1. VIRUSES AND NUCLEAR ORGANIZATION IN ONCOGENESIS Yegor VASSETZKY, CNRS UMR 8126, Institut de Cancérologie Gustave Roussy

2. VIRAL THEORY OF CANCER: UPS AND DOWNS Peyton Rouss 1911: discovery of RSV Denis Burkitt 1957: discovery of Burkitt’s lymphoma 1966: Nobel Prize Anthony Epstein Yvonne Barr 1964: discovery of EBV in Burkitt’s lymphoma samples

3.  A non-Hodgkin Lymphoma  Three forms:  An endemic form in Afrtica is 100% associated with EBV  A sporadic form in Europe and North America, rare and non-associated with EBV  A form associated with HIV is frequent in Europe and is found in up to 2% (!) of AIDS patients  In ~90% of the cases BL is linked to the translocation t(8;14)(q24;q32) of the CMYC gene locus next to the IGH gene locus leading to activation of the CMYC gene. BURKITT’S LYMPHOMA

4. HIGH OCCURRENCE OF BURKITT’S LYMHOMA IN HIV PATIENTS: WHY? Shen,2011 CancerFrequency in the general poplation Frequency in AIDS patients Ratio Burkitt’s Lymphoma 1:200 0001: 400050 Mantle Cell Lymphoma 1:200 000 1 gp-120 can interact with CD21 expressed on B cells (Moir et al 2000) HIV-1 causes B-cell hyperactipvation (Schnittan et al,1984) Elevated class switch in B lymphocytes Induces B cell to proliferate (Nair MPN and al 1988) Causes B cell abnormal response The production of autoantibodies Aberrant B-cell surface markers: Change in B cell receptors.  Three events are necessary to produce a translocation: DNA double strand breaks (Vilenchik et Knudson, 2003). Double strand breaks repair via NHEJ (Abeysinghe et al., 2003). Spatial proximity (colocalization) of the two translocation partners. (Nikiforova et al., 2000, Misteli, 2003).

5. CHROMOSOMAL TERRITORIES Bolzer et al., Plos Biology (2005), 3 (5) e207  The chromosomes are organized in the nucleus:  In a tissue specific manner  The organization is transmitted though the cell divisions  This organization is evolutionarily conserved  The gene-rich regions occupy more central position in the nucleus Theodor Boveri (1862-1915) Carl Rabl (1853-1917) Bolzer et al., PlosBiology (2005), 3 (5) e207

6. NUCLEAR ARCHITECTURE

7. HIV: A ROLE IN RELOCALIZATION OF C-MYC INTO REGIONS PROXIMAL TO IgH? Normal B-Lymphocytes: IGH/CMYC proximity in ~3% of cells C-MYC IGH Topro 3 Periphery Centre

8. HIV PROVOKES A RELOCALIZATION OF A CMYC LOCUS IN THE NUCLEAR SPACE AND ITS COLOCALIZATION WITH IgH 3µm CMYC IGH Topro 3

9.  9 genes encoding 3 structural, 2 envelope, and 6 regulatory proteins in addition to 3 enzymes  TAT – transactivator of transcription encoded by 2 different exons. The 102 aa Tat is responsible for activation of viral Tat is secreted into the circulation and is capable to penetrate into cells. Produced in excess in infected cells. Tat is present in blood of AIDS patients. HIV GENOME

10. Tat induces a dose-dependent CMYC/IGH colocalization Tat does not induce IGH/  -globin or IGH/CCND1 colocalization in B lymphocytes Tat does not induce CMYC/IGH colocalization in macrophages and T cells HIV Tat PROVOKES A SPECIFIC COLOCALIZATION OF THE IGH AND CMYC LOCI IN THE NUCLEAR SPACE OF B CELLS CancerFrequency in the general poplation Frequency in AIDS patients Ratio Burkitt’s Lymphoma 1:200 0001: 400050 Mantle Cell Lymphoma 1:200 000 1

11. IGH/CMYC COLOCALIZATION IN AIDS PATIENTS

12. 30 TRANSACTIVATION DOMAIN OF HIV Tat IS REQUIRED TO INDUCE COLOCALIZATION OF IGH AND CMYC AND ACTIVATE CMYC TRANSCRIPTION RNA pol2 CMYC IGH

13. IGH/CMYC COLOCALIZATION IS NHEJ-DEPENDENT Nucleus IGH CMYC MRE11 MERGE

14. HIV TAT INDUCES DNA DAMAGE IN B-LYMPHOCYTES

15. HIV TAT INDUCES DNA DAMAGE IN THE CMYC LOCUS % of cells with gH2AX foci @ 6h % of cells with gH2AX/CMYC Colocalization *** *** *** ns Nucleus CMYC  H2ax Merge

16. RAG1 IS INDUCED BY TAT AND IS ESSENTIAL FOR IGH/CMYC COLOCALIZATION RAG1/2 AID ns * ns

17. NFkB PATHWAY AND ITS REGULATION BY HIV TAT HIV Tat ikBa phosphorylation @ 1h ** ns Li & Verma, 2002

18. IgH c-myc HIV EBV HIV: A ROLE IN INTRANUCLEAR REORGANIZATION AND IN GENERATION OF SPECIFIC TRANSLOCATIONS HIV Tat  NFkB  RAG  DSB  NHEJ  CMYC relocalization Tat C22 RAGi Mirin Transription NU7026 T T T

19. Nuclear Organization and Pathologies Lab (UMR-8126, IGR, Villejuif, France ):  Marc Lipinski, DR CNRS  Chrystèle Bilhou-Nabera, MCU-PH  Diego Germini, postdoctorant  Tatiana Tsfasman, postdoctorante  Yara Bou Saada, doctorante UPS  Shirmoné Botha, doctorante  Anatasia Sukhanova, M1  Rawan El-Amine, doctorante (cotutelle Liban)  Carla Dib, doctorante  Eric Oksenhendler, HSL, Paris  Sergey Razin, IBG, Moscow  Olga Iarovaya, IBG, Moscow  Evgeny Sheval, MSU, Moscow  S. Bury-Moné, ENS Cachan