RAG-mediated DNA Damage Responses in Immune Development and Function

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.

Grant Number: 4R01AI173077-04
NIH Institute/Center: NIH
Principal Investigator: Jeffrey Bednarski