Modelli murini di linfomagenesi. Roberto Chiarle, M.D. Firenze, 24/11/2011

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1 Modelli murini di linfomagenesi Roberto Chiarle, M.D. Firenze, 24/11/2011

2 Chromosomal translocations in tumors Translocation are common in many types of tumors and are often considered primary oncogenic events. Clonal translocations are the hallmark of most B and T cell tumors, but are often present in solid tumors, such as carcinomas and sarcomas. Translocations cause aberrant expression of oncogenes which confers a strong selective advantage but factors influencing frequency of translocations before establishment of cellular selection have not been fully elucidated Mouse Chr12 IgH c-myc Mouse Chr15 Campbell et al. Cell 2011

3 Factors that influence the occurrence of translocations in tumors Mechanistic Factors 1- DSBs formation and frequency 2- Spatial proximity 3- Intra- or Inter-chromosomal joining (DSB repair pathways) Cellular Selection Oncogenic translocations are subject to cellular selective forces, through elements that influence expression of translocated loci and activation of oncogenic pathways

4 Mechanistic Factors: 1- DSBs formation and frequency Bone marrow Peripheral lymphoid organs (lymph nodes, spleen) DSBs DSBs DSBs BCL2-IgH BCL1-IgH BCL2-IgH c-myc-igh

5 Mechanistic Factors: 2- Spatial proximity Folding principles of the human genome Lieberman-Aiden, Science 2009

6 CONTACT-FIRST MODEL DSBs Repair within proximal chromosomes Chromosomal territories DSBs-FIRST MODEL DSBs Repair between distant chromosomes DSBs movement Gostissa, Alt and Chiarle. Annual Rev Immunol, 2011

7 Mechanistic Factors: 3- Intra- or Inter-chromosomal joining (DSB repair pathways) Gostissa, Alt and Chiarle. Annual Rev Immunol, 2011

8 IgH Class Switch Recombination

9 The Yeast I-SceI Meganuclease as a tool to study DSB repair, CSR and Translocations

10 A ΔSγ1 model (chr. 12) ieµ C µ C δ C γ3 * * 0.5kb C γ1 C γ2b C γ2a C ε C α S µ S γ3 ΔS γ1 S γ2b S γ2a S ε S α B c-myc model (chr. 15) Ex1 **** **** Ex2 Ex3 25x I-SceI Ex1 * Ex2 Ex3 1x I-SceI * I-SceI target sequence Sequencing primer LoxP

11 High Throughput Genomic Translocation Sequencing (HTGTS)

12 High Throughput Genomic Translocation Sequencing (HTGTS) I-SceI Induction of DSB Translocation Intramolecular ligation 1 st PCR (enrich) DNA fragmentation by frequent cutter Ligate linker 1 st PCR (enrich) 2 nd nested-pcr (specificity) 2 nd nested-pcr (specificity, sequencing primers) 3 rd PCR (sequencing primers) 454 sequencing 1 st PCR primers 2 nd PCR primers adapter primers method ad-pcr ad-pcr ad-pcr circ-pcr mouse DNA 25x I-Sce I c-myc WT 25x I-Sce I c-myc ΔSγ1 AID-/- - WT 2x I-Sce I 25x I-Sce I c-myc WT huma n DNA K562 K562 K562 K562 mouse junc huma n junc background 0.53% 0.56% 0.33% 0.63%

13 High Throughput Genomic Translocation Sequencing (HTGTS) Control Retrovirus I-SceI Retrovirus

14 High Throughput Genomic Translocation Sequencing (HTGTS) c-myc locus IgH locus Chiarle et al. Cell, 2011

15 High Throughput Genomic Translocation Sequencing (HTGTS) from the c-myc locus 1a) Arid5a 1b) Aff3 Minus strand Plus strand Cen 1a 1b 2a) Rapgef1 2b) Traf1 2c) Mmp24 2d) Bcl2l1 2a 2b 4a) Pax5 4b) Hivep3 4a 6a) Clec2d 7a) Il4i1 7b) Apbb1 7c) Il4ra 7d) Il21r 8a) Fcer2a 8a 9a) Fli1 9b) Kirrel3 10a) Tnfaip3 10b) Socs2 10a 11a 11a) Sfi1 12a) 11b) Bcl11a Rad51l1 11c) Ebf1 12b) 11d) GrapDync1h1 11e) Mpdu1 12c) IgH 13a) CD83 13b) Mef2c 14a) Fermt2 16a) Lrrc33 18a) Zfp608 17a) Pim1 17b) mir-715 Tel 2cd 4b 6a 7a 7b 7cd 9a 9b 10b 11b 11c 11d 11e 12 a 12 b 13 a 13 b 14 a 16 a 17 a 17 b 18 a 19 a 19a) Scd2 X a Xa) Gucy2f X c-myc Chiarle et al. Cell, 2011

16 High Throughput Genomic Translocation Sequencing (HTGTS) from the IgH locus (489) (282) 10kb WT * ΔSγ1/ 2x I-SceI Iµ Sµ-Cµ Sγ3 Sγ2a Sγ2b Iε Sε-Cε Sα AID-/- Iγ1 Sγ1-Cγ1 Chiarle et al. Cell, 2011

17 High Throughput Genomic Translocation Sequencing (HTGTS) Hotspots Iµ-Sµ-Cµ Iε-Sε-Cε Iγ1-Sγ1-Cγ1 Mmp24 Il4ra CD83 Pim1 mir-715 region Fermt2 Gm12493 Aff3 Kirrel3 Apbb1 Jκ Chr1: Il21r Rapgef1 Fcer2a Arid5a Il4i1 Chr4: Pax5 Clec2d Gucy2f Sfi1 Grap Fli1 Traf1 Chr1: Bcl11a Rad51l1 Tnfaip3 Scd2 Zfp608 Sh3bp5 Socs2 Hivep3 Ebf1 Mef2c Dync1h1 Lrrc33 Bcl2l1 Mpdu1 Itpk1 WT AID-/- Chiarle et al. Cell, 2011

18 Promoter and transcription dependency of translocations WT AID-/- Chiarle et al. Cell, 2011

19 Promoter and transcription dependency of translocations Chr7: 89Kb Chr13: 27Kb Chr1: 63Kb AID / Junc=ons (1) (0) (6) (1) WT Junc=ons (35) (6) (30) (19) sense An= sense Il4ra Il21r CD83 Aff3 Chiarle et al. Cell, 2011

20 CERMS Center for Experimental Research and Medical Studies University of Torino, Italy Giorgio Inghirami Claudia Voena Cinzia Martinengo Chiara Ambrogio Cristina Mastini Mara Compagno Matteo Menotti Roberta Pulito Roberto Piva Ramesh Choudhari Silvia Peola Maria Stella Scalzo PCMM Children s Hospital IDI - Immune Disease institute Fred W. Alt Monica Gostissa Richard Frock Darienne Myers Susanna Lewis Daniel Malkin Yu Zhang Molecular Biotechnology Center University of Torino, Italy Guido Forni Federica Cavallo Emilio Hirsch Silvio Aime Dario Longo Lorenzo Silengo Fiorella Altruda