAP4 overexpression was also observed in tumors of the Eμ-TCL1 mouse model of CLL suggesting a potential means of investigation (
      • Johnston H.E.
      • Carter M.J.
      • Cox K.L.
      • Dunscombe M.
      • Manousopoulou A.
      • Townsend P.A.
      • Garbis S.D.
      • Cragg M.S.
      Integrated Cellular and Plasma Proteomics of Contrasting B-cell Cancers Reveals Common, Unique and Systemic Signatures.
      ). Targeting CKAP4 with either ligands or immunotherapy may offer means of treating CLL.
      INPP5F mRNA overexpression in CLL has previously been linked with low progression-free survival for patients undergoing fludarabine-based therapy (
      • Palermo G.
      • Maisel D.
      • Barrett M.
      • Smith H.
      • Duchateau-Nguyen G.
      • Nguyen T.
      • Yeh R.F.
      • Dufour A.
      • Robak T.
      • Dornan D.
      • Weisser M.
      • investigators R.
      Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia.
      ). The proteomics and Western blot validation presented here indicate that the INPP5F protein is commonly overexpressed in CLL. Together these observations suggest INPP5F may present a therapeutic target in CLL, especially in treatment-resistant cases and its mechanism in CLL warrants further investigation.
      The identification of several consistently upregulated membrane proteins in CLL versus HDBs (Fig. 4, supplemental Fig. S4) highlighted the potential of proteomics approaches to identify novel immunotherapy targets for selective targeting with monoclonal antibodies. Additionally, identification of proteins linked with BCR signaling should enable a better understanding of this process in CLL which may enable improved therapeutic targeting of this currently incurable disease (
      • Zhu M.
      • Janssen E.
      • Leung K.
      • Zhang W.
      Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling.
      ,
      • Chen J.
      • McLean P.A.
      • Neel B.G.
      • Okunade G.
      • Shull G.E.
      • Wortis H.H.
      CD22 attenuates calcium signaling by potentiating plasma membrane calcium-ATPase activity.
      ,
      • Breiman A.
      • Lopez Robles M.D.
      • de Carne Trecesson S.
      • Echasserieau K.
      • Bernardeau K.
      • Drickamer K.
      • Imberty A.
      • Barille-Nion S.
      • Altare F.
      • Le Pendu J.
      Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin).
      ,
      • Satpathy S.
      • Wagner S.A.
      • Beli P.
      • Gupta R.
      • Kristiansen T.A.
      • Malinova D.
      • Francavilla C.
      • Tolar P.
      • Bishop G.A.
      • Hostager B.S.
      • Choudhary C.
      Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation.
      ). LAX1 was shown to be phosphorylated upon BCR stimulation by Src and Syk (
      • Zhu M.
      • Janssen E.
      • Leung K.
      • Zhang W.
      Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling.
      ), with B-cell hyper-responsiveness observed in LAX1−/− mice suggesting a regulatory role in BCR signaling (
      • Zhu M.
      • Granillo O.
      • Wen R.
      • Yang K.
      • Dai X.
      • Wang D.
      • Zhang W.
      Negative regulation of lymphocyte activation by the adaptor protein LAX. J.
      ). ATP2B4 has a role in BCR-induced calcium efflux (
      • Chen J.
      • McLean P.A.
      • Neel B.G.
      • Okunade G.
      • Shull G.E.
      • Wortis H.H.
      CD22 attenuates calcium signaling by potentiating plasma membrane calcium-ATPase activity.
      ) and Prolectin, also known as CLEC17A, is expressed in germinal center B-cells where expression correlates with proliferation (
      • Graham S.A.
      • Jegouzo S.A.
      • Yan S.
      • Powlesland A.S.
      • Brady J.P.
      • Taylor M.E.
      • Drickamer K.
      Prolectin, a glycan-binding receptor on dividing B cells in germinal centers.
      ). Prolectin also has a potential role in BCR signaling, through an association with BLNK (
      • Satpathy S.
      • Wagner S.A.
      • Beli P.
      • Gupta R.
      • Kristiansen T.A.
      • Malinova D.
