How are V(D)J recombination events involved in generating oncogenic chromosome translocations underlying some leukemias and lymphomas? Many proposals have been advanced. Some events appear to involve joining of RAG-generated DSBs to breaks made by other mechanisms at the partner loci. A few years ago, we proposed that errors the choice of repair pathway to heal the DSB could lead to aberrant joining events. Our recent data now reveal that specific mutations in either RAG1 or RAG2 abrogate pathway choice and also destabilize the post-cleavage complex in vitro. Our preliminary data implicate some of these mutations in RAG-induced oncogenic translocations in murine lymphomas. These same mutations are associated with increased alternative NHEJ in lymphocytes in vivo. Together, these data support the following hypotheses, which we will test in the proposed experiments. Hypothesis 1: "Pathway choice control" plays a critical role in maintaining genomic stability, and is maintained in V(D)J recombination by the RAG post-cleavage complex. Decreased stability of the RAG postcleavage complex (resulting from any of a number of causes) allows increased availability of the coding and/or signal ends to inappropriate joining pathways, facilitating formation of aberrant V(D)J recombination products, including oncogenic chromosome translocations. Hypothesis 2: Abrogating pathway choice control allows alternative NHEJ to emerge as a mutagenic repair pathway. Understanding control of pathway choice and the consequences of disabling this regulatory mechanism in the context of V(D)J recombination will illuminate a question that has remained unanswered for almost 30 years: how do the oncogenic translocations that occur in developing lymphocytes arise? The knowledge we gain from the proposed studies is also likely to help us approach this important question in other contexts. If, in the proposed studies, our RAG mutants which deregulate pathway choice allow us to observe frequent oncogenic rearrangements in the context of an intact classical pathway, we will be able to establish that alternative NHEJ can indeed compete with the classical joining mechanisms. This result would imply that control of pathway choice is imposed, by some mechanism, in other DSB repair situations that do not involve the RAG proteins.

Public Health Relevance

Although the exact steps that lead to malignant transformation in leukemia and lymphoma remain unknown, a large fraction appear to result from mistakes in the physiologic DNA rearrangement process responsible for diversifying antigen receptor genes in B and T lymphocytes. We have recently generated recombinase mutants that prematurely release the DNA ends. Interestingly, these mutants cause lymphomas in mice;the proposed experiments will use these mice and other molecular tools generated during the last funding period to explore the mechanisms by which disruption of the recombinase-DNA end complex leads to lymphomagenesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA104588-08
Application #
8197240
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Howcroft, Thomas K
Project Start
2004-04-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
8
Fiscal Year
2012
Total Cost
$274,813
Indirect Cost
$103,055
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Gigi, Vered; Lewis, Susanna; Shestova, Olga et al. (2014) RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'. Nucleic Acids Res 42:6352-64
Coussens, Marc A; Wendland, Rebecca L; Deriano, Ludovic et al. (2013) RAG2's acidic hinge restricts repair-pathway choice and promotes genomic stability. Cell Rep 4:870-8
Chaumeil, Julie; Micsinai, Mariann; Ntziachristos, Panagiotis et al. (2013) The RAG2 C-terminus and ATM protect genome integrity by controlling antigen receptor gene cleavage. Nat Commun 4:2231
Deriano, Ludovic; Chaumeil, Julie; Coussens, Marc et al. (2011) The RAG2 C terminus suppresses genomic instability and lymphomagenesis. Nature 471:119-23
Arnal, Suzzette M; Holub, Abigail J; Salus, Sandra S et al. (2010) Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways. Nucleic Acids Res 38:2944-54
Deriano, Ludovic; Stracker, Travis H; Baker, Annalee et al. (2009) Roles for NBS1 in alternative nonhomologous end-joining of V(D)J recombination intermediates. Mol Cell 34:13-25