Glucocorticoids are
critical components of combination chemotherapy regimens in
paediatric acute
lymphoblastic leukaemia (ALL), although resistance is frequently encountered at
relapse. Restoring glucocorticoid sensitivity could have a significant impact
in improving disease outcome. The proapoptotic BIM protein is an important
mediator of glucocorticoid-induced apoptosis in normal and malignant
lymphocytes, while the antiapoptotic BCL2 confers resistance. The signalling
pathways regulating
BIM and
BCL2 gene expression in
glucocorticoid-treated lymphoid cells remain unclear. In this study, paediatric
ALL patient-derived xenografts (
PDXs) inherently sensitive or
resistant to glucocorticoids were expanded and maintained in immunodeficient
NOD/SCID mice. Eight hours following dexamethasone treatment of mice, human leukaemia
cells were harvested from spleens. Microarray analysis showed that
KLF13 and
MYB gene expression changes were significantly greater in
dexamethasone-sensitive than -resistant PDXs. ChIP analysis detected
glucocorticoid receptor (GR) binding at the
KLF13
promoter to trigger
KLF13 expression
only in sensitive PDXs. Next, KLF13 bound to the
MYB promoter in competition with SP1, deactivating
MYB expression only in sensitive PDXs.
Sustained
MYB expression in resistant
PDXs resulted in maintenance of
BCL2
expression and inhibition of apoptosis. ChIP-seq analysis revealed a novel GR
binding site in a
BIM intronic region
(IGR) that was engaged only in dexamethasone-sensitive PDXs. Two conserved GR
binding elements were identified at the
BIM
IGR and were shown to enhance
BIM
transcription in response to dexamethasone treatment by luciferase reporter
assays. Analysing the abundance of DNaseI
Hypersensitive Sites, we found that the
BIM IGR exists in an open chromatin
conformation exclusively in lymphoid cells, which indicates a critical role of
chromatin conformation in glucocorticoid-induced apoptosis of lymphocytes. The
absence of GR binding at the
BIM IGR
was associated with
BIM silencing and
dexamethasone resistance.
This study has identified novel mechanisms of
opposing BCL2 and BIM gene regulation that control
glucocorticoid-induced apoptosis in paediatric ALL cells in vivo.