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Germinal Centers and Lymphomagenesis

V(D)J Recombination. IgV hypermutation. Immature B-cells. Germinal Center B-cells. Memory B-cells. Ag. B-CLL. Plasma cells. Bone Marrow. Mantle. Germinal Center. ALL. MCL. BL FL DLBCL. MM. Apoptosis. Germinal Centers and Lymphomagenesis.

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Germinal Centers and Lymphomagenesis

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  1. V(D)J Recombination IgV hypermutation Immature B-cells Germinal Center B-cells Memory B-cells Ag B-CLL Plasma cells Bone Marrow Mantle Germinal Center ALL MCL BL FL DLBCL MM Apoptosis Germinal Centers and Lymphomagenesis Klein and Dalla-Favera, Nature Immunology, 2008 Ig isotype switch Naive B-cells

  2. AID Two major mechanisms of genetic lesion Germinal Center CSR SHM physiologic Hyper proliferation Chromosomal Translocations (Ig-cMYC, Ig-BCL6) Aberrant SHM (multiple oncogenes) pathologic DLBCL/BL DLBCL

  3. Diffuse Large B-cell Lymphoma (DLBCL) • Most common and aggressive subtype of B-NHL in the adult (~40% of all diagnoses) • Poor response to therapy; ~30% of patients not cured by currently available therapeutic strategies • Marked heterogeneity in morphologic, molecular, and clinical features • Several phenotypic subgroups recognized by gene expression profile studies

  4. Role of BCL6 and the “BCL6 pathway” in DLBCL pathogenesis

  5. Gene expression profiling can classify DLBCL into at least two distinct subtypes Germinal Centre IgV somatic hypermutation Ig class switch Plasma cell Dark zone Light zone Naïve B cell Antigen Germinal Centre-like (GCB) DLBCL Activated B cell-like (ABC) DLBCL Memory B cell Alizadeh A et al, Nature 2000; Staudt LM, N Engl J Med 2003

  6. BCL6 BCL6 protein Trans-repression K 1 706 P P Zn+ Zn+ Zn+ Zn+ Zn+ Zn+ COOH NH2 POZ (protein-protein interaction) PEST (inactivation) Zinc Fingers (DNA binding) Role in Germinal Center Translocations & mutations (DLBCL) Breakpoints (35% of cases) BCL6 +/+ BCL6 / PNA 1 2 3 4 5 6 7 8 9 10  BCL6 5' 3' Activating mutations (13% of cases)

  7. Targets directly bound, dynamically connected, and down-regulated in GC Tools to identify physiologic BCL6 direct target genes Network relationship Transcriptional repression in GC Direct binding + - - ChIP-chip - + - ARACNe - - + GEP ChIP-chip: BCL6 ChIP from normal GC B cells ARACNe: a bioinformatic tool able to predict transcriptional relationships between genes starting from GEP GEP: Genes down-regulated in normal GC B cells compared to Naïve & Memory B cells

  8. BCL6, Germinal Center and Lymphomagenesis Centroblast Memory B cell BCL6 Naive B cell Niu et al., J Exp Med 2003 Phan et al., Nature 2004 Basso et al., Blood 2010 Ranuncolo et al., Nat Immunol 2007 Saito et al., Proc Natl Acad Sci USA 2009 Tunyaplin et al., J Immunol 2004 Plasma cell Germinal Center

  9. BCL6, Germinal Center and Lymphomagenesis Centrocyte Centroblast Memory B cell BCL6 Naive B cell Niu et al., J Exp Med 2003 Phan et al., Nature 2004 Basso et al., Blood 2010 Ranuncolo et al., Nat Immunol 2007 Saito et al., Proc Natl Acad Sci USA 2009 Tunyaplin et al., J Immunol 2004 Plasma cell Germinal Center

  10. BCL6, Germinal Center and Lymphomagenesis Centrocyte Centroblast Memory B cell BCL6 Naive B cell Niu et al., J Exp Med 2003 Phan et al., Nature 2004 Basso et al., Blood 2010 Ranuncolo et al., Nat Immunol 2007 Saito et al., Proc Natl Acad Sci USA 2009 Tunyaplin et al., J Immunol 2004 Follicular Lymphoma Burkitt Lymphoma Plasma cell Germinal Center

