1、TheRoleofAntigenic Targets / Stimuli of the B-cell Receptors in Lymphoid malignanciesMichael PfreundschuhNathalie Fadle, Evi Regitz, Sandra Grass, Klaus-Dieter PreussJose-Carreras-Center for Immuno- and Gene TherapyInnere Medizin I, Universitt des SaarlandesHomburg (Saar), GermanyBackground:Backgrou
2、nd:n nBCR of B-cell neoplasias = (monoclonal) antibodyBCR of B-cell neoplasias = (monoclonal) antibodyn nantigenic targets of BCR: chronic antigenic antigenic targets of BCR: chronic antigenic stimulation?stimulation?n nidentification of BCR targets: clues as to the identification of BCR targets: cl
3、ues as to the pathogenesis of B-cell neoplasms ?pathogenesis of B-cell neoplasms ?n nproblem: few antigens identifiedproblem: few antigens identifiedAntigenic Targets of ParaproteinsEntities studiedEntities studiedn nMGUS/MMMGUS/MMn nCLLCLLn nFollicular lymphomaFollicular lymphoman nPCNSLPCNSLn nNLP
4、HL (nodular pragranuloma)NLPHL (nodular pragranuloma)Antigenic Targets of ParaproteinsProtein Macro-Array (fetal brain): Identification of Paraprotein Targets: MethodsPreuss et al., Int J Cancer 2009; 125:656-61 Protein Macro-Array (fetal brain): - 192 paraproteins screened Preuss et al., Int J Canc
5、er 2009; 125:656-61 Identification of Paraprotein Targets: MethodsProtein Macro-Array (fetal brain): - 192 paraproteins screened - 29 (15%): Paratarg-7Preuss et al., Int J Cancer 2009; 125:656-61 Identification of Paraprotein Targets: MethodsPreuss et al., Int J Cancer 2009; 125:656-61 Protein Macro
6、-Array (fetal brain): - 192 paraproteins screened - 29 (15%): Paratarg-7- IgA: 13%- IgG: 16% IgG1: 0% IgG2: 0% IgG3: 42% IgG4: 0% Identification of Paraprotein Targets (II)Demonstration of paraprotein-mediated reaction: Titer: 1:108 to 1:1010 Absorption Studies Cloning of the B-cell receptor Competi
7、tion assay with paraprotein Identification of Paraprotein Targets (II)Preuss et al., Int J Cancer 2009; 125:656-61 Paratarg-7 : CharacterizationParatarg-7 : CharacterizationGrass et al. Lancet Oncology 2009; 10: 950-956P-7 in patients and controls:Paratarg-7 : CharacterizationParatarg-7 : Characteri
8、zationnSequence analysis: P-7 in patients and controls:Grass et al. Lancet Oncology 2009; 10: 950-956Paratarg-7 : CharacterizationParatarg-7 : CharacterizationnSequence analysis: nno mutations nno SNPsP-7 in patients and controls:Grass et al. Lancet Oncology 2009; 10: 950-956Paratarg-7 : Characteriz
9、ationParatarg-7 : CharacterizationnSequence analysis: nno mutations nno SNPsnGel electrophoresis: identical bandsP-7 in patients and controls:Grass et al. Lancet Oncology 2009; 10: 950-956pHMWGIEFGel electro-phoresis1: untreated2: 24h, 37C, only buffer 3: 24h, 37C +APPatientControlParatarg-7: Charac
10、terizationParatarg-7: CharacterizationPhosphatase Treatment Grass et al. Lancet Oncology 2009; 10: 950-956pHMWGIEFGel electro-phoresis1: untreated2: 24h, 37C, only buffer 3: 24h, 37C +APPatientControlParatarg-7: CharacterizationParatarg-7: CharacterizationPhosphatase Treatment Grass et al. Lancet On
11、cology 2009; 10: 950-956pHMWGIEFGel electro-phoresisPhosphatase Treatment 1: untreated2: 24h, 37C, only buffer 3: 24h, 37C +APPatientControlParatarg-7: CharacterizationParatarg-7: CharacterizationGrass et al. Lancet Oncology 2009; 10: 950-956 Prevalence of pP-7 :Prevalence of pP-7 :Paratarg-7 : Char
12、acterizationParatarg-7 : Characterization Prevalence of pP-7:Prevalence of pP-7: hyperphosphorylated hyperphosphorylated (pP-7) (pP-7) in in all all patients patients with with P-7-specific P-7-specific paraproteinparaproteinParatarg-7 : CharacterizationParatarg-7 : Characterization Prevalence of pP
