174 | Chapter 7 In summary, we demonstrated that RAG1/2 can introduce DNA breaks in non-Ig regions. While the previous studies implicated RAG1/2 activity in the formation of genomic lesions based on the composition of the breakpoint sequences and in-vitro cleavage assays, our study provides more specific evidence for RAG1/2 activity outside of the Ig loci and maps the genome-wide location of RAG-dependent DNA breaks in mouse v-Abl pre-B cell line. The presence of simple sequence repeat motifs, especially CA and GA repeats, and GC-rich regions around RAG-dependent NBS1 peaks may reflect the affinity of the RAG’s off-target activity to non-B DNA structures. DNA breaks are important regulators of RAG1/2 expression and activity In B-ALL, numerous secondary genetic events bear characteristics arising from aberrant RAG activity, implicating RAG as a key contributor to genomic instability. Aberrant RAG activity also plays a role in the sub-clonal diversification of B-ALL, leading to the emergence of therapy-resistant subclones45,46. Despite available insights into the regulation of Ig gene recombination, regulation of RAG1/2 in pre-B cells exposed to excessive DNA damage remains poorly understood. In normal B-cell development, pre-B cell proliferation and RAG expression/activity are strictly separated, maintaining genomic stability. This separation is upheld by multiple layers of regulation, which may be disrupted in proliferating leukemic cells expressing RAG continuously. In Chapter 4 we showed that in pre-B cells undergoing V(D)J recombination, Rag1 and Rag2 mRNA and protein expression rapidly respond to genotoxic stress induced by DSBs. DNA breaks activate ATM, a protein kinase that plays a crucial role in cellular responses to DNA damage47. ATM acts as a sensor of DSBs and orchestrates activation of signaling pathways, such as checkpoint kinase 2 (CHK2), p53, and breast cancer 1 (BRCA1), which in turn regulate processes like DNA repair, cell cycle checkpoint activation, and apoptosis. In the context of pre-B cells, ATM ensures the fidelity of this process by coordinating the repair of DSBs introduced during V(D)J recombination, thereby preventing genomic instability and the potential development of leukemia or lymphoma. In our study, treatment of mouse pre-B cells, undergoing light chain recombination, with ionizing radiation mimetic neocarzinostatin (NCS), resulted in ATM autophosphorylation at Ser1981 and RAG downregulation. The ATM autophosphorylation at Ser1981 has previously been shown to stabilize ATM at the site of the DSBs and it is required for proper DNA damage response (DDR)48. In our study, we demonstrated an efficient activation of ATM/ DDR following the NCS treatment. The pre-treatment with an ATM kinase inhibitor prevented the NCS-induced RAG downregulation. In addition, RAG levels in pre-B cells derived from Atm-/- mouse bone marrow did not respond to NCS treatment. RAG2 was shown to accumulate in the G1 phase of the cell cycle, but it degraded quickly when the cell entered the G1-S transition and the levels remained low until the
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