132 | Chapter 5 transformed B220+IgM– cells.7 We show that the specific AKTi GSK-69069329 increased RAG activity in Abl cells. Interestingly, the AKT-mediated RAG repression may operate by direct inhibition of FOXO1 and by feeding into NF-kB–mediated FOXO1 regulation, because simultaneous inhibition of the AKT and the NF-kB pathways resulted in increased levels of nuclear FOXO1, a concomitant decrease in FOXO1 phosphorylation, and a synergistic effect on RAG activity. In support, we show that AKT is involved in NF-kB activation by phosphorylating IKKα on serine 23, as was shown previously.38 In addition, we demonstrate that expression of IkBaSR had a stimulatory effect on RAG activity in Abl cells and in primary mouse pre-B cells, albeit to a lesser extent. Several studies addressed the role of NF-kB in early B-cell development with discordant results. NF-kB was shown to be involved in Igk germline transcription but not RAG activity.36 Other studies suggest that NF-kB is dispensable for B-cell development.39,40 More recently, it was shown that NF-kB is active in pre-B cells and in immature B cells engaged in receptor editing during Igl recombination.41,42 Strikingly, in an earlier study, it was shown that NF-kB1/p50-deficient B cells had elevated RAG expression and activity.43 To add to the complexity, kinase-dead IKKa knockin mice showed defective B-cell development because of decreased PAX5 and IRF4 gene expression.44 It is likely that different modalities of NF-kB signaling exist in normal and transformed developing B cells that determine the outcome of this pleiotropic signaling pathway. Recently, it was shown that CDK4 suppresses RAG expression in B-cell lymphomas in Em-Myc-transgenic mice. In that model, CDK4 deficiency accelerated lymphomagenesis in a RAG-dependent fashion by regulating FOXO1 levels. The authors showed that CDK4 phosphorylates FOXO1 on residue serine 329, which provokes nuclear exclusion.28 Of interest, CDK4 activity is regulated by NF-kB,45 and it was shown that IkBα binds to CDK4 thereby inhibiting its kinase function.46 We demonstrated that simultaneous inhibition of the NF-kB and the AKT pathways resulted in diminished Cdk4 mRNA and CDK4 protein levels and decreased FOXO1-serine 329 phosphorylation. In addition, specific inhibition of CDK4 increased RAG activity. On the basis of these observations, we speculate that IκBα stabilization could promote RAG expression by inhibition of CDK4, which acts as a negative regulator of FOXO1 (Figure 7). The NF-kB pathway is one of the major regulators of antiapoptotic gene expression during B-cell development, which makes it intrinsically difficult to assess other functions of this pathway because perturbation of NF-kB has detrimental effects on cell survival. It is possible that the antiapoptotic function of NF-kB is (partly) uncoupled from its other functions in Abl cells because Abl kinase activity can prevent apoptosis independently of NF-kB.47 Moreover, the mouse Abl cells used in this study harbor the Em-Bcl2 transgene and express the antiapoptotic BCL2 protein, which may have helped to uncover the role of NF-kB in the regulation of RAG expression. In support of this, treatment of the mouse and human BCR-ABL-positive cell lines with the IKKbi and AKTi resulted in only a minor increase in cell death (<10% specific cell death) over
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