13、-7:Prevalence of pP-7: hyperphosphorylated hyperphosphorylated (pP-7) (pP-7) in in all all patients patients with with P-7-specific P-7-specific paraproteinparaprotein pP-7 carrier state:pP-7 carrier state: 15% in IgA/IgG-MGUS / multiple myeloma15% in IgA/IgG-MGUS / multiple myeloma 2% in healthy co
14、ntrols 2% in healthy controlsParatarg-7 : CharacterizationParatarg-7 : Characterization Prevalence of pP-7:Prevalence of pP-7: hyperphosphorylated hyperphosphorylated (pP-7) (pP-7) in in all all patients patients with with P-7-specific P-7-specific paraproteinparaprotein pP-7 carrier state:pP-7 carr
15、ier state: 15% in IgA/IgG-MGUS / multiple myeloma15% in IgA/IgG-MGUS / multiple myeloma 2% in healthy controls 2% in healthy controls RR of pP-7 carrier for IgA/IgG-MGUS/MM: 7.9 xRR of pP-7 carrier for IgA/IgG-MGUS/MM: 7.9 xParatarg-7 : CharacterizationParatarg-7 : Characterization pP-7 Family Studi
16、espP-7 Family Studies(Grass et al. Lancet Oncology 2009; 10: 950-956) Families analyzed:Families analyzed: 1616Relatives analyzed:Relatives analyzed: 9090 pP-7 Family StudiesFamilies analyzed:Families analyzed: 1616Relatives analyzed:Relatives analyzed: 9090Conclusion: autosomal-dominant traitConclu
17、sion: autosomal-dominant traitpP-7 Family StudiesFamilies analyzed:Families analyzed: 1616Relatives analyzed:Relatives analyzed: 9090Conclusion: autosomal-dominant traitConclusion: autosomal-dominant traitExplanation Explanation for for cases cases with with familial familial MGUS MGUS / / MM and WM
18、MM and WMpP-7 Family StudiesPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%MGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%36%MGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Dif
19、ferent Ethnic GroupsAfroamericansEuropeansJapanese15%36%4.5%MGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%36%4.5%2%MGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%3
20、6%4.5%2%6%MGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%36%4.5%2%0.3%6%MGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%36%4.5%2%0.3%6%8xMGUS/MMControlsRel.RiskPreva
21、lence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%36%4.5%2%0.3%6%8x5xMGUS/MMControlsRel.RiskPrevalence of pP-7 Carrier State in Different Ethnic GroupsAfroamericansEuropeansJapanese15%36%4.5%2%0.3%6%8x5x13xMGUS/MMControlsRel.RiskZwick et al., Blood 2013 (under re
22、vision)Co-Immunoprecipitation*FLAGPP2ACunitPKC-+-+-+- neg control: SMCHD1-FLAG - ATG13-FLAG *Immunoprecipitationwithanti-FLAG,SDS-PAGEfollowedbyimmunodetectionPreuss et al., Blood 2011ParatargAntigenPatientShiftinIEFPhosphorylationPKCPP2A1Cylicin22TPP2+3IGFBP-2?4Porcinekinesin5Microtubass.prot.+6LAP
23、TM5+7SLP-2+8ATG13+9RSP16+10SPAG7+11SIVA+Phosphorylation of Paraprotein TargetsPreuss et al., Blood 2011Antigenic Targets of ParaproteinsPhosphorylation state of P-7:Hyperphosphorylation of P-7 inducible in healthy controlsDefect in de-phosphorylation responsible for persistant pP-7Dominant-negative
24、effect of heterotrimeric PP2A possiblePKC and PP2A:-no SNP-no mutation-no posttranslational modificationPP2A: Inactivated in pP7 carriers (by phosphorylation of Y307 in catalytic subunit C)Antigenic Targets of ParaproteinsPhosphorylation state of P-7:Hyperphosphorylation of P-7 inducible in healthy
25、controlsDefect in de-phosphorylation responsible for persistant pP-7Dominant-negative effect of heterotrimeric PP2A possiblePKC and PP2A:-no SNP-no mutation-no posttranslational modificationPP2A: Inactivated in pP7 carriers (by phosphorylation of Y307 in catalytic subunit C)Antigenic Targets of Para
