Reversal of glucocorticoid resistance in paediatric acute lymphoblastic leukaemia is dependent on restoring BIM expression

Abstract

Background: Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Glucocorticoids form a critical component of chemotherapy regimens and resistance to glucocorticoid therapy is predictive of poor outcome. We have previously shown that glucocorticoid resistance is associated with upregulation of the oncogene C-MYC and failure to induce the proapoptotic gene BIM.

Methods: A high-throughput screening (HTS) campaign was carried out to identify glucocorticoid sensitisers against an ALL xenograft derived from a glucocorticoid-resistant paediatric patient. Gene expression analysis was carried out using Illumina microarrays. Efficacy, messenger RNA and protein analysis were carried out by Resazurin assay, reverse transcription-PCR and immunoblotting, respectively.

Results: A novel glucocorticoid sensitiser, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide (GCS-3), was identified from the HTS campaign. The sensitising effect was specific to glucocorticoids and synergy was observed in a range of dexamethasone-resistant and dexamethasone-sensitive xenografts representative of B-ALL, T-ALL and Philadelphia chromosome-positive ALL. GCS-3 in combination with dexamethasone downregulated C-MYC and significantly upregulated BIM expression in a glucocorticoid-resistant ALL xenograft. The GCS-3/dexamethasone combination significantly increased binding of the glucocorticoid receptor to a novel BIM enhancer, which is associated with glucocorticoid sensitivity.

Conclusions: This study describes the potential of the novel glucocorticoid sensitiser, GCS-3, as a biological tool to interrogate glucocorticoid action and resistance.

Fig. 1. Ex vivo efficacy of GCS-3 in combination with dexamethasone or prednisolone against ALL-19 xenograft cells.

a Chemical structure of GCS-3, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide. b–d ALL-19 xenograft cells were exposed to GCS-3, glucocorticoid or both in combination at a fixed ratio of concentrations for 48 h, and cell viability was then assessed by Resazurin assay (b, c) or flow cytometry (d). e After 12 h drug incubation, drug-containing media were removed and cells were incubated in fresh media for 36 h, and cell viability was assessed by Resazurin assay. f ALL-19 xenograft cells were treated with 10 µM GCS-3, 10 µM dexamethasone or both in combination. Cell viability was determined by flow cytometry at various time points up to 72 h. Each data point represents the mean ± SEM of at least three independent experiments.

Fig. 2. Ex vivo efficacy of GCS-3 in combination with dexamethasone against ALL xenograft cells.

ALL-7 (a), ALL-54 (b), ALL-4 (c), ALL-31 (d) and ETP-2 (e) xenograft cells were exposed to GCS-3, dexamethasone or both in combination at a fixed ratio of concentrations for 48 h, and cell sensitivity was then assessed by Resazurin cytotoxicity assay. Each data point represents the mean ± SEM of at least three independent experiments.

Fig. 3. GCS-3 requires a functional GR to induce caspase-dependent apoptosis.

HAL-01 (a), UoC-B1 (b) or ALL-4CL (c) cells were exposed to GCS-3, dexamethasone or both in combination at a fixed ratio of concentrations for 48 h, and cell sensitivity was then assessed by Resazurin cytotoxicity assay. Each data point represents the mean ± SEM of at least three independent experiments. d, e ALL-19 xenograft cells were treated with 10 µM GCS-3 and 1 µM dexamethasone (DEX) for 1 h before separation into nuclear and cytoplasmic fractions. d Equal amounts of protein (10 µg) from each fraction were immunoblotted for GR, Topo I (nuclear loading control) and α-tubulin (cytoplasm loading control). e Dexamethasone-induced binding of the GR to a GRE motif was assessed using a DNA-binding ELISA, with equal amounts of nuclear lysates and HeLa control lysate (5 µg). The OD450 nm of each sample minus blank is shown. Each data point represents the mean ± SEM of three independent experiments. f ALL-19 xenograft cells were pre-treated with 10 µM QVD-OPh (QVD) or vehicle control for 2 h. Cells were then exposed to 10 µM GCS-3, 10 µM dexamethasone (DEX) or both for 48 h and cell sensitivity was assessed by flow cytometry. Each data point represents the mean ± SEM of four independent experiments. Significance was calculated using the unpaired t test with Welch’s correction.

Fig. 4. Effects of GCS-3 on dexamethasone-induced gene expression changes.

ALL-19 cells were treated with 10 µM dexamethasone (DEX), 10 µM GCS-3 or both in combination and gene expression profiling was performed. The top differentially expressed genes between the dexamethasone and combination treatment groups are shown after 12 h (a) and 24 h (b). Expression levels were normalised for each gene, where the mean is 0, higher than the mean are shown in red and lower in blue. c C-MYC mRNA expression was analysed by qRT-PCR and calculated relative to the 12 and 24 h vehicle-treated controls. Each data point represents the mean ± SEM of three independent experiment. Significance was calculated using the unpaired t test with Welch’s correction. d C-MYC protein expression was analysed after 12 and 24 h treatment by immunoblotting and a representative blot is shown.

Fig. 5. Expression levels of candidate genes in ALL-19 cells.

a Microarray analysis of the effects of 10 µM GCS-3, 10 µM dexamethasone and the combination on gene expression in ALL-19 cells. Two biological replicates were analysed at 12 and 24 h post treatment, where red represents high and blue low gene expression. GR (b), KLF13 (c), MYB (d), BCL-2 (e) and BIM (f) mRNA expression was analysed by qRT-PCR and calculated relative to the 12 and 24 h vehicle-treated controls. Each data point represents the mean ± SEM of four independent experiments. Significance was calculated using the unpaired t test with Welch’s correction. g BIM protein expression was analysed after 12 and 24 h treatment by immunoblotting and a representative blot is shown.

Fig. 6. BIM upregulation correlates with GCS-3/dexamethasone efficacy.

a ALL-19 xenograft cells were treated with 10 µM dexamethasone, 10 µM GCS-3 or both in combination for 8 h. Conventional ChIP of GR binding at the BIM-IGR, fold change was calculated relative to the IgG control. b Nalm6 cells that were stably transduced with a BIM promoter-reporter construct were treated with 1 µM dexamethasone, 10 µM GCS-3 or both in combination for 16 h. Fold change in luciferase induction was calculated. c–e ALL-19 cells were pre-treated with 1 µM RU486, then treated with 10 µM GSC-3, 10 µM dexamethasone and the combination for 24 h (qRT-PCR) or 48 h (cell sensitivity). c Cell sensitivity was assessed by flow cytometry. d C-MYC and e BIM mRNA expression was analysed by qRT-PCR and calculated relative to the control. Each data point represents the mean ± SEM of three independent experiments. Significance was calculated using the unpaired t test with Welch’s correction.