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Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia.

Real PJ, Tosello V, Palomero T, Castillo M, Hernando E, de Stanchina E, Sulis ML, Barnes K, Sawai C, Homminga I, Meijerink J, Aifantis I, Basso G, Cordon-Cardo C, Ai W, Ferrando A - Nat. Med. (2008)

Bottom Line: Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo.GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation.In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.

ABSTRACT
Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2-like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

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Inhibition of NOTCH1-HES1 signaling restores glucocorticoid receptor autoregulation. (a) Microarray gene expression changes in CUTLL1 cells at 24 h treated with DMSO, CompE, dexamethasone and CompE plus dexamethasone. Relative expression levels are color coded as indicated at the bottom. (b) Quantitative RT-PCR analysis of the glucocorticoid receptor gene (NR3C1) and Western blot analysis and quantitation of glucocorticoid receptor protein levels in CUTLL1 cells treated with dexamethasone and CompE compared with vehicle only (DMSO). (c) Western blot analysis of NR3C1 levels and induction of apoptosis by dexamethasone and CompE in CUTLL1 cells infected with retroviruses expressing the glucocorticoid receptor (pMSCV NR3C1). (d) Analysis of apoptosis induction by dexamethasone plus CompE in CUTLL1 cells infected with shRNA lentiviruses targeting the glucocorticoid receptor (pGIPZ NR3C1) (e) Quantitative ChIP analysis of HES1 binding to NR3C1 promoter sequences. (f) Effects of HES1, MYB and dexamethasone (1 µM) in the activity of a human NR3C1 A1 reporter. Luciferase activity is shown relative to an internal control expressing Renilla luciferase. (g,h) NR3C1 expression (g) and analysis of apoptosis (h) in CUTLL1 cells treated with dexamethasone and CompE after lentiviral shRNA knockdown of HES1 (HES1 shRNA). A shRNA targeting the luciferase gene (shRNA LUC) was used as control. Drug concentrations in a–c were CompE 100 nM and dexamethasone 1 µM. Bars represent means ± SD of triplicate experiments. Statistical significance was assessed with Student’s t-test. HES1 and NR3C1 relative protein levels are indicated at the bottom of corresponding lanes in the Western blot.
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Figure 2: Inhibition of NOTCH1-HES1 signaling restores glucocorticoid receptor autoregulation. (a) Microarray gene expression changes in CUTLL1 cells at 24 h treated with DMSO, CompE, dexamethasone and CompE plus dexamethasone. Relative expression levels are color coded as indicated at the bottom. (b) Quantitative RT-PCR analysis of the glucocorticoid receptor gene (NR3C1) and Western blot analysis and quantitation of glucocorticoid receptor protein levels in CUTLL1 cells treated with dexamethasone and CompE compared with vehicle only (DMSO). (c) Western blot analysis of NR3C1 levels and induction of apoptosis by dexamethasone and CompE in CUTLL1 cells infected with retroviruses expressing the glucocorticoid receptor (pMSCV NR3C1). (d) Analysis of apoptosis induction by dexamethasone plus CompE in CUTLL1 cells infected with shRNA lentiviruses targeting the glucocorticoid receptor (pGIPZ NR3C1) (e) Quantitative ChIP analysis of HES1 binding to NR3C1 promoter sequences. (f) Effects of HES1, MYB and dexamethasone (1 µM) in the activity of a human NR3C1 A1 reporter. Luciferase activity is shown relative to an internal control expressing Renilla luciferase. (g,h) NR3C1 expression (g) and analysis of apoptosis (h) in CUTLL1 cells treated with dexamethasone and CompE after lentiviral shRNA knockdown of HES1 (HES1 shRNA). A shRNA targeting the luciferase gene (shRNA LUC) was used as control. Drug concentrations in a–c were CompE 100 nM and dexamethasone 1 µM. Bars represent means ± SD of triplicate experiments. Statistical significance was assessed with Student’s t-test. HES1 and NR3C1 relative protein levels are indicated at the bottom of corresponding lanes in the Western blot.