26、proteinsPhosphorylation state of P-7:Hyperphosphorylation of P-7 inducible in healthy controlsDefect in de-phosphorylation responsible for persistant pP-7PKC and PP2A:-no SNP-no mutation-no posttranslational modificationPP2A: Inactivated in pP7 carriers (by phosphorylation of Y307 in catalytic subun
27、it C)Antigenic Targets of ParaproteinsPhosphorylation state of P-7:Hyperphosphorylation of P-7 inducible in healthy controlsDefect in de-phosphorylation responsible for persistant pP-7PKC and PP2A:-no SNP-no mutation-no posttranslational modificationPP2A: Inactivated in pP7 carriers (by phosphorylat
28、ion of Y307 in catalytic subunit C and substitution of B55 by B56)MHC-II-restrictedT-cellResponsesagainstParatarg-7BulkStimulation(ELISpotd27):Neumann et al., Int J Cancer 2014 (in press)BulkStimulation(ELISpotd27):verystrongT-cellresponseforanon-viralpeptideNeumann et al., Int J Cancer 2014 (in pre
29、ss)BulkStimulation(ELISpotd27):verystrongT-cellresponseforanon-viralpeptidespecificreactionagainstp13-27withphosphorylatedserine17Neumann et al., Int J Cancer 2014 (in press)NaturalProcessingofp14-28Ser17pNeumann et al., Int J Cancer 2014 (in press)NaturalProcessingofp14-28Ser17pNeumann et al., Int
30、J Cancer 2014 (in press)T-cellresponseHLA-DRB1*1301restrictedNaturalProcessingofp14-28Ser17pNeumann et al., Int J Cancer 2014 (in press)T-cellresponseHLA-DRB1*1301restrictedNoresponseagainstp14-28Ser17wtNaturalProcessingofp14-28Ser17pNeumann et al., Int J Cancer 2014 (in press)Working hypothesis:pP-
31、7 in MGUS/MM/MW?inactivePP2AwtP-7pP-7 CD4 +*pP-7Phosphory-lationofcatalyticsubunit3B-cellpP-7 ClonepP-7*ifMHC-IIhaplotypecapableofreocognizingMHC-II-presentedphosphoepitope Single Nucleotide Polymorphisms and Microsatellite-based Genome-wide Linkage AnalysisLinkage analysisIllumina SentrixR BeadChip
32、 Array, the HumanHap300-DuoBead Chipcomprising 318.000 tag single nucleotide polymorphisms to perform a genome-wide linkage analysis(spacing of 100kb and an allele frequency 0.15 )pP-7: LOD-ScorepP-7: LOD-ScoreLinkage at Chromosome:Linkage at Chromosome: 4q354q35n nflanking SNP: rs2545281-rs6854448f
33、lanking SNP: rs2545281-rs6854448n nGenetic distance.:178.94-191.96 =13 cM Genetic distance.:178.94-191.96 =13 cM (deCODE)(deCODE)n nphysical distance:physical distance: 181.89-186.18 Mb = 4.3 Mb 181.89-186.18 Mb = 4.3 Mb (NCBI.36.3)(NCBI.36.3)pP-7: Search for associated genepP-7: Search for associat
34、ed geneNext generation sequencing 4q35: no shared SNP or mutation identified Whole exome sequencing (2x): - no shared SNP or mutation identified Whole genome methylation analysis- no specific shared pattern identified Paratarg-7:BACTransferpP-7intowtP-7LCLwtP-7intopP-7LCLDifferentPointMutationsinDif
35、ferentFamilies All mutations in non-coding regions !ABGS7ExtractP69ExtractpP7PointMutations:MechanismsofactionpP-7: Search for associated genepP-7: Search for associated gene responsiblemutationsidentified mutationsarefamily-specific differentmutationsbindidenticalprotein mutations responsible for o
36、ther hyper-phosphorylatedparatargs?Searchforotherposttranslationallymodifiedantigens:HSP90-SUMOasaParaproteinTargetKubuschok et al. J Clin Invest 2014 (in press)HSP90-SUMOasaParaproteinTargetPrevalence of HSP90-SUMO Carrier in Different Ethnic GroupsAfroamericansEuropeansJapanese11%11%4.5%2%1%2%15x6
37、x7xMGUS/MMControlsRel.RiskKubuschok et al. J Clin Invest 2014 (in press)ParaproteinTargetsinMGUS/MMSummary (pP-7 & HSP-90-SUMO): upto50%:inheritedriskfactormarkersforindividualsatriskprophylacticstrategies:-alteredpeptideligands-MHC-II/peptide-specificantibodiesforpermissiveMHC-IIhaplotypesEntities
38、studiedEntities studiedn nMGUS/MMMGUS/MMn nCLLCLLn nFollicular lymphomaFollicular lymphoman nPCNSLPCNSLn nNLPHL (nodular pragranuloma)NLPHL (nodular pragranuloma)Antigenic Targets of ParaproteinsAntigenictargetsof50CLL-derivedBCR#AntigenAcc.No.EiptopePat.#Function1Pyruvate carboxylase (PC)NM_000920a