      • Francavilla C.
      • Tolar P.
      • Bishop G.A.
      • Hostager B.S.
      • Choudhary C.
      Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation.
      ).
      The analysis of drug targets further highlighted the potential of proteomics in the identification of putative clinical tools (Fig. 5). Wee1 overexpression, for instance (Fig. 3B), suggests a potential target to inhibit the cell cycle, with the inhibitor MK1775 shown to have therapeutic benefit in other cancers (
      • Zhang M.
      • Dominguez D.
      • Chen S.
      • Fan J.
      • Qin L.
      • Long A.
      • Li X.
      • Zhang Y.
      • Shi H.
      • Zhang B.
      WEE1 inhibition by MK1775 as a single-agent therapy inhibits ovarian cancer viability.
      ). The upregulation of HMOX1 and 2 suggested an increased degradation of free heme in CLL, combined inhibition of which could induce apoptosis (
      • Zhou Y.
      • Hileman E.O.
      • Plunkett W.
      • Keating M.J.
      • Huang P.
      Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents.
      ). A trend of HDACs upregulated in these results highlighted the potential of targeted HDAC inhibitors (HDACi). HDAC1 and HDAC3 were both observed upregulated and specifically targetable, for instance, with Entinostat; previously identified to induce pro-apoptotic effects in CLL cells (
      • Lucas D.M.
      • Davis M.E.
      • Parthun M.R.
      • Mone A.P.
      • Kitada S.
      • Cunningham K.D.
      • Flax E.L.
      • Wickham J.
      • Reed J.C.
      • Byrd J.C.
      • Grever M.R.
      The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.
      ). HDAC7 additionally exhibited consistent upregulation, observed previously at the mRNA level (
      • Van Damme M.
      • Crompot E.
      • Meuleman N.
      • Mineur P.
      • Bron D.
      • Lagneaux L.
      • Stamatopoulos B.
      HDAC isoenzyme expression is deregulated in chronic lymphocytic leukemia B-cells and has a complex prognostic significance.
      ), suggesting the possibility of more targeted means of HDAC interference, with fewer off-target effects compared with those seen in previous pan-HDACi trials in CLL (
      • Van Damme M.
      • Crompot E.
      • Meuleman N.
      • Mineur P.
      • Bron D.
      • Lagneaux L.
      • Stamatopoulos B.
      HDAC isoenzyme expression is deregulated in chronic lymphocytic leukemia B-cells and has a complex prognostic significance.
      ,
      • Garcia-Manero G.
      • Yang H.
      • Bueso-Ramos C.
      • Ferrajoli A.
      • Cortes J.
      • Wierda W.G.
      • Faderl S.
      • Koller C.
      • Morris G.
      • Rosner G.
      • Loboda A.
      • Fantin V.R.
      • Randolph S.S.
      • Hardwick J.S.
      • Reilly J.F.
      • Chen C.
      • Ricker J.L.
      • Secrist J.P.
      • Richon V.M.
      • Frankel S.R.
      • Kantarjian H.M.
      Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes.
      ,
      • Byrd J.C.
      • Marcucci G.
      • Parthun M.R.
      • Xiao J.J.
      • Klisovic R.B.
      • Moran M.
      • Lin T.S.
      • Liu S.
      • Sklenar A.R.
      • Davis M.E.
      • Lucas D.M.
      • Fischer B.
      • Shank R.
      • Tejaswi S.L.
      • Binkley P.
      • Wright J.
      • Chan K.K.
      • Grever M.R.
      A phase 1 and pharmacodynamic study of depsipeptide (FK228) in chronic lymphocytic leukemia and acute myeloid leukemia.
      ).
      The strong signature of upregulated mRNA processes highlighted by the bioinformatics analyses (Fig. 6) suggests a general underlying defect in CLL, independent of subtype. SF3B1 mutations (
      • Quesada V.
      • Conde L.
      • Villamor N.
      • Ordonez G.R.
      • Jares P.
      • Bassaganyas L.
      • Ramsay A.J.
      • Bea S.
      • Pinyol M.
      • Martinez-Trillos A.
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      • Colomer D.
      • Navarro A.
      • Baumann T.
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      • Rozman M.
      • Delgado J.
      • Gine E.
      • Hernandez J.M.