Mentions: Both NOTCH1 and the glucocorticoid receptor function as ligand activated transcription factors, suggesting that crosstalk between NOTCH1 signaling and glucocorticoid therapy may enhance the transcriptional response to dexamethasone in glucocorticoid resistant T-ALL. To analyze the effects of glucocorticoid treatment in the transcriptional regulatory network controlled by NOTCH1 in T-ALL 19 and to explore the mechanisms mediating the reversal of glucocorticoid resistance upon NOTCH inhibition, we performed gene expression profiling with oligonucleotide microarrays in CUTLL1 cells treated with vehicle (DMSO), dexamethasone (1 µM), CompE (100 nM) or cotreated with dexamethasone plus CompE for 24 hours. This analysis demonstrated a more efficient glucocorticoid response in cells treated with CompE plus dexamethasone with synergistic upregulation of glucocorticoid regulated genes such as TSC22D3/GILZ, CD53, SOCS1 and BTG2 (Fig. 2a and Supplementary Fig. 6 online). Importantly, the glucocorticoid receptor gene (NR3C1) ranked among the top genes synergistically upregulated by dexamethasone plus GSI cotreatment in our microarray analysis (Fig. 2a). Numerous studies on the mechanisms of glucocorticoid resistance have established that an effective upregulation of the glucocorticoid receptor gene in response to glucocorticoids is required for the activation of apoptosis in human leukemias 21–26. Quantitative RT-PCR and Western blot analysis showed a moderate increase in glucocorticoid receptor levels in CUTLL1 cells and primary T-ALL lymphoblasts treated with GSI and dexamethasone (Fig. 2b and Supplementary Figure 3 online). In contrast, dexamethasone plus CompE cotreatment resulted in a significantly higher (5 fold) upregulation of glucocorticoid receptor transcripts and protein levels (Fig. 2b). These results suggest that inhibition of NOTCH1 signaling enhance glucocorticoid receptor auto-up-regulation and glucocorticoid sensitivity in otherwise glucocorticoid resistant T-ALL cells. Consistent with this hypothesis, and in agreement with previous reports in T-ALL 27–29, retroviral expression of the glucocorticoid receptor gene restored glucocorticoid sensitivity and enhanced the apoptotic response of CUTLL1 cells to dexamethasone (Fig. 2c). Conversely, shRNA knockdown of the glucocorticoid receptor resulted in complete abrogation of apoptosis induced by dexamethasone plus CompE (Fig. 2d)


Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia.

Real PJ, Tosello V, Palomero T, Castillo M, Hernando E, de Stanchina E, Sulis ML, Barnes K, Sawai C, Homminga I, Meijerink J, Aifantis I, Basso G, Cordon-Cardo C, Ai W, Ferrando A - Nat. Med. (2008)

Inhibition of NOTCH1-HES1 signaling restores glucocorticoid receptor autoregulation. (a) Microarray gene expression changes in CUTLL1 cells at 24 h treated with DMSO, CompE, dexamethasone and CompE plus dexamethasone. Relative expression levels are color coded as indicated at the bottom. (b) Quantitative RT-PCR analysis of the glucocorticoid receptor gene (NR3C1) and Western blot analysis and quantitation of glucocorticoid receptor protein levels in CUTLL1 cells treated with dexamethasone and CompE compared with vehicle only (DMSO). (c) Western blot analysis of NR3C1 levels and induction of apoptosis by dexamethasone and CompE in CUTLL1 cells infected with retroviruses expressing the glucocorticoid receptor (pMSCV NR3C1). (d) Analysis of apoptosis induction by dexamethasone plus CompE in CUTLL1 cells infected with shRNA lentiviruses targeting the glucocorticoid receptor (pGIPZ NR3C1) (e) Quantitative ChIP analysis of HES1 binding to NR3C1 promoter sequences. (f) Effects of HES1, MYB and dexamethasone (1 µM) in the activity of a human NR3C1 A1 reporter. Luciferase activity is shown relative to an internal control expressing Renilla luciferase. (g,h) NR3C1 expression (g) and analysis of apoptosis (h) in CUTLL1 cells treated with dexamethasone and CompE after lentiviral shRNA knockdown of HES1 (HES1 shRNA). A shRNA targeting the luciferase gene (shRNA LUC) was used as control. Drug concentrations in a–c were CompE 100 nM and dexamethasone 1 µM. Bars represent means ± SD of triplicate experiments. Statistical significance was assessed with Student’s t-test. HES1 and NR3C1 relative protein levels are indicated at the bottom of corresponding lanes in the Western blot.
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Related In: Results  -  Collection