  11. Bcl6- Blimp1+ Pathways downregulating BCL6 in the germinal center CD40L CD40 NF-kB IRF4 Blimp1 Bcl6 Plasma cell Late GC B cell Saito et al., Cancer Cell 2007 Klein et al., Nature Immunology 2007

  12. IRF4 IRF4 IRF4 IRF4 E1 Translocations and mutations of BCL6 prevent CD40-induced downregulation by IRF4 Germinal Center B cells off IRF4 IRF4 E1 BCL6 Saito et al., Cancer Cell 2007 DLBCL on Chromosomal translocations E1 BCL6 Gene X Somatic Mutations on   BCL6

  13. Lymphoproliferative disorders and DLBCL in IµHABCL6 mice DLBCL LPD Histology: Clonality: Monoclonal (10/10 cases) Mutated IgV genes (9/10 cases) Oligo/Polyclonal (8/8 cases) Cytogenetics: Normal Multiple clonal lesions Trisomy 15 (7/10 cases) Cattoretti et al., Cancer Cell 2005

  14. Bcl6- Blimp1+ DLBCL The CD40/NF-kB/IRF4/Blimp1 pathway can be blocked at multiple levels in ABC-DLBCL CD40L CD40 NF-kB IRF4 Blimp1 Bcl6 Plasma cell Late GC B cell Saito et al., Cancer Cell 2007

  15. BLIMP1 is inactivated by mutations in ABC-DLBCL a. Gene rearrangements Splice site mutations Frameshift deletions Nonsense mutations b. c. tonsil DLBCL#56 DLBCL#93 IRF4 BLIMP1 % Mutated cases merge Pasqualucci et al., J Exp Med 2006

  16. Blimp1CD19KO mice have reduced survival LogRank p value <0.0001

  17. BCL6 R B6BS M BLIMP1 M Bcl6- Blimp1+ DLBCL The CD40/NF-kB/IRF4/Blimp1 pathway can be blocked at multiple levels in ABC-DLBCL CD40L CD40 NF-kB IRF4 Blimp1 Bcl6 Plasma cell Late GC B cell Pasqualucci et al., J Exp Med 2006 Tam et al., Blood 2006 Saito et al., Cancer Cell 2007

  18. Bcl6- Blimp1+ DLBCL The CD40-NFkB-IRF4-Blimp1 pathway can be blocked at multiple levels in ABC-DLBCL: role of NF-kB CD40L CD40 NF-kB targets NF-kB L. Staudt group, JEM 2001, Nature 2007 IRF4 Blimp1 Bcl6 Plasma cell Late GC B cell Pasqualucci et al., J Exp Med 2006 Tam et al., Blood 2006 Saito et al., Cancer Cell 2007

  19. 100% 80% p50 p52 p50+p52 none 60% % of NFkB enriched cases 40% 20% 0% ABC (N=21) GCB (N=28) NC (N=9) Immunohistochemistry identifies active nuclear NF-kB complexes in >50 % of ABC-DLBCL p105/p50 (classical) p100/p52 (alternative) nuclear cytoplasmic nuclear cytoplasmic Fraction of nuclear + cases ABC (n=6) GCB (n=12) ABC (n=24) GCB (n=28) NC (n=9) not profiled (n=76) Cell lines Biopsies Compagno et al, Nature 2009

  20. Multiple NF-kB pathway components are mutated in ABC-DLBCL Total: 19/37 (51%) ABC-DLBCL 6/31 (19%) GCB-DLBCL

  21. P The NF-kB negative regulator A20 ZF ZF ZF ZF ZF ZF ZF OTU domain 1 790 K63 Deubiquitination of K63-linked chains Ubiquitin ligase activity (K-48 linked) Ub Ub Ub Ub TNF RIP RIP RIP Ub Ub Ub Ub K63Ub: signal for activation K48Ub: signal for degradation K48 • Cytoplasmic Zinc finger protein • Dual-function ubiquitin modifying enzyme • Expressed predominantly in B/T lymphoid tissues during development • Induced by TNF, CD40, TLR and LMP1 stimulation • Involved in termination of NF-kB responses in vivo • Located on chromosomal band 6q23, frequently deleted in cancer

