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Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GRgene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

Boos: European Erwinase Providers (EUSAPharm): Speakers Bureau; Medac: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lanvers-Kaminsky:Medac: Speakers Bureau.

Abstract 2458This icon denotes a clinically relevant abstract

L-Asparaginase (ASNase) is a key component of multi-agent chemotherapy regimens used in the treatment of pediatric Acute Lymphoblastic Leukemia (ALL). This bacterially-derived enzyme lowers blood asparagine levels by catalyzing the hydrolysis of L-asparagine, leading to specific killing of leukemic blasts. However, relapses and associated therapy resistance occur in about 20% of the patients. Although the molecular mechanisms that contribute to ASNase resistance are not well understood, increased expression of asparagine synthethase (ASNS), the enzyme responsible for asparagine synthesis, has been linked to ASNase resistance both in ALL cell lines and in primary leukemias. We recently reported that micro-deletions affecting the B-cell translocation gene 1 (BTG1) occur in about 10% of pediatric pre-B ALL cases. Here we show that BTG1 loss contributes to ASNase resistance by up-regulation of ASNS.Using RNA interference, we show that loss of BTG1 expression promotes cell growth and renders pre-B ALL cells completely refractory to ASNase induced cell death. Resistance to ASNase in the BTG1 knockdown cells is accompanied by increased expression of ASNS, while knockdown of this metabolic enzyme is sufficient to reverse therapy resistance, indicating that upregulation of ASNS is required for the observed resistance phenotype. We further show that BTG1 associates with and regulates the activity of the transcription factor ATF4, a key regulator of metabolic stress responses and a central component in the regulatory network that controls ASNS expression.Together, our experiments identify BTG1 as an important regulator of ASNS expression which acts by modulating ATF4 function. We expect that a detailed molecular understanding of how BTG1 loss contributes to ASNase resistance, will lead to the identification of pharmacological targets that can be used to improve treatment responses in therapy-resistant ALL.No relevant conflicts of interest to declare.

Abstract 1028

No relevant conflicts of interest to declare.

By genome wide profiling we have found that about 10 % of pediatric pre-B ALL cases contain a (single copy) deletion of the B cell translocation gene 1 (BTG1) gene. BTG1 belongs to a family of potential tumor suppressor genes, which include BTG2, BTG3, TOB and TOB2. Proteins encoded by members of this gene family have been implicated in the induction of growth arrest or apoptosis in a variety of model systems. Moreover, BTG1 associates with and regulates the activity of the arginine methyl transferase PRMT1, a coactivator of nuclear receptor-mediated transcription. Hence we hypothesized that loss of BTG1 function, for instance due to deletion, may affect glucocorticoid induced therapy responses in ALL.Using RNA interference, we find that loss of BTG1 expression decreases sensitivity of pre-B ALL cells to the apoptosis-inducing effects of synthetic GCs about 10,000 fold (Figure). This acquired GC resistance is accompanied by a greater than 10 fold reduction in GR protein expression as well as a (near complete) loss of GC-induced gene expression. Conversely, re-expression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression. By chromatin immunoprecipitations using anti PRMT1 antibodies we show that PRMT1 is recruited to the GR gene promoter in a BTG1-dependent manner, consistent with a role for this arginine methyl transferase in the regulation of GR-mediated gene expression.Together, our results demonstrate the importance of the BTG1/PRMT1 complex in regulating GR-mediated gene expression and reveal how deregulation of the this complex can give rise to GC resistance. Targeting of these coactivators as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC based therapies in ALL as well as other hematological malignancies.No relevant conflicts of interest to declare.

Abstract 3458

Abstract 3244

Glucocorticoids (GCs) are global regulators of gene transcription that act by binding to the glucocorticoid receptor, a ligand-activated transcription factor belonging to the nuclear receptor family. As synthetic GCs such as prednisolone or dexamethasone specifically induce cell cycle arrest and apoptosis in lymphoid cells, these drugs are widely used in the treatment of (childhood) acute lymphoblastic leukemia (ALL). GC resistance is a strong independent predictor of poor prognosis in the treatment of childhood ALL, but the underlying mechanisms remain poorly understood. In a previous study (Kuiper et al. Leukemia 2007) we determined by array-based SNP analysis that microdeletions affecting the anti-proliferative gene B-cell translocation gene 1 (BTG1) occurred in 4 out of 33 pre-B ALL cases. In our current study we show that prednisolone causes a 3-fold induction of BTG1 mRNA expression both in pre-B ALL cell lines and primary ALL, implicating BTG1 in cellular responses to GCs. Moreover, RNA-interference-mediated knockdown of BTG1 renders RS4;11 pre-B leukemia cells refractory to prednisolone-induced apoptosis. We show by genome-wide mRNA profiling that loss of BTG1 completely abrogates prednisolone-induced gene expression by downregulation of the glucocorticoid receptor NR3C1. Moreover, we present evidence showing that BTG1 is part of a nuclear receptor complex that regulates expression of NR3C1 and consequently GC responsiveness in ALL. In conclusion, we have identified BTG1 as an important determinant of GC sensitivity in ALL.

Glucocorticoids (GCs) are global regulators of gene transcription that act by binding to the glucocorticoid receptor, a ligand-activated transcription factor belonging to the nuclear receptor family. As synthetic GCs such as prednisolone or dexamethasone specifically induce cell cycle arrest and apoptosis in lymphoid cells, these drugs are widely used in the treatment of (childhood) acute lymphoblastic leukemia (ALL). GC resistance is a strong independent predictor of poor prognosis in the treatment of childhood ALL, but the underlying mechanisms remain poorly understood. In a previous study (Kuiper et al. Leukemia 2007) we determined by array-based SNP analysis that microdeletions affecting the anti-proliferative gene B-cell translocation gene 1 (BTG1) occurred in 4 out of 33 pre-B ALL cases. In our current study we show that prednisolone causes a 3-fold induction of BTG1 mRNA expression both in pre-B ALL cell lines and primary ALL, implicating BTG1 in cellular responses to GCs. Moreover, RNA-interference-mediated knockdown of BTG1 renders RS4;11 pre-B leukemia cells refractory to prednisolone-induced apoptosis. We show by genome-wide mRNA profiling that loss of BTG1 completely abrogates prednisolone-induced gene expression by downregulation of the glucocorticoid receptor NR3C1. Moreover, we present evidence showing that BTG1 is part of a nuclear receptor complex that regulates expression of NR3C1 and consequently GC responsiveness in ALL. In conclusion, we have identified BTG1 as an important determinant of GC sensitivity in ALL.