5 NF-κB and AKT signaling prevent DNA damage by suppressing RAG1/2 | 131 Discussion In B-ALL, many secondary genetic events bear the hallmarks of being derived from aberrant RAG activity, suggesting that RAG is an important instigator of genomic instability.14,15 Aberrant RAG activity also contributes to subclonal diversification of B-ALL, which results in the development of therapy-resistant subclones.34 Despite these recent insights, the (mis) regulation of RAG expression in B-ALL remains poorly understood. During B-cell development, pre-B cell proliferation and RAG expression and/or activity are strictly separated, preventing genomic instability. Multiple layers of regulation safeguard this separation, which may be disrupted in proliferating leukemic cells that constitutively express RAG. In BCR-ABL-positive B-ALL, large cycling pre-B cells are transformed by the Abl kinase activity, preventing their differentiation toward the immature B-cell stage. Typically, proliferating BCR-ABL-positive B-ALL cells do not show RAG activity, indicating that the separation between proliferation and recombination is (still) enforced.19,35 Inhibition of the Abl kinase with STI571 leads to induction of RAG expression and activity accompanied by cell cycle exit, similar to that in normal pre-B or immature B cells.18,19 In proliferating pre-B cells, RAG expression is repressed by AKT signaling that negatively regulates FOXO1. Our results show that the NF-kB pathway contributes to the repression of RAG activity in cycling-transformed pre-B cells by regulating FOXO1. NF-kB was implicated in regulating Igk locus accessibility36; however, it is unlikely that increased RAG activity upon NF-kB inhibition can be ascribed to effects on locus accessibility because the RAG reporter used in this study measured RAG activity independently of Igk accessibility. The role of AKT in the regulation of RAG expression in Abl cells is not entirely clear; previous results with an Abl cell line generated from a RAG1GFP knock-in mouse showed that AKT inhibition by the AKTVIII inhibitor did not result in increased RAG1GFP expression,37 whereas the expression of constitutively active AKT suppressed RAG1GFP expression in un- Figure 6. Inhibition of AKT and NF-kB signaling induces RAG1 protein expression in primary human B-ALL cells, and NF-kB gene expression signature negatively correlates with RAG1, RAG2, and TdT mRNA expression in untreated B-ALL patients. (A) Immunoblot analysis of whole-cell extracts from primary (BCR-ABL-negative) B-ALL blasts (n = 3) cultured for 48 hours with 2.5 mM IKKbi and 2.5 mM AKTi or vehicle (DMSO; untreated). (B) FACS plots of primary human B-ALL cells from 3 patients cultured for 48 hours with 2.5 mM IKKbi and 2.5 mM AKTi or vehicle (DMSO; untreated). FACS plots show CD34 vs surface IgM (sIgM) expression within the CD19+ gate; gated events were CD10+ (data not shown). Numbers above outlined gates indicate percentage of cells. (C) Heatmap showing NF-kB low (yellow squares) and NF-kB high (purple squares) B-ALL patient subgroups based on a 19 NF-kB pathway/target classifier gene expression profile. Each column represents a B-ALL patient, and each row represents a unique gene classifying the 2 subgroups. (D) Relative RAG1, RAG2, and TdT mRNA expression in NF-kB low and NF-kB high signature B-ALL patients. Each circle represents an individual patient, 2log expression is depicted, and gray bars indicate means. The ANOVA F-statistic and P value are given below each graph.
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