  22. A20 mutations lead to truncated A20 proteins exons ZF ZF ZF ZF ZF ZF ZF 790 1 OTU domain K48 Ub ligase activity frameshift deletions (n=5) frameshift insertions (n=7) splice site (n=2) nonsense (n=12) Ex2 In2 TUMOR NORMAL

  23. DLBCL-2062 DLBCL-2053 allele A B Mutated Deleted Normal 6q23 A20 is biallelically inactivated in ~30% of ABC/non-GC DLBCL Affymetrix SNP6.0 heterozygous deletion homozygous deletion DLBCL N OLIG3 A20 CEP 6 A20 Biallelic deletion Mutation + deletion Biallelic mutation Monoallelic mutation Monoallelic deletion Normal

  24. ≥60% ≥18% Bcl6- Blimp1+ DLBCL ≥30% Pathway Lesions in ABC-DLBCL Ag CD40L CD40 BCR CD79A,B CD40 21% NF-kB IRF4 MAPK/ERK MTOR/AKT Blimp1 apoptosis Bcl6 Plasma cell Late GC B cell

  25. Therapeutic targets

  26. SUDHL2 SUDHL7 Days after seeding Therapeutic targeting: A20-null DLBCL are “addicted” to NF-KB a. b. c. pWPI pWPI-HAA20 pWPI pWPI-HAA20 18% 2.4% SUDHL2 (A20m/m) absorbance 11% 1.5% RCK8 (A20m/m) absorbance 1.1% 1% SUDHL4 (A20wt/wt) d. pWPI HA-A20 p50 1.3% 0.3% SUDHL7 (A20wt/wt) DAPI 7AAD % apoptotic cells AnnexinV AnnV+7AAD+ AnnV+7AAD- SUDHL2

  27. Co-repressor complex Ac SAHA HDAC inhibitor Small molecules BCL6 P Acetylation DNA damage Ac P p53 Sir2 inhibitor NIACINAMIDE ETOPOSIDE DEATH The BCL6:p53 network: a therapeutic target

  28. How many genetic lesions to make a DLBCL?

  29. Somatic Mutation + Copy Number Variation Analysis Somatic mutations (SM) Whole Exome Sequencing (7 cases, T versus N DNA) Identification of candidate SM n=1396 Copy number variation (CNV) SNP array analysis (72 cases) Identification of CNV loci n=2143 Validation of candidate SV (Sanger Sequencing, same cases) n=87 Identification of MCR (<20 candidate target genes) n=3068 N=16 List of candidate target genes n=84 List of candidate target genes n=244 Mutation screening of pathway-linked genes in large panel (n=98)

  30. Whole exome sequencing of DLBCL and CLL Sonication Linker ligation 5µg of human genomic DNA (N=7DLBCL and 5CLL, paired Tumor/Normal) whole genome fragment pool (500-700 bp each) Hybridize Enriched target DNA High Throughput Sequencing Elute Wash 2.1M Human Exome Array 34Mb of sequence 165,000 coding exons* 551 miRNA exons (*~1300 genes not included) 454 Genome Sequencer ≥1 million reads/plate ~400bp/read ≥400 Mb/plate (2 plates/sample) Mapping to Human Genome (hg18) Identification of sequence variants Validation by direct sequencing

  31. Somatic non-synonymous mutation load in DLBCL and CLL DLBCL B-CLL N of somatic mutations Total N of somatic mutations confirmed by direct sequencing: DLBCL = 63 (average 15/case) B-CLL = 30 (average 6/case)

  32. Columbia University Ryan Phan Masumichi Saito Ulf Klein Marta Crespo Jie Gao Urban Novak Katia Basso Yukiko Kitagawa Govind Bhagat Mara Compagno Adina Grunn Laura Pasqualucci VVVS Murty Vladimir Trifonov Raul Rabadan Andrea Califano University of Novara, Italy Davide Rossi Gianluca Gaidano Cornell University, New York Amy Chadburn IOSI, Lugano, Switzerland Francesco Bertoni

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