180 | Chapter 7 gether referred to as Ikkbi) resulted in an increase in RAG-reporter activity, and strikingly, we observed a clear synergistic effect of concomitant Ikkb and PI3K/AKT inhibition on the increase in RAG activity and expression, and on the upregulation of RAG transcription factor FOXO1. Importantly, unlike upon the treatment of the v-Abl transformed pre-B cells with Abelson kinase inhibitor STI571, the cells treated with AKT and NF-kB inhibitor did not exit the cell cycle and showed RAG activity in the S-phase, which has previously been linked to increase in genomic instability and lymphomagenesis80. This can be partially attributed to the fact that during S phase of the cell cycle, when DNA replication takes place, the chromatin structure becomes more accessible, potentially allowing RAG1/2 to access DNA sequences they wouldn’t normally have access to. Also, RAG-induced DSBs occurring during this phase can coincide with DNA replication forks, leading to the formation of complex DNA structures that are prone to errors in repair. Also, the non-homologous end-joining (NHEJ) pathway, used to resolve RAG-induced DSBs is known to be error-prone, which in the G1 phase is beneficial as it further increases the diversity of the recombined V(D)J regions, however in the S phase, the use of the error-prone DNA repair pathway might result in translocations, deletions and an overall increase in genomic instability96–98. In line with this observation, our data show that Ikkb inhibition resulted in a RAG-dependent increase in genomic instability, measured by the levels of intranuclear gH2AX in cycling v-Abl wildtype as compared to Rag2- /- pre-B cells. AKT inhibition alone did not increase the intranuclear gH2AX. Notably, the gH2AX staining was only observed in large cells, in further support of the notion that induction of RAG expression and activity was provoked in cycling cells. Previously, the cell cycle was shown to be an important regulator of RAG expression: in the Eμ-Myc transgenic mouse model of B-cell lymphoma, the cyclin-dependent kinase 4 (CDK4) phosphorylated FOXO1, which resulted in FOXO1 degradation and abolished the FOXO-dependent induction of Rag1 and Rag2 expression99. In our study we observed that the levels of cyclin-A and CDK2 were significantly decreased following the IKKbi and AKTi treatment of v-Abl transformed cells; in fact, the degree of cyclin-A and CDK2 downregulation was similar to the one observed following the treatment with Abelson kinase inhibitor. Also, CDK4 protein and mRNA expression were decreased following the treatment with IKKbi and AKTi. Inversely, treatment with the small molecule inhibitor of CDK4/6 kinase activity PD-0332991 resulted in increased RAG activity. Previous studies have shown that CDK4 is regulated by NF-kB and also, IkBa binding to CDK4 was demonstrated to inhibit its kinase function90,100. We therefore hypothesize that IkBa might be involved in RAG regulation through CDK4 inhibition, which in turn acts as a negative regulator of FOXO1. We further hypothesize that the synergistic effect of combined inhibition of AKT and IKKb might be explained by the fact that both pathways impinge on the expression of RAG transcription factor FOXO1.
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