Abstract
Abstract 11
Glucocorticoids (GC) play a fundamental role in the treatment of all lymphoid tumors because of their capacity to induce apoptosis in lymphoid progenitor cells. The importance of GC therapy in lymphoid malignancies is underscored by the strong association of GC response with prognosis in childhood acute lymphoblastic leukemia (ALL). Thus, resistance to glucocorticoids is a well recognized feature of poor prognosis in the treatment of childhood ALL. A number of different mechanisms contributing to GC resistance in T-ALL have been proposed including increased expression of antiapoptotic factors such as MCL1, insufficient expression of the glucocorticoid receptor gene (NR3C1), expression of NR3C1 splice variants and loss of NR3C1 auto up-regulation. The development of strategies to reverse glucocorticoid resistance could have a profound impact on the treatment of ALL. Recently, we have showed that inhibition of NOTCH1 with gamma secretase inhibitors (GSIs) sensitized GC-resistant T-ALL cell lines and primary samples to GC-induced apoptosis, supporting a role for GC plus GSIs in the treatment of GC-resistant T-ALLs. Here, we have used a Systems Biology approach to uncover additional pathways involved in the regulation of glucocorticoid receptor activity and glucocorticoid resistance in ALL. In this approach we used Master Regulator Inference analysis (MaRInA), a novel algorithm designed to identify critical regulators of complex biological traits, to analyze the gene expression profiles of pre-treatment T-ALL samples determined to be either sensitive (IC50<150μ g/ml) or resistant (IC50>150μ g/ml) to GC-induced apoptosis in vitro. This analysis identified a transcriptional module controlled by the AKT pathway as the most highly enriched gene set in the resistant samples, strongly suggesting a critical role for PI3K-AKT signaling in the regulation of glucocorticoid resistance in T-ALL. Notably, loss PTEN and constitutive activation of AKT are highly prevalent in T-ALL. Analysis of the effects of AKT in the transcriptional activity of the glucocorticoid receptor and in glucocorticoid receptor induced apoptosis revealed that constitutive activation of AKT results in impaired glucocorticoid receptor activity. Based on these results we hypothesize that AKT could have a direct inhibitory effect on the glucocorticoid receptor. Consistent with this hypothesis, mass spectrometry analysis demonstrated high levels of phosphorylation of the NR3C1 Ser134 in cells harbouring constitutively active AKT. Notably, this residue is within an AKT consensus sequence, XRXXS, and is highly conserved amongst species. Moreover, protein pull down and in vitro kinase assays demonstrated that AKT can directly interact with NR3C1 and mediates its phosphorylation on Ser134. Analysis of the biological relevance of this posttranslational modification showed that constitutively active AKT induces: (i) marked decrease in NR3C1 stability; (ii) retention of NR3C1 in the cytosol in the presence of dexamethasone and (iii) impaired glucocorticoid receptor autoupregulation; all of which can be blocked by the expression of a phosphorylation-deficient serine to alanine (S134A) NR3C1 mutant. Analysis of the effects of AKT activation on the interaction between NOTCH1 signaling and glucocorticoid induced apoptosis showed that AKT can effectively block the reversal of glucocorticoid resistance induced by inhibition of NOTCH1 signaling with GSIs in a Ser134 phosphorylation dependent manner. Finally, we demonstrate that pharmacologic inhibition of AKT can effectively reverse glucocorticoid resistance in T-ALL primary human samples and cell lines. Overall these results identify a direct interaction between the PI3K/AKT pathway and NR3C1 signaling and provide a strong rationale for the clinical testing the combination of AKT inhibitors and glucocorticoids in T-ALL.
Disclosures:
No relevant conflicts of interest to declare.
Clinical implications of glucocorticoid receptor studies in childhood acute lymphoblastic leukemia
