V(D)J recombinaten and chromosomal translocations

Research project P 13984 V(D)J recombination and chromosomal translocations Ulrich JÄGER 11.10.1999 Lymphoid neoplasms (non-Hodgkin`s lymphomas and acute lymphoblastic leukemias) are frequently associated with reciprocal translocations of proto-oncogenes into one of the immunoglobulin (Ig) or T-cell receptor (TCR) loci. This illegitimate recombination which constitutes the molecular basis of malignant transformation is supposedly associated with the mechanism of normal Ig- or TCR rearrangement (V(D)J-recombination). However, the evidence for the involvement of V(D)J-recombinase is primarily based on "post-translocation" sequence information of breakpoints and chromosomal junctions of reciprocal translocations. These observations will now have to be tested in functional systems. The most frequent translocation, the t(14; 18)/BCL2-IgH translocation in follicular lymphoma will serve as a model. We have recently generated a comprehensive sequence library of t(14; 18) direct (BCL2-JH) and reciprocal (DH-BCL2) junctions and found compelling evidence for the involvement of V(D)J-recombinase on the IgH breakpoints and clear evidence for a different mechanism coupled to error-prone repair generating the breaks in the BCL2 breakpoint region. There is increasing evidence that the translocation takes place in the lymph node and not in the bone marrow as previously postulated. Recent reports also point to the existence of a transpostional activity of the Ig recombination signal sequences. We now aim to further cahracterize and dissect the mechanisms and conditions of chromosomal breakage and religation of the IgH and BCL2 genes in transient and stable transfection systems containing the translocation partners. The goal of these experiments is (1) to estimate the frequency of BCL2-IgH recombination; (2) to define the minimal DNA-elements required; (3) to define external and internal factors (enzymes, DNA-binding proteins, chemotherapeutic agents, pesticides, cytokines and receptors, antigenic stimuli, radiation) involved; (4) to define the stage of development at which the translocation takes place using lymphoid and non-lymphoid cell lines of various differentiation status. Finally, this technology shall be made applicable to other translocations in leukemia and lymphoma and may serve to test new agents which influence these processes.

We have established a test system for carcinogenesis as well as a novel mechanism leading to leukemia and lymphoma. The results of this project may be translated into methods for functional cancer diagnostics, cancer prevention and therapy. Chromosomal translocations are the first event in the development of malignant diseases of the blood and lymphatic system (leukemias and lymphomas). The aim of this project was to investigate the most frequent translocations in leukemia and lymphoma in patient samples. Subsequently, an artificial system was developed which allows the investigation cellular and environmental influences in vitro. In the first year, chromosomal breakpoints from 93 patients with lymphomas were studied by molecular methods. The involvement of a novel DNA-repair mechanism was discovered in translocations specific for follicular lymphomas and mantle cell lymphomas. This error-prone DNA-polymerase becomes active when other repair mechanisms fail. The major goal of this project was the establishment of the functional in vitro recombination assay. For this reason, breakpoint regions from various chromosomes and genes were cloned into plasmid vectors and studied after transfection into cell lines. In the second year we applied the system to various translocations which could be classified into 2 types depending on the translocation mechanism. Our in vitro results also predicted the existence of translocations in normal thymus. In the third year we compared the genetic differences between these translocations in normal individuals with those observed in patients with acute T-cell leukemia. This led to the discovery of a novel translocation mechanism which may be of general relevance ("signal joint reactivity"). The results of these studies were published in Cancer Research, Journal of Experimental Medicine and Nature Genetics.