Figure 1.
Generation of isogenic CCRF-CEM cell lines with the BIM deletion polymorphism.
(A) Structure of the BIM gene and major splice isoforms. The BIM deletion polymorphism lies within intron 2 and upstream of exon 3, as indicated by the dashed line. Exon 4 contains the crucial BH3 domain required for apoptosis. Exon 3 (E3) and 4 (E4) are spliced in a mutually exclusive fashion, leading to the generation of either E4-containing isoforms with the BH3 domain (BIM EL, BIM L and BIM S) or E3-containing isoforms without the BH3 domain (BIM γ). When present, the deletion biases splicing towards E3-containing non-apoptotic isoforms. (B) Agarose gel of the products from a PCR reaction to detect the polymorphism in zinc finger nuclease (ZFN)-treated CCRF-CEM subclones, with the lower band indicating the presence of the deletion. Parental CCRF-CEM and KCL-22 cells (a CML cell line known to be heterozygous for the BIM deletion polymorphism) were included as controls. (C) The ratio of exon 3 to exon 4-containing transcripts (E3:E4) in CCRF-CEM BIMi2+/+, BIMi2+/− and BIMi2−/− clones as measured by qPCR. Error bars indicate mean ± SEM (n = 3). A student's t-test was performed for pairwise comparisons of E3:E4 ratio between genotypes. * indicates a significant difference with P<0.05.
Figure 2.
The BIM deletion confers dexamethasone resistance in CCRF-CEM cells.
(A) Cell viability following exposure of CCRF-CEM subclones to increasing concentrations of dexamethasone. Viability was measured by MTS assay at 48 h. Error bars indicate mean ± SEM (n = 3) of 3 independent replicates. (B) Western blot of cell lysates from CCRF-CEM BIMi2+/+, BIMi2+/− and BIMi2−/− clones following treatment with increasing doses of dexamethasone for 48 h. The induction of cleaved PARP and cleaved caspase 3 were used as readouts for apoptosis. An antibody that recognizes pro-apoptotic exon-4 containing BIM isoforms (BIM EL, L and S) was used to show the extent of BIM upregulation following GC exposure. β-actin was used as a loading control. (C) Western blot showing phosphorylation of the glucocorticoid receptor (Phospho-GR S211) in CCRF-CEM BIMi2+/+, BIMi2+/− and BIMi2−/− clones upon treatment with dexamethasone.
Table 1.
Biological and clinical features of patients from the Ma-Spore ALL 2003 trial genotyped for the BIM polymorphism.
Figure 3.
Retrospective analysis of the Ma-Spore ALL 2003 cohort according to the presence or absence of the BIM deletion polymorphism.
Kaplan-Meier curves comparing event-free survival (A) or overall survival (B) in patients with or without the BIM deletion polymorphism are shown.
Figure 4.
Methotrexate, vincristine, and L-asparaginase activate apoptosis in a BIM-independent manner, and overcome BIM deletion-mediated GC resistance.
CCRF-CEM clones were treated with dexamethasone (DEX) (0.1 µM) with or without (A) methotrexate (MTX) (1 µM), (B) vincristine (VCR) (2 ng/ml), or (C) L-asparaginase (ASP) (0.5 IU/ml) for 48 h. Following incubation, cell lysates were obtained and analyzed for cleaved PARP and caspase 3, as well as BIM induction. β-actin was used as a loading control.
Figure 5.
The addition of methotrexate, vincristine or L-asparaginase resensitizes BIM deletion-containing CCRF-CEM clones to dexamethasone.
Cell viability was measured by MTS assay after (A) methotrexate (MTX) (1 µM), (B) vincristine (VCR) (2 ng/ml), or (C) L-asparaginase (ASP) (0.5 IU/ml) was used singly or in combination with dexamethasone (DEX) (0.1 µM) for 48 h. Values obtained for treated cells were normalized to the untreated control for the same genotype. Error bars indicate SEM (n = ) of 3 independent replicates.