Figure 5
Figure 5. Kinase-inactive IKK mutants attenuate B-lymphoid leukemogenesis by BCR-ABL1 in mice. (A) Kaplan-Meier survival curve for B-ALL induced in mice by BCR-ABL1/GFP, BCR-ABL1/IKKαKM, or BCR-ABL1/IKKβKM. The number of individual mice in each arm is indicated. All recipients developed B-ALL. Coexpression of either IKKαKM or IKKβKM significantly prolonged the survival of mice with BCR-ABL1–induced B-ALL (P < .0001, Mantel-Cox test). In addition, mice with B-ALL induced by BCR-ABL1/IKKαKM survived significantly longer than those induced by BCR-ABL1/IKKβKM (P = .0007, Mantel-Cox test). (B) Total cell number collected from malignant pleural effusions in leukemic mice from the cohorts in panel A. Compared with B-ALL induced by BCR-ABL1/GFP, recipients with B-ALL induced by BCR-ABL1/IKKαKM (P = .0039) or BCR-ABL1/IKKβKM (P = .0393, Student t test) have significantly fewer leukemic cells in malignant pleural effusions. (C) Immunoblot analysis of protein lysates from pleural effusion lymphoblasts from mice with B-ALL induced by BCR-ABL1/GFP (lanes 2-5), BCR-ABL1/IKKαKM (lanes 6-12), or BCR-ABL1/IKKβKM (lanes 13-17). BM from an untransplanted mouse was loaded in lane 1 as a control. Lysates were analyzed with antibodies against c-Abl, FLAG, and actin. (D) Representative confocal micrographs of nuclear RelA expression in B-lymphoid leukemic cells from recipients of BM transduced with BCR-ABL1/GFP, BCR-ABL1/IKKαKM, or BCR-ABL1/IKKβKM. Cells were stained with antibody against RelA (Red) and counterstained with Hoechst dye (blue) as described in “Materials and methods.” Scale bars = 10 μm. (E) Quantification of nuclear RelA expression from the data in panel D (mean + SE). Leukemic cells expressing BCR-ABL1/GFP showed significantly higher nuclear RelA than cells expressing BCR-ABL1/IKKαKM (*P = .0003, Student t test) or BCR-ABL1/IKKβKM (**P < .0001, Student t test). (F) Analysis of genomic DNA from leukemic tissues of mice with B-ALL induced by BCR-ABL1/GFP (lanes 1-6), BCR-ABL1/IKKαKM (lanes 7-12), or BCR-ABL1/IKKβKM (lanes 13-19) by Southern blot with IRES probe to detect distinct proviral integration events. Two control DNAs (Con, lanes 20-21) were from cell lines that each contain a single BCR-ABL1 provirus. B-ALLs induced by BCR-ABL1/IKKαKM (P = .001, Student t test) and BCR-ABL1/IKKβKM (P = .0057, Student t test) showed significantly decreased frequency of leukemia-initiating cells as compared with BCR-ABL1/GFP. Because of the lower titers consistently obtained for BCR-ABL1 retroviruses coexpressing IKKα/βKM (supplemental Figure 1), we used more dilute BCR-ABL1/GFP retrovirus to match titers, resulting in a lower average number of proviral clones in the GFP control arm than in Figure 2B.

Kinase-inactive IKK mutants attenuate B-lymphoid leukemogenesis by BCR-ABL1 in mice. (A) Kaplan-Meier survival curve for B-ALL induced in mice by BCR-ABL1/GFP, BCR-ABL1/IKKαKM, or BCR-ABL1/IKKβKM. The number of individual mice in each arm is indicated. All recipients developed B-ALL. Coexpression of either IKKαKM or IKKβKM significantly prolonged the survival of mice with BCR-ABL1–induced B-ALL (P < .0001, Mantel-Cox test). In addition, mice with B-ALL induced by BCR-ABL1/IKKαKM survived significantly longer than those induced by BCR-ABL1/IKKβKM (P = .0007, Mantel-Cox test). (B) Total cell number collected from malignant pleural effusions in leukemic mice from the cohorts in panel A. Compared with B-ALL induced by BCR-ABL1/GFP, recipients with B-ALL induced by BCR-ABL1/IKKαKM (P = .0039) or BCR-ABL1/IKKβKM (P = .0393, Student t test) have significantly fewer leukemic cells in malignant pleural effusions. (C) Immunoblot analysis of protein lysates from pleural effusion lymphoblasts from mice with B-ALL induced by BCR-ABL1/GFP (lanes 2-5), BCR-ABL1/IKKαKM (lanes 6-12), or BCR-ABL1/IKKβKM (lanes 13-17). BM from an untransplanted mouse was loaded in lane 1 as a control. Lysates were analyzed with antibodies against c-Abl, FLAG, and actin. (D) Representative confocal micrographs of nuclear RelA expression in B-lymphoid leukemic cells from recipients of BM transduced with BCR-ABL1/GFP, BCR-ABL1/IKKαKM, or BCR-ABL1/IKKβKM. Cells were stained with antibody against RelA (Red) and counterstained with Hoechst dye (blue) as described in “Materials and methods.” Scale bars = 10 μm. (E) Quantification of nuclear RelA expression from the data in panel D (mean + SE). Leukemic cells expressing BCR-ABL1/GFP showed significantly higher nuclear RelA than cells expressing BCR-ABL1/IKKαKM (*P = .0003, Student t test) or BCR-ABL1/IKKβKM (**P < .0001, Student t test). (F) Analysis of genomic DNA from leukemic tissues of mice with B-ALL induced by BCR-ABL1/GFP (lanes 1-6), BCR-ABL1/IKKαKM (lanes 7-12), or BCR-ABL1/IKKβKM (lanes 13-19) by Southern blot with IRES probe to detect distinct proviral integration events. Two control DNAs (Con, lanes 20-21) were from cell lines that each contain a single BCR-ABL1 provirus. B-ALLs induced by BCR-ABL1/IKKαKM (P = .001, Student t test) and BCR-ABL1/IKKβKM (P = .0057, Student t test) showed significantly decreased frequency of leukemia-initiating cells as compared with BCR-ABL1/GFP. Because of the lower titers consistently obtained for BCR-ABL1 retroviruses coexpressing IKKα/βKM (supplemental Figure 1), we used more dilute BCR-ABL1/GFP retrovirus to match titers, resulting in a lower average number of proviral clones in the GFP control arm than in Figure 2B.

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