Irradiation Promotes V(D)J Joining and RAG-Dependent Neoplastic Transformation in SCID T-Cell Precursors

Abstract

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinaseactivating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.Lymphocytes require the faithful execution of DNA repair processes to generate a highly diverse repertoire of antigen receptors. The variable region exon of each T-cell receptor (TCR) and B-cell antigen receptor (BCR) gene is assembled by site-specific cleavage and rejoining of variable (V), diversity (D), and joining (J) gene segments in developing T and B lymphocytes. Tandem genomic arrays of dozens to hundreds of V, D, or J gene segments allow combinatorial diversification of the available germline repertoire during lymphocyte development to create a large number of clonally distinct VDJ or VJ genes. In addition, terminal deoxynucleotidal transferase (TdT), a lymphocyte-specific polymerase, inserts non-germline-encoded nucleotides at V(D)J junctions, providing additional somatic diversification of the available germline repertoire (30,44,67). Thus, V(D)J recombination endows T and B lymphocytes with the capacity to specifically recognize and destroy an almost infinite array of pathogens. However, DNA breaks are highly recombinogenic and must be repaired efficiently to maintain genomic stability and minimize the risk of oncogenic transformation (31,33,43,49,90,104). A high frequency of lymphoid tumors have chromosomal translocations involving antigen receptor genes (reviewed in reference 101), raising the question as to whether the V(D)J recombination process threatens genomic stability in lymphoid lineages.The molecular mechanism of V(D)J cleavage has recently been elucidated by elegant biochemical studies (reviewed in references 93 and 106). This process is mediated by the lymphoid-specific RAG-1 and RAG-2 proteins, which bind recombination signal sequences fl...

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