Kinase-inactive IKK mutants attenuate CML-like MPN induced by BCR-ABL1 in mice. BM from 5-FU–treated donors was transduced with retrovirus expressing BCR-ABL1/GFP, BCR-ABL1/IKKαKM, or BCR-ABL1/IKKβKM, and subsequently either plated in methylcellulose culture (A) or transplanted into irradiated recipients to induce CML-like MPN (B). (A) Myeloid colony assay. Transduced BM cells (1 × 10⁴) were seeded per plate in triplicate. Colony number was determined at day 14. The differences in colony number for BCR-ABL1/IKKαKM– or BCR-ABL1/IKKβKM–transduced progenitors were significant (*P = .022 and **P = .024, Student t tests). (B) Kaplan-Meier survival curve for recipients of BCR-ABL1–transduced BM in the CML model. The number of mice from the different arms is indicated, with the phenotype of disease indicated by the shading of the symbol; mice with mixed hematologic disease are indicated by mixed shading. Recipients of BM transduced by BCR-ABL1/IKKαKM (P < .0001, Mantel-Cox test) or BCR-ABL1/IKKβKM (P < .0001, Mantel-Cox test) survived significantly longer than BCR-ABL1/GFP recipients. (C) Kinase-inactive IKK mutants inhibit BCL-X expression induced by BCR-ABL1–expressing myeloid progenitors. Immunoblot analysis of protein lysates from spleens of mice with CML-like MPN induced by BCR-ABL1/GFP (lanes 4-8), BCR-ABL1/IKKαKM (lanes 9-11), and BCR-ABL1/IKKβKM (lanes12-14). Lysates from spleens of 3 untransplanted mice (lanes 1-3) were loaded as controls. The blot was analyzed with antibodies against BCR-ABL1, FLAG, IκBα, c-MYC, and BCL-X, and actin. (D) Analysis of genomic DNA from leukemic tissues of mice with CML-like MPN induced by BCR-ABL1/GFP (lanes 3-10), BCR-ABL1/IKKαKM (lanes 11-17), or BCR-ABL1/IKKβKM (lanes 18-23) by Southern blot with an IRES probe to detect distinct proviral integration events. Two control DNAs (Con, lanes 1-2) were from cell lines that each contain a single BCR-ABL1 provirus. CML-like MPN induced by BCR-ABL1/IKKαKM (P < .0001, Student t test) and BCR-ABL1/IKKβKM (P = .0003, t test) showed a significant reduction in leukemia-initiating cell frequency 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 3B. (E) Limiting dilution secondary transplantation analysis of resident BM leukemia stem cell frequency in primary mice with CML-like MPN induced by BCR-ABL1/GFP (black), BCR-ABL1/IkBαSR (red), and BCR-ABL1/IKKαKM (green) retroviruses. The frequency of secondary recipients who did not develop CML-like MPN (log scale) is indicated by the closed symbols; open symbols indicate all recipients developed MPN at that cell dose. Dashed lines indicate 95% confidence intervals.