B-lymphoid progenitors outgrowth by Bcr-Abl KD mutants. BM from BALB/c mice was transduced with the indicated BCR-ABL construct and plated at the indicated cell numbers per well in each of three wells. For stromal support, nontransduced cells were added to bring the total number of cells to 10⁶/well. A well was scored as positive when the viable nonadherent cell number reached 10⁶/well. The x axis indicates the number of days after plating, and the y axis indicates the number of wells, from 1 to 3, that are scored as positive. (A) Native Bcr-Abl; (B) Y253F; (C); E255K; (D) T315I; (E) M351T; (F) H396P; (G) vector control.

In vitro kinase assays. (A) Plots of velocity versus peptide concentration using isolated KDs of the indicated construct fused to GST. Assays were performed in triplicate, with the standard deviation of the velocities shown as error bars. (B) Plots of velocity versus peptide concentration using native and T315I expressed as full c-Abl constructs. Assays were performed in triplicate with the standard deviation of the velocities shown as error bars. (C) Plots of velocity versus peptide concentration using native and mutants expressed in full-length Bcr-Abl. Assays were performed in triplicate with the standard deviation of the velocities shown as error bars. (D) Anti-Abl and anti-phospho-tyrosine blots of purified c-Abl and Bcr-Abl and a Coomassie gel of purified full-length Bcr-Abl.

In vitro proliferation competition experiments. (A) Representative chromatograms from the direct sequencing of competition cellular growth experiments. 50/50 mixtures of Ba/F3 cells expressing Bcr-Abl and the indicated KD mutant were grown for 18 days in RPMI medium supplemented with either 10 or 1% FBS as indicated. Row 1 shows native sequence, and row 2 shows the starting conditions with a 50/50 mixture of native and the indicated mutant; rows 3 and 4 show the day 18 chromatograms for the 10 and 1% FBS conditions, respectively. (B) Combined results of the percentage of mutant allele after 18 days in 10% FBS. The percentage of mutant allele was determined by measurement of the area under the curve of mutant allele and native allele taken from chromatograms normalized to a starting condition of 50% mutant. Error bars represent the standard deviation. (C) Combined results of the percentage of mutant allele after 18 days in 1% FBS. The percentage of mutant allele was determined by measurement of the area under the curve of mutant allele, and native allele taken from chromatograms normalized to a starting condition of 50% mutant. Errors bars represent the standard deviation. (D) Anti-Abl immunoblots of Ba/F3 cell lines used for the competition experiments showing comparable expression levels of Bcr-Abl. c-Abl expression is used as an internal control.

Myeloid colony formation by Bcr-Abl kinase domain mutants, and Kaplan-Meyer survival curves. (A) BM from BALB/c mice infected with the indicated BCR-ABL construct was plated in triplicate in methylcellulose supplemented with 10% FBS in the presence or absence of IL-3, IL-6, and SCF in two independent trials with the results combined. Colony formation was scored on day 10 with colonies containing 50 or more cells scored as positive. The results are plotted as a percentage of WT BCR-ABL colonies that formed in the presence of cytokines. An asterisk indicates a statistically significant (P < 0.05) deviation from native Bcr-Abl. (B) BM from BALB/c mice infected with the indicated BCR-ABL construct was plated in triplicate in methylcellulose supplemented with 1% FBS in the presence or absence of IL-3, IL-6, and SCF in two independent trials with the results combined. Colony formation was scored on day 10 with colonies containing 50 or more cells scored as positive. The results are plotted as a percentage of native BCR-ABL colonies that formed in the presence of cytokines. An asterisk indicates a statistically significant (P < 0.05) deviation from native Bcr-Abl. (C) Kaplan-Meyer survival curve for recipients of BM from 5-fluoruracil-treated donors transduced with MSCV-p210^Bcr-Abl-internal ribosome entry site-GFP harboring native Bcr-Abl, Y253F, E255K, T315I, M351T, H396P, or control retrovirus. None of the mutants tested exhibited statistical difference in survival compared to the wild type (P > 0.05).

Signaling of Bcr-Abl in cell lines expressing wild type or KD mutants. Ba/F3 cells were serum starved for 24 h, and lysates were immunoblotted with the indicated phospho-specific antibody. Specific antibodies were used to assure equal loading. (A) Whole-cell lysates probed with anti-phosphotyrosine (4G10); arrows indicate potential differences in phosphorylation. All lanes contained 50 μg of total cell lysate as measured by a bicinchoninic acid assay. The molecular weight (MW) is indicated. (B) A total of 200 μg of the same lysates were probed with SHIP1, phospho-SHIP1, Akt, phospho-Akt (Thr 308) and phospho-Akt (Ser 473), with actin used as a loading control.