7 General discussion | 179 that in cycling v-Abl transformed pre-B cells, inhibition of AKT kinase activity led to an increase in RAG-reporter activity and similar results were obtained when inhibiting PI3K with small molecule inhibitors in these cells, suggesting that in proliferating pre-B cells, RAG expression is actively suppressed by the PI3K/AKT pathway. The PI3K/AKT-mediated suppression of RAG was mediated by the RAG1/2 transcription factor FOXO1. Our data showed that AKT inhibition resulted in upregulation of nuclear FOXO1 and concomitant decrease of the phosphorylated forms of FOXO1 on Ser256 and Ser329, which are associated with FOXO1 destabilization50,85. The NF-κB signaling pathway has been demonstrated to play a role in B-cell development. It is intricately regulated by various upstream signaling pathways, including the B-cell receptor (BCR), toll-like receptors (TLRs), cytokine receptors, and co-stimulatory molecules86. The NF-kB family of transcription factors consists of 5 members, NF-kB1 (p50 and its precursor p105), NF-kB2 (p52 and its precursor p100), which are synthesized as precursor molecules and catalytically processed into the active form, and RelA (p65), RelB, and c-Rel which are synthesized in their mature form and interact in the cytoplasm with inhibitors until they receive activating signals87. Generally, there are 2 ways of NF-kB pathway activation: the classical and alternative. In the classical pathway, IKK-b phosphorylates the inhibitory subunits of IkB-α, IkB-β, or IkB-ε, leading to their degradation and causing the accumulation of RelA, c-Rel, and p50 in the nucleus where it delivers mainly survival and proliferation signals. In the alternative pathway, NF-κB–inducing kinase (NIK) activates IKK-α, which phosphorylates NF-kB2. This results accumulation of p52/RelB heterodimers in the nucleus. The alternative NF-κB pathway is regulated mainly through the control of NIK turnover, with TNF receptor-associated factor 3 (TRAF3), TNF receptor-associated factor 2 (TRAF2), and Baculoviral IAP repeat-containing protein 1 and 2 (cIAP1/2) critically involved in this process88,89. Numerous mouse and cell line knock-in and knock-out studies have highlighted the complexity of the role of NF-kB in B-cell development, and the results provided sometimes contradicting conclusions, probably due to the many redundancies in NF-kB subunits. For instance, mice expressing a dominant negative form of IkappaB alpha (IkBα) exhibited reduced pre-B cell numbers, potentially as a result of altered PAX5 and IRF4 expression90. Additionally, constitutive NF-κB activation via p100 deletion resulted in arrested B-cell development at the pro-B cell stage91. However, the constitutive activation of alternative NF-κB pathway, achieved by B-cell specific knock out of Traf3 (CD19Traf3−/− mice), or constitutive activation of the classical pathway in mice expressing IKK2ca in B cells, did not affect B-cell development92,93. Also, a study of mice simultaneously carrying inactivating mutations in RelA and c-Rel showed inhibition in germline Igk transcription but not RAG94, whereas another study demonstrated that NF-kB1/p50-deficient B cells do show an increase in RAG1/2 expression and activity95. In our study, using an inducible RAG recombination-reporter cell line, we show that two structurally different Ikkb small molecule inhibitors (BMS-345541 and MLN120B, to-
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