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

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.


SUPPLEMENTARY TABLES Supplementary
Nearly Additive 12h, then removal

Supplementary Table S3. Ex vivo efficacy of GCS-3 in combination with dexamethasone against non-leukemic, human cells. Human
PBMCs and CD34+ cells were exposed to GCS-3, dexamethasone, or both in combination at a 1:1 fixed-ratio of concentrations for 48 h. Cell sensitivity was assessed by Resazurin cytotoxicity assay. Deviation from Bliss-additivity (BA) was calculated at each tested concentration.

Cell
Deviation from BA at each tested ratio;

Supplementary Table S4. Ex vivo efficacy of GCS-3 in combination with non-glucocorticoids against ALL-19 xenograft cells. ALL-19
xenograft cells were exposed to a GCS-3, non-glucocorticoid, or both in combination at a fixed-ratio of concentrations for 48 h. Cell sensitivity was then assessed by Resazurin cytotoxicty assay. The ratio of GCS-3 to drug (G/D ratio) is determined from single agent assays. Deviation from Bliss-additivity (BA) was calculated at each tested concentration.  Table S5. Ex vivo efficacy of GCS-3 in combination with dexamethasone against ALL xenograft cells. Xenograft cells were exposed to GCS-3, dexamethasone (DEX), or both in combination at a fixed-ratio of concentrations for 48 h. Cell sensitivity was then assessed by Resazurin cytotoxicty assay. The ratio of GCS-3 to dexamaethasone (G/D ratio) is determined from single agent assays. Deviation from Bliss-additivity (BA) was calculated at each tested concentration.

Supplementary Table S6. In vitro efficacy of GCS-3 in combination with dexamethasone against glucocorticoid resistant cell lines.
HAL-01, UoC-B1 or ALL-4CL cells were exposed to a GCS-3, dexamethasone, or both in combination at a fixed-ratio of concentrations for 48 h. Cell sensitivity was then assessed by Resazurin cytotoxicty assay. The ratio of GCS-3 to drug (G/D ratio) is determined from single agent assays. Deviation from Bliss-additivity (BA) was calculated at each tested concentration. For each treatment cells were exposed to dexamethasone for 48 h. Cell sensitivity was assessed by Resazurin assay. Each data point represents the mean ± SEM of three independent experiments. Figure S3. Ex vivo efficacy of GCS-3 in combination with dexamethasone against human PBMC cells. Human PBMC cells from 3 separate donors 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 three independent experiments.

Supplementary
Supplementary Figure S5. Ex vivo efficacy of GCS-3 in combination with non-glucocorticoids against ALL-19 xenograft cells. ALL-19 xenograft cells were exposed to GCS-3, non-glucocorticoid, 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.
Supplementary Figure S6. Ex vivo efficacy of GCS-3 in combination with dexamethasone against ALL xenograft cells. ALL 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.
Supplementary Figure S6 continued. Ex vivo efficacy of GCS-3 in combination with dexamethasone against ALL xenograft cells. ALL 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. Figure S7. Effect of GCS-3/dexamethasone on nuclear and cytoplasmic GR expression. ALL-19 xenograft cells were treated with 10 µM GCS-3, 1 µM dexamethasone (DEX) for 1 h before separation into nuclear and cytoplasmic fractions. Equal amounts of protein (10 µg) from each fraction were immunoblotted for GR, topoisomerase 1 (nuclear loading control) and α tubulin (cytoplasm loading control). GR expression relative to loading control is shown. Each data point represents the mean ± SEM of three independent experiments.

Supplementary
Supplementary Figure S8. Effect of the pan-caspase inhibitor, QVD-OPh, on response of ALL-19 cells to vincristine. ALL-19 xenograft cells were pre-treated with 10 µM QVD-OPh (QVD) or vehicle control for 2 h. Cells were then exposed to 23 nM of vincristine (VCR) for 48 h and cell sensitivity was assessed by flow cytometry. Each data point represents the mean ± SEM of four independent experiments.
Supplementary Figure S9. Gene expression profiling treatment groups. ALL-19 xenograft cells were treated with 10 µM dexamethasone, 10 µM GCS-3, or both in combination for 12 or 24 h. Gene expression profiling was performed on each of the 8 treatment groups in duplicate. Supplementary Figure S14. GR binding at GILZ. Conventional ChIP of GR binding at GILZ R1/2. ALL-19 xenograft cells were treated with 10 µM dexamethasone, 10 µM GCS-3, or both in combination for 8 h. Fold change was calculated relative to the IgG control. Each data point represents the mean ± SEM of three independent experiments. Significance was calculated using unpaired t-test with Welch's correction.