84 | Chapter 4 of Thr490 of RAG2 by the CyclinA/CDK2 complex, which is active at the G1/S transition, signals the periodic destruction of RAG2. A threonine-to-alanine mutation at this residue (T490A) rendered RAG2 insensitive to this regulation and allowed its persistence in S- and G2/M-phases14. Such unrestricted expression of RAG2 throughout the cell cycle resulted in formation of aberrant recombinations15. In addition, on a p53-deficient background, the T490A RAG2 mutation led to the development of lymphoid malignancies harboring chromosomal translocations involving the antigen receptor loci16. Proper termination and restriction of RAG expression and activity are necessary to prevent formation of potentially dangerous recombinations and genomic instability. A recent study suggested that aberrant RAG activity is a dominant driver of secondary genetic hits in ETV6-RUNX1-positive childhood acute lymphoblastic leukemia (ALL) cases, where many breakpoints map near RSS motifs17. Moreover, a large portion of B-ALL cases display constitutive RAG expression, which has been associated with ongoing Igh rearrangements even after the malignant clone transformation18, thereby contributing to secondary genetic hits that may be at the basis of therapy resistance19. RAG-dependent chromosomal translocations occur as a result of the aberrant joining of RAG-induced DSBs with DSBs in other chromosomes. Therefore, B cells that are actively engaged in V(D)J recombination (pro-B cells and small pre-B cells) are especially at risk to procure such aberrant joining events when exposed to genotoxic stress. It is not very likely that cell cycle-dependent RAG2 degradation is the only protective mechanism that limits RAG-associated DNA damage. Rather, it is conceivable that mechanisms exist that down-regulate RAG activity when DNA damage is detected, irrespective of cell-cycle stage. However, it is not known whether B-lineage cells undergoing V(D)J recombination possess protective mechanisms that limit the formation of aberrant chromosomal rearrangements under such conditions. Given its oncogenic potential, mechanisms controlling RAG expression and activity in cells that have initiated V(D)J recombination need to be better understood. In this study we show that the DNA damage response limits the expression of RAG1/2 in pre-B cells, involving the Ataxia Telangiectasia mutated kinase (ATM) and FOXO1, thus preventing further formation of potentially dangerous lesions. Materials and methods Cell culture and small molecule compounds The v-Abl transformed mouse pre-B cell lines were generated from wild type (WT) (A70) and Rag2-/- (R2K3) mice that harbor the Em-Bcl2 transgene and were kindly provided by Dr. Craig Bassing (University of Pennsylvania School of Medicine, Philadelphia, PA). The human BCR-ABL+ B-ALL cell lines BV173 and SUP-B15 were obtained from DSMZ (Braunschweig, Germany) and cultured in RPMI 1640 (Gibco Life Technologies, Bleiswijk The Netherlands) supplemented with 2mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 20%
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