5 NF-κB and AKT signaling prevent DNA damage by suppressing RAG1/2 | 117 Introduction The adaptive immune system plays a crucial role in the defense against pathogens, functioning by virtue of highly specific antigen receptors expressed on B and T cells. Effective immunity requires a diverse repertoire of these antigen receptors, which is achieved by recombination of variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin (Ig) and T-cell receptor (Tcr) loci.1 VDJ recombination requires the recombination activation gene proteins 1 and 2 (RAG1 and RAG2) to instigate DNA breaks in recombination signal sequences (RSSs) that flank the recombining gene segments.2,3 At the pro-B-cell stage, RAG1/2 initiates Ig heavy chain (Igh) recombination after which RAG is downregulated, followed by several rounds of cell division at the large pre-B-cell stage. Subsequently, pre-B cells exit the cell cycle, and RAG expression is upregulated resulting in Ig light chain (Igl) recombination. The functional expression of a tolerant (non-self) B-cell receptor (BCR) switches off RAG, whereas expression of an autoreactive BCR leads to prolonged RAG expression, thereby allowing secondary Igl recombinations in a process known as receptor editing.4,5 Signals emanating from the interleukin-7 receptor (IL7R) and the pre-B-cell receptor (pre-BCR) regulate the dynamic pattern of RAG expression, which involves phosphoinositide-3 kinase (PI3K) and protein kinase B (PKB, also known as AKT) impinging on forkhead box O (FOXO) transcription factors that are required for RAG expression.6,7 The interplay between these signals ensures a sharp demarcation between proliferation and Ig gene recombinations in order to conserve genomic stability in pre-B cells. Additionally, RAG2 protein is phosphorylated at threonine 490 (T490) by the cyclin A/cyclin-dependent kinase 2 (CDK2) complex, eliciting S phase kinase-associated protein 2 (SKP2) –mediated ubiquitination and protein degradation in S phase.8,9 A breach of this regulation results in genomic instability that activates a p53-dependent checkpoint, as was shown by the increased lymphomagenesis in p53-deficient RAG2-T490A mice.10 There is ample evidence for the involvement of RAG in chromosomal aberrations in lymphomas and leukemias, which underscores the importance of proper regulation of this potentially harmful recombination mechanism.11 Moreover, B-cell acute lymphoblastic leukemias (B-ALLs) show a developmental block at the pro- to pre-B cell stage and frequently display constitutive RAG, terminal deoxy-transferase (TdT) expression, and ongoing Ig gene recombinations.12,13 Recent genome-wide analyses of BCR-ABL-positive and ETV6-RUNX1-positive B-ALL have shown that breakpoints of secondary genetic events frequently map near RSS motifs, suggesting the involvement of RAG.14,15 Given its oncogenic potential, a deeper understanding of the regulation of RAG expression and activity is warranted. About 25% of adult B-ALL and 5% of childhood B-ALL patients carry the BCRABL1 fusion gene,16 a tyrosine kinase that mimics IL7R and pre-BCR signaling.17 Here, we made use of human BCR-ABL-positive B-ALL cell lines, Abelson-transformed (Abl) mouse
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