Reversal of glucocorticoid resistance in human lymphoid malignancies with histone deacetylase inhibitors

Abstract

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Glucocorticoids (GCs) are a crucial component in all protocols for the treatment of human lymphoid malignancies, including childhood acute lymphoblastic leukemia (ALL). A central role in GC-mediated apoptosis is played by the proapoptotic BH3-only proteins, most notably Bim, Puma and, to a lesser extent, Noxa. Following exposure to GCs, these genes are upregulated at the mRNA and protein level. Silencing of Bim using siRNA technology correlates with protection from GC induced apoptosis in ALL cell lines. To dissect the molecular mechanisms leading to GC resistance in vivo, we developed a model of childhood ALL, using patient biopsies established as xenografts in immune-deficient (NOD/SCID) mice. Using this model, we demonstrated that resistance is typically associated with failure to induce Bim mRNA and protein in response to GC treatment. Using a methylated DNA immunoprecipitation analysis of the entire Bim CpG island, we found that increased methylation of Bim 5’ Untranslated Region (5’UTR) is associated with high-level GC resistance in 3/3 xenografts. In contrast, 4/4 xenografts previously shown to induce Bim had low levels of DNA methylation. Here we show that resistance to GCs is also associated with deacetylation of histone-H3K9 at the Bim locus. The deacetylation was specific for Bim: in fact, Noxa and Puma were highly acetylated in all the resistant xenografts. The degree of Bim histone-H3 acetylation correlated with total Bim protein induced after treatment of a panel of 11 xenografts with dexamethasone in vitro (R = 0.95; p < 0.0001). The concomitant presence of histone-H3 deacetylation and Bim 5’UTR methylation was detectable in a subset of highly resistant xenografts. Treatment with the histone deacetylase inhibitor Vorinostat (SAHA) re-induced Bim acetylation in resistant cells; thus, we tested the effect of combined treatment of SAHA and GCs in vitro. The combined treatment partially restored the sensitivity to GCs where the Bim locus was only deacetylated (e.g. in xenograft ALL-2). Histone deacetylase inhibition alone, however, was not sufficient to modulate GC responsiveness where the Bim locus was also highly methylated (e.g. ALL-19). To investigate the effect of these findings in vivo, NOD/SCID mice were inoculated with cells from ALL-2 and treated with SAHA, dexamethasone (DEX), saline control or SAHA + DEX for 4 weeks. The Kaplan-Meyer event-free survival (EFS) curves demonstrated that SAHA + DEX synergistically prolonged mouse EFS compared with mice treated with SAHA or DEX alone (logrank P value < 0.0001). These data indicate that modulation of histone acetylation and, possibly, DNA demethylation at the Bim locus, may provide a novel therapeutic approach for circumventing GC resistance in lymphoid malignancies.