RAG-mediated DNA Damage Responses in Immune Development and Function

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PROJECT SUMMARY Lymphocyte development is precisely controlled to enable clonal expansion and expression of a diverse immunoglobulin receptor repertoire, which proceeds through DNA double-stranded breaks (DSBs) generated by the RAG endonuclease. These two dichotomous, but interdependent processes, are managed through the cooperation of diverse cellular signals to prevent cells with DSBs from entering cell cycle where they could be aberrantly repaired as translocations. During early B cell development, the pre-B cell receptor (pre-BCR), through activation of the SYK kinase, coordinates both the proliferative expansion of pre-B cells and the assembly of immunoglobulin receptor genes. Negative regulation of the pre-BCR is required to ensure cell cycle arrest and limit the number of DNA breaks generated during immunoglobulin receptor gene assembly. Indeed, unopposed pre-BCR signaling, particularly increased SYK activity, drives proliferation and leukemic transformation. However, the mechanisms that repress SYK and pre-BCR signaling are not known and remain a critical gap in our understanding of B cell maturation. We have identified a novel cell-type specific program activated by signals from RAG DSBs that suppresses SYK and inhibits pre-BCR signaling. Deficiencies in this DNA damage- mediated feedback circuit result in initiation of pre-B cell leukemia. Surprisingly, this signaling network is not triggered by all DNA injury but, rather, is specific to RAG DSBs generated during immunoglobulin receptor gene assembly. Our goal is to determine how signals from RAG DSBs integrate with developmental programs to coordinate B cell maturation and prevent leukemic transformation. We propose that RAG DSBs suppress SYK to enforce cell cycle arrest and, thereby prevent B cells with DNA breaks from re-entering cell cycle. This DNA damage-mediated checkpoint program would permit iterative attempts at generation of a mature antigen receptor to promote B cell differentiation while preventing leukemic initiation. Further, we propose that these DNA damage signals are activated through distinct domains of the RAG endonuclease that interact with proteins at sites of DSBs to modulate signaling pathways. This RAG-specific mechanism in B cells discriminates between normal and errant DSBs to activate appropriate cellular responses. Utilizing an innovative experimental approach that allows interrogation of DSB signals within the context of B cell developmental programs, our proposed studies will define how RAG DSB signals maintain pre-B cell checkpoint and will resolve the mechanisms that distinguish RAG-mediated from non-RAG-mediated DNA damage. Completion of these studies will delineate pathways critical for dampening proliferative signals in early B cells, establish signals that restrict leukemogenesis, and define novel functions of the RAG endonuclease in regulating DNA damage responses.