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Figure 2: Inhibition of NOTCH1-HES1 signaling restores glucocorticoid receptor autoregulation. (a) Microarray gene expression changes in CUTLL1 cells at 24 h treated with DMSO, CompE, dexamethasone and CompE plus dexamethasone. Relative expression levels are color coded as indicated at the bottom. (b) Quantitative RT-PCR analysis of the glucocorticoid receptor gene (NR3C1) and Western blot analysis and quantitation of glucocorticoid receptor protein levels in CUTLL1 cells treated with dexamethasone and CompE compared with vehicle only (DMSO). (c) Western blot analysis of NR3C1 levels and induction of apoptosis by dexamethasone and CompE in CUTLL1 cells infected with retroviruses expressing the glucocorticoid receptor (pMSCV NR3C1). (d) Analysis of apoptosis induction by dexamethasone plus CompE in CUTLL1 cells infected with shRNA lentiviruses targeting the glucocorticoid receptor (pGIPZ NR3C1) (e) Quantitative ChIP analysis of HES1 binding to NR3C1 promoter sequences. (f) Effects of HES1, MYB and dexamethasone (1 µM) in the activity of a human NR3C1 A1 reporter. Luciferase activity is shown relative to an internal control expressing Renilla luciferase. (g,h) NR3C1 expression (g) and analysis of apoptosis (h) in CUTLL1 cells treated with dexamethasone and CompE after lentiviral shRNA knockdown of HES1 (HES1 shRNA). A shRNA targeting the luciferase gene (shRNA LUC) was used as control. Drug concentrations in a–c were CompE 100 nM and dexamethasone 1 µM. Bars represent means ± SD of triplicate experiments. Statistical significance was assessed with Student’s t-test. HES1 and NR3C1 relative protein levels are indicated at the bottom of corresponding lanes in the Western blot.
Mentions: Both NOTCH1 and the glucocorticoid receptor function as ligand activated transcription factors, suggesting that crosstalk between NOTCH1 signaling and glucocorticoid therapy may enhance the transcriptional response to dexamethasone in glucocorticoid resistant T-ALL. To analyze the effects of glucocorticoid treatment in the transcriptional regulatory network controlled by NOTCH1 in T-ALL 19 and to explore the mechanisms mediating the reversal of glucocorticoid resistance upon NOTCH inhibition, we performed gene expression profiling with oligonucleotide microarrays in CUTLL1 cells treated with vehicle (DMSO), dexamethasone (1 µM), CompE (100 nM) or cotreated with dexamethasone plus CompE for 24 hours. This analysis demonstrated a more efficient glucocorticoid response in cells treated with CompE plus dexamethasone with synergistic upregulation of glucocorticoid regulated genes such as TSC22D3/GILZ, CD53, SOCS1 and BTG2 (Fig. 2a and Supplementary Fig. 6 online). Importantly, the glucocorticoid receptor gene (NR3C1) ranked among the top genes synergistically upregulated by dexamethasone plus GSI cotreatment in our microarray analysis (Fig. 2a). Numerous studies on the mechanisms of glucocorticoid resistance have established that an effective upregulation of the glucocorticoid receptor gene in response to glucocorticoids is required for the activation of apoptosis in human leukemias 21–26. Quantitative RT-PCR and Western blot analysis showed a moderate increase in glucocorticoid receptor levels in CUTLL1 cells and primary T-ALL lymphoblasts treated with GSI and dexamethasone (Fig. 2b and Supplementary Figure 3 online). In contrast, dexamethasone plus CompE cotreatment resulted in a significantly higher (5 fold) upregulation of glucocorticoid receptor transcripts and protein levels (Fig. 2b). These results suggest that inhibition of NOTCH1 signaling enhance glucocorticoid receptor auto-up-regulation and glucocorticoid sensitivity in otherwise glucocorticoid resistant T-ALL cells. Consistent with this hypothesis, and in agreement with previous reports in T-ALL 27–29, retroviral expression of the glucocorticoid receptor gene restored glucocorticoid sensitivity and enhanced the apoptotic response of CUTLL1 cells to dexamethasone (Fig. 2c). Conversely, shRNA knockdown of the glucocorticoid receptor resulted in complete abrogation of apoptosis induced by dexamethasone plus CompE (Fig. 2d)

Bottom Line: Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo.GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation.In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.

ABSTRACT
Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2-like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

Show MeSH
Related in: MedlinePlus