MECHANISMS OF V(D)J RECOMBINASE MEDIATED GENOMIC INSTABILITY AND LYMPHOMAGENESIS

A child has a 1 in 2000 chance of developing leukemia by the age of 15. The most common types of childhood leukemia, acute lymphoblastic leukemia and acute myeloid leukemia, typically exhibit gross chromosomal abnormalities. While it is known how some of these abnormalities contribute to tumor development by altering the activities of protooncogenes or tumor suppressors, much less is known about how these genomic derangements are initiated. Tumor-associated chromosomal translocations result from destabilizing DNA transactions, including a form of programmed DNA rearrangement during which antigen receptors encoded in discrete DNA segments are brought together, V(D)J recombination. V(D)J recombination is initiated by the cleavage of participating gene segments at recombination signal sequences (RSSs) by the lymphoid-specific proteins RAG-1 and RAG-2. The destruction of RAG-2 is regulated by its phosphorylation and polyubiquitylation at the G1-S transition by Cyclin A/Cdk2 and SKP2-SCF, respectively, coupling V(D)J recombination to the cell cycle. Accumulation of RAG-2 is uncoupled from the cell cycle in a mouse model by introducing a T490A mutation (RAG-2(T490A)). The RAG-2(T490A) mutation, in the absence of p53, promotes development of B and T lymphomas bearing complex, clonal chromosomal translocations. Analogous to the RAG-2 T490A mutation, deficiency in SKP2 results in accumulation of RAG-2 throughout the cell cycle. In Skp2-/- and RAG-2(T490A) mice, recombination intermediates, in the form of broken signal ends, persist throughout cell cycle. Further, both mouse models harbor aberrant recombination products with templated additions, a feature consistent with repair of RAG mediated DNA breaks by Non-homologous End Joining (NHEJ) in combination with abortive Break Induced Replication. Similar templated additions are found at translocation junction breakpoints in some forms of human lymphoma, lending support to the notion that dysregulated V(D)J recombination underlies their formation. A survey of the RAG-2 sequence in 40 samples of pediatric T cell Acute Lymphoblastic Leukemia (ALL) uncovered 3 missense mutations. Two of these are within an autoinhibitory region, under investigation for its role in regulating the interaction of the recombinase with its DNA substrate. Following initiation of recombination by RAG-1 and RAG-2 and formation of post-cleavage complexes, ubiquitous DNA repair factors act to repair the DNA double strand breaks. One of the DNA repair factors, Ataxia Telangectasia Mutated (ATM), phosphorylates hundreds of substrates, promoting cell cycle checkpoint activation and either NHEJ or Homologous Recombination (HR). Among its substrates is p53 Binding Protein 1 (53BP1) which is recruited to divalent chromatin marks surrounding double strand breaks. 53BP1 protects against end resection in a RIF-1 dependent manner and promotes repair by NHEJ. While ATM and 53BP1 are recognized tumor suppressors, little is know about how they interact in the suppression of oncogenic chromosomal translocations. We report that loss of 53BP1 markedly decreased latency of V(D)J recombinase-mediated T cell lymphomas in ATM-deficient mice. The proposed mechanism for decreased tumor latency is aggravation of the repair defect previously reported for ATM-deficient mice. In mice doubly deficient for ATM and 53BP1, a switch from classical NHEJ to alternative forms of DNA repair was not observed, rather there were defects in cell cycle checkpoints, resulting in more frequent chromosomal breaks and translocations. Finally, 53BP1 suppressed large deletions in a subset of V(D)J recombinase-mediated hybrid joints (a result of defective end joining) in ATM-deficient thymocytes. Taken together, these observations support a role for 53BP1 in the protection of genomic stability that, during oncogenic stress, is partially independent of ATM