39、) aa 1031-1084b) aa 1124-118157Gluconeogenesis, lipogenesis, insulin secretion; located in the mitochondrial matrix (Jitrapakdee et al., 2008)2LLP homolog, long-termsynaptic facilitationNM_032338aa 38-911Unknown3Fam32ANM_014077a) aa 1-32b) aa 69-7923Hypothetical protein4Notch2NM_024408aa 495-5059Mem
40、ber of the Notch family which regulates interactions between physically adjacent cells (Kojika and Griffin, 2001)5Calcyclin-bindingProteinNM_014412a) aa 92-102b) aa 1-10246Involved in calcium-dependent ubiquitination and subsequent proteosomal degradation of target proteins (Ning et al., 2007)6Struc
41、tural maintenance of chrom. hinge domain 1 (SMCHD1)NM_015295aa 1897-19078Has a role in the maintenance of X inactivation (Blewitt et al., 2008) 7MAZ (synonym PUR1, SAF1)myc-associated zinc fingerprotein isoform 2NM_00104253910Purine-binding transcription factor (Bossone et al., 1992a; Bossone etal.,
42、 1992b)8ACTGActin-gammaNM_001614.21119MTUS1microtubule associatedtumor suppressor 1NM_001001924aa 1146-116212Tumor suppressor (Di Benedetto et al., 2006)Specific Binding of Autoantigenic Targets ofSpecific Binding of Autoantigenic Targets ofCLL-BCRCLL-BCR Specific activation of CLL Cells by Autoanti
43、genic BCR Target1234c-mycactinCytoplasmicCa2+ProliferationC-mycInductionPreuss et al, Blood 2013 (e-pub April 14)Agathangelidis A,., Stamatopoulos K. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood.
44、 2012 May 10;119(19):4467-75. doi: 10.1182/blood-2011-11-393694. Epub 2012 Mar 13. PubMed PMID: 22415752; PubMed Central PMCID: PMC3392073. Known antigenTo be identifiedCACYBPFam32oxLDLStereotyped Subsets: Identification of Antigens Stereotyped Subsets: Identification of Sub-SubsetsConclusions: anti
45、genic targets of 12/50 BCR and all major stereotyped subsets identified first experimental proof of subset hypothesis definition of epitope-defined sub-subsets molecular basis of epitope-spreading direct activation of CLL cells by BCR targets no hyperphosphorylation, no SNPs, no mutations found to d
46、ate Antigenic Targets of CLL-BCREntities studiedEntities studiedn nMGUS/MMMGUS/MMn nCLLCLLn nFollicular lymphomaFollicular lymphoman nPCNSLPCNSLn nNLPHL (nodular pragranuloma)NLPHL (nodular pragranuloma)Antigenic Targets of ParaproteinsReactivity of CNS-DLBCL BCR againsthyperglycosylated SAMD14/Neur
47、abinW-471W-471W-143W-143BCR:SAMD14specificity-+PatientEndo-Htreatment-+-+150kDa120kDa50kDaReactivity of CNS-DLBCL BCR againsthyperglycosylated SAMD14/NeurabinW-471W-471W-143W-143BCR:SAMD14specificity-+Patient-+-+150kDa120kDa50kDaEndo-HTreatmentHyperglycosylation!Antibody Reactivity of CNS-DLBCL Pati
48、entsagainst hyperglycosylated SAMD14/Neurabin8/12 CNS-DLBCL patients: carriers of SAMD14/Neurabin1mod Malignant clone evolves from poylclonal B-cell responseRole of BCR Targets in the Pathogenesis of B-Cell Neoplasms (I)BCRAntigen (SAMD14 Neurabin-1)Germinal CenterGerminal CenterRole of BCR Targets
49、in the Pathogenesis of B-Cell Neoplasms (II)Antigenic Targets of PCNSL BCRConclusions:1. PCNSL-BCR Target: SAMD14/Neurabinmod in 2/32. Chronic antigenic stimulation pathogenic ?3. Serum antibody diagnostic ?4. Novel therapeutic / prophylactic approaches ?Entities studiedEntities studiedn nMGUS/MMMGU
50、S/MMn nCLLCLLn nFollicular lymphomaFollicular lymphoman nPCNSLPCNSLn nNLPHL (nodular pragranuloma)NLPHL (nodular pragranuloma)Antigenic Targets of ParaproteinsNLPHL:PyruvateCarboxylaseasatargetAn infectious agent, at last .19832586 3024 145619881827961PyruvatcarboxylaseStreptococcus pyogenesStaphylo
