7 General discussion | 181 Also, following the NF-kB and AKT inhibition in in vitro cultures of BCR-ABL negative primary human B-ALL cells, we observed an increase in RAG protein levels, and an increase in surface IgM expression as assessed by FACS, suggesting that in addition to murine cells, this regulatory pathway is also active in human cells. Our re-analysis of the publicly available mRNA expression profiles from previously published childhood B-ALL cohort showed that NF-kB was negatively correlated with Rag1, Rag2, and TdT expression. These findings suggest that NF-κB may possess tumor-suppressive properties in B-ALL, aligning with previous observations in different cell types. It is noteworthy that several emerging therapies targeting NF-κB, PI3K-AKT, and CDK4 are being tested in leukemias and lymphomas101–103. Given our results, it is conceivable that such treatments could inadvertently stimulate the oncogenic activity of RAG in leukemia patients, potentially exacerbating clonal diversification and genomic instability in pre-B cells. In Chapter 6 we uncovered an additional novel mechanism that regulates RAG1/2 expression upon induction of DNA damage. We found that the RAG protein and mRNA downregulation in response to DNA damage is also dependent on p53. This regulatory mechanism acts as a negative feedback loop, likely involving microRNA-34a (miR-34a), which is a p53 downstream target, and the RAG transcription factor FOXP1, which is one of the established targets of miR-34a104,105. TP53, a well-known tumor suppressor gene, plays a pivotal role in preventing cancer development by regulating cell growth and apoptosis. Alterations in p53, such as mutations, deletion, or dysregulation in signaling upstream from p53 have been associated with tumorigenesis, which underscores the tumor-suppressor role of p53106. In the context of lymphoid malignancies, aberrant functioning of p53 was clearly shown to contribute to tumor formation, as previously extensively reviewed107. Because p53 is a very well-established substrate of activated ATM108, we explored the role of p53 in the regulation of RAG1/2 expression upon induction of DNA damage. Downregulation of Rag1 and Rag2 mRNA expression was partially prevented in p53-/- primary pre-B cells exposed to g-irradiation in contrast to their wild-type counterparts.. Moreover, in the absence of p53, primary mouse pre-B cells undergoing light chain rearrangement, showed increased surface IgM expression, emphasizing the functional consequences of p53 modulation. In the severe combined immunodeficiency (scid) mouse model, the mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs) results in a developmental block of B and T cells at a very early stage109. Notably, p53 deficiency in the scid model, achieved by crossing the scid mice with Tp53-/- mice, was able to partially overcome the scid-conferred developmental block and allowed TCRb protein production110. In germinal center B cells and pre-B cells111, the proto-oncogene BCL6 was shown to repress p53 expression by binding to two specific sites within p53 promoter112. In our study, the overexpression of BCL6 in mouse v-Abl pre-B cells was sufficient to produce productive Igk recombination and partially overcome the developmental block imposed by v-Abl, similar to the results observed
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