      • Gonzalez-Diaz M.
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      • Royo R.
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      • Orozco M.
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      • Zamora J.
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      • Valencia A.
      • Himmelbauer H.
      • Bayes M.
      • Heath S.
      • Gut M.
      • Gut I.
      • Estivill X.
      • Lopez-Guillermo A.
      • Puente X.S.
      • Campo E.
      • Lopez-Otin C.
      Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.
      ) and observations of major dysregulation of splicing patterns (
      • Ferreira P.G.
      • Jares P.
      • Rico D.
      • Gomez-Lopez G.
      • Martinez-Trillos A.
      • Villamor N.
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      • Gonzalez-Perez A.
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      • Colomer D.
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      Transcriptome characterization by RNA sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic leukemia.
      ) have previously indicated aberrant spliceosome activity in CLL. Additionally, inhibition of SF3B1 by spliceostatin-A was toxic to CLL, independent of SF3B1 mutational status, suggesting a broader role for aberrant splicing in CLL biology (
      • Larrayoz M.
      • Blakemore S.J.
      • Dobson R.C.
      • Blunt M.D.
      • Rose-Zerilli M.J.
      • Walewska R.
      • Duncombe A.
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      The SF3B1 inhibitor spliceostatin A (SSA) elicits apoptosis in chronic lymphocytic leukaemia cells through downregulation of Mcl-1.
      ). The observation of broadly consistent overexpression of spliceosomal proteins (Fig. 7) may therefore offer some explanation for these previous observations. This reinforces the notion that interference with aberrant splicing activity could offer a means of better understanding and potentially treating CLL.
      A limitation to our study was the observation of non-B-cell contaminants in the HDB samples resulting from the substantial difference in percentage of non-B-cells and platelets between healthy donor and CLL patient PBMCs; varying from 95% in healthy PBMCs to below 10% in CLL patient PBMCs. Emphasis was therefore placed upon overexpressed proteins in CLL and conclusions based upon downregulated proteins - potentially attributable to contamination in the HDB samples - were avoided.
      In summary, these results offer the first comprehensive insight into the molecular composition of CLL compared with healthy donor B-cells at the proteome level. They demonstrate the potential of proteomics to identify protein-specific differences between cancers and healthy tissues. The application of such approaches on a larger scale promises the elucidation of putative therapeutic targets and prognostic and diagnostic indicators, in addition to the dissection of the underlying cancer biology.

      DATA AVAILABILITY

      The raw data and processed outputs have been deposited in the PRIDE archive: https://www.ebi.ac.uk/pride/archive/projects/PXD002004.

      Acknowledgments

      We thank all patients who contributed to this study. We would like to thank Roger Allsopp and Derek Coates for raising funds the FT-MS proteomics platform at the University of Southampton. Thanks are due to Professor Paul Townsend for his role in securing this funding. Karen Kimpton and Dr Zadie Davis, PhD, are acknowledged for technical assistance and for collating the clinical and laboratory data. We would like to acknowledge the use of the IRIDIS High Performance Computing Facility and associated support services at the University of Southampton and the PRIDE team in the submission of our data to the PRIDE database. We would also like to thank Cory White for SPIQuE development, Dr. Xunli Zhang for HPLC access and Sofia Macari for assistance with HPLC. Also, thanks to Graham Packham and Chris Sutton for critical feedback on this work.

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