128 | Chapter 5 mouse pre-B cells were transduced with the IkBaSR and the RAG-reporter constructs and analyzed by flow cytometry 7 days after transduction. In the presence of IL7 and Flt3L, RAG activity was approximately twofold higher in the IkBaSR-transduced cells compared with the control cell population (15% vs 7% GFP+ cells) (Figure 5C). Withdrawal of IL7 increased RAG activity, upon which inhibition of NF-kB signaling had no substantial effect (60% vs 67% GFP+ cells) (Figure 5D). NF-kB and AKT suppress RAG expression in primary human pre-B ALL cells To confirm that the NF-kB and AKT pathways regulate RAG in primary human B-ALL cells, we stimulated B-ALL blasts in vitro from 3 (BCR-ABL–) patients with the combination of IKKbi and AKTi. RAG1 protein expression was increased in all 3 patients (Figure 6A). In addition, surface IgM expression on B-ALL blasts (CD19+CD10+CD34+/−) from these patients was increased (ranging from 1.8-fold to 13-fold) after treatment (Figure 6B), which may suggest increased RAG activity resulting in productive IgM expression in these cells. To further explore whether the activity of the NF-kB pathway is related to RAG expression in primary B-ALL patients, we reanalyzed the gene expression profiles of a previously published cohort of BCR-ABL-negative childhood B-ALL patients.26,27 This heterogeneous patient group consists of 207 untreated high-risk B-ALL patients. We performed unsupervised clustering based on the expression of 19 NF-kB pathway/target genes by using a k-means clustering algorithm that identified an NF-kB low signature B-ALL patient group (n = 106) and an NF-kB high signature patient group (n = 101) (Figure 6C). Although there is considerable overlap between the patient groups that reflects their heterogeneous nature, the NF-kB low signature group displayed significantly increased RAG1, RAG2, and TdT mRNA expression compared with the NF-kB high signature group (Figure 6D), highlighting the Figure 5. NF-kB superrepressor augments RAG activity in mouse Abl pre-B cells and in IL7-dependent untransformed mouse pre-B cell cultures. (A) FACS plots of WT mouse Abl pre-B cells transduced with IkBaSR-YFP and RAG-reporter (GFP) IRES-RFP. Scatter plots show YFP (x-axis) vs GFP (y-axis) within RFP-positive gate. Histograms show RAG-reporter (GFP) in IkBaSR-YFP–negative cells (middle row) and in IkBaSR-YFP–positive cells (bottom row). Cells were treated for 96 hours as indicated above FACS plots. Numbers above outlined gates indicate percentage of cells. (B) Graph depicting percentage of RAG-reporter activity (GFP) in IkBaSR-YFP–positive cells and IkBaSR-YFP–negative cells. Horizontal lines represent means, and 4 independent experiments were performed. Statistical significances were determined by twoway ANOVA. (C) FACS scatter plots of untransformed mouse pre-B cells cultured for 7 days with IL7. Pre-B cells (B220+, IgM–CD43+) were retrovirally transduced with IkBaSR-YFP and RAG-reporter (GFP) IRES-RFP constructs. Gating strategy is shown (B220+RFP+). FACS histograms show RAGreporter activity (GFP) in IkBaSR-YFP–negative and IkBaSR-YFP–positive cells. A representative example of 3 independent experiments is shown. (D) RAG-reporter activity (GFP) 2 days after IL7 withdrawal. Numbers above outlined gates indicate percentage of cells. A representative example of 2 independent experiments is shown. ***P < .001.
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