Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia - PubMed (original) (raw)
. 2009 Nov;158(5):1183-95.
doi: 10.1111/j.1476-5381.2009.00389.x. Epub 2009 Sep 23.
C Ware, C Efferson, J O'Neil, S Rao, X Qu, J Gorenstein, M Angagaw, H Kim, C Kenific, K Kunii, K J Leach, G Nikov, J Zhao, X Dai, J Hardwick, M Scott, C Winter, L Bristow, C Elbi, J F Reilly, T Look, G Draetta, Lht Van der Ploeg, N E Kohl, P R Strack, P K Majumder
Affiliations
- PMID: 19775282
- PMCID: PMC2782329
- DOI: 10.1111/j.1476-5381.2009.00389.x
Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia
J Tammam et al. Br J Pharmacol. 2009 Nov.
Abstract
Background and purpose: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy.
Experimental approach: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours.
Key results: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis.
Conclusions and implications: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.
Figures
Figure 1
Daily administration of γ-secretase inhibitor (GSI) was not tolerated at effective concentrations. (A) DND-41 cells were implanted subcutaneously in female CD1 nu/nu mice. Tumour-bearing mice were randomized into various groups of equal average tumour volume (∼200 mm3). Different doses of GSI (50 and 100 mg·kg−1) or vehicle (0.5% methyl cellulose) were given orally once daily as indicated. Tumour size was measured (see Methods) and recorded biweekly. (B) Body weight of mice treated with GSI or vehicle were measured biweekly.
Figure 2
Intermittent dosing of γ-secretase inhibitor (GSI) was effective in T-cell acute lymphoblastic leukaemia (T-ALL) xenograft models. (A) Secretory epithelial cells (dark pink) in the mouse intestine were visualized by periodic acid Schiff (PAS) staining following treatment with either vehicle (Veh) or GSI. Upper panel shows response to vehicle; middle and lower panels show intestine from GSI-treated mice (100 mg·kg−1) treated with three daily doses followed by 2 or 8 days of no treatment. Scale bar, 100 µm. (B) The intensity of PAS staining was scored manually in different dose groups and plotted against time as indicated. (C and D) Mice with subcutaneous T-ALL tumour xenografts were treated with two different GSI doses on two different schedules. Using the GSI at 100 mg·kg−1, with a 3 days on/4 days off dosing schedule (three times a week) caused significant (P < 0.05) anti-tumour effects as measured by tumour volume in DND-41 (C) and TALL-1 xenografts (D). GSI caused tumour regression in the TALL-1 xenografts when given once a week at 300 mg·kg−1 (D). Body weight (mean ± SEM) of DND-41 (E) and TALL-1 (F) xenograft tumour-bearing mice were measured twice a week during GSI treatment and plotted against time as indicated.
Figure 3
γ-Secretase inhibitor (GSI) inhibited Notch signalling, decreased tumour cell proliferation and induced apoptosis in T-cell acute lymphoblastic leukaemia (T-ALL) xenograft models. (A) Mice with TALL-1 xenograft tumours were treated with a single dose of GSI at 300 mg·kg−1; (B) APP-YAC mice were treated with either vehicle (0.5% methyl cellulose) or GSI at 75 mg·kg−1 and 300 mg·kg−1 doses. (A,B) Plasma Aβ40 levels were measured as a surrogate for inhibition of γ-secretase at 0 h (vehicle), 4 h, 1 day, 3 days, 4 days and 7 days after treatment. Serum was isolated and Aβ40 level was determined. Percent of Aβ40 remaining in GSI treated mice compared with vehicle treated mice. (C) TALL-1 tumour sections were stained with anti-HES1 antibody at 4 h, 1 day, 3 days, 4 days and 7 days post dose of GSI at 300 mg·kg−1. Scale bar, 50 µm. TALL-1 xenograft tumours were harvested 4 h after receiving a dose of GSI at 300 mg·kg−1 (lower panel). Notch pathway activity was determined by the expression of DTX1, HES5, HES1 and ASCL1 by quantitative polymerase chain reaction analysis. (D) Mice with TALL-1 xenografts were treated with either vehicle (0.5% methyl cellulose) or GSI at 100 mg·kg−1 thrice weekly for 3 weeks. Tumours were harvested 4 h after the third dose in week 3, dissected and stained with TUNEL, or with antibodies against Ki67 and activated caspase-3. Scale bar, 50 µm.
Figure 4
An effective dose of γ-secretase inhibitor (GSI) increased apoptosis as measured by activated caspase-3. Mice bearing TALL-1 tumour xenografts were treated with three consecutive daily doses of either vehicle (Veh), 50, 100 or 150 mg·kg−1 of GSI. Mice were killed 4 h after the last dose, and tumours were collected. Activation of caspase-3 was measured by Western blot analysis in tumour lysates and band intensity measured by densitometric analysis.
Figure 5
γ-Secretase inhibitor (GSI) altered mitochondrial transmembrane potential (ΔΨm) and facilitated cytochrome c release in TALL-1 cells. Cells were treated with either dimethyl sulphoxide (DMSO) or 1 µM GSI for 2 days and harvested on days 1, 3 and 7 after the last addition of drug. (A) Cells treated with GSI or DMSO were stained with JC1 dye and analysed by flow cytometry. (B) Cytosol without mitochondria (S-100 fraction) was prepared from cells treated with either DMSO or 1 µM of GSI, and immunoblot analysis was performed using antibodies against cytochrome c and β-actin. Lower panel, ratio of cytochrome c to β-actin was determined by densitometric analysis.
Figure 6
NOTCH-dependent inhibition of mitochondrial dysfunction genes by γ-secretase inhibitor (GSI). RNA profiling from 13 human T-cell acute lymphoblastic leukaemia (T-ALL) cells treated with either dimethyl sulphoxide (DMSO) or GSI at 0.1 or 1 µM for 3 days. (A) Unsupervised profiling analysis: genes with log10 expression ratios that correlated (P ≤ 0.05) with GSI sensitivity following GSI treatment were analysed for pathway association (left panel). Down-regulated genes associated with mitochondrial dysfunction are shown (right panel). (B) Correlation between GSI-induced mitochondrial gene response (average of genes shown in A) versus GI50 for each cell line indicated. Blue and red symbols represent gene response derived from 3 day treatment with 0.1 and 1 µM GSI respectively. Correlation of GSI-induced mitochondrial gene response (log10 ratio average of genes shown in A) versus GSI-induced change in NOTCH1 target genes (log10 ratio average of 10 NOTCH1 target genes: HES1, HES4, HES5, HEYL, HEY2, DTX1, MYC, NRARP, PTCRA, SHQ1) (left panel). Blue and red symbols represent gene response derived from 3 day treatment with 0.1 and 1 µM GSI respectively. The cell line Loucy is indicated by the abbreviation L; MOLT-16 by M; SKW-3 by SW; SUPT-11 by S; BE-13 by B; HSB-2 by HS; CCRF-CEM by C; PF-382 by P; RPMI-8402 by R; HPB-ALL by H; DND-41 by D; KOPTK-1 by K and TALL-1 by T. (C) DND-41 cells containing vector (MigR1) or NOTCH intracellular domain (ICN) were treated with either DMSO or GSI at 1 µM for 3 days. Average score of NOTCH1 target genes and mitochondrial genes are shown. (D) TALL-1 cells were treated with either DMSO or GSI for three consecutive days and harvested at 6, 24 and 48 h after the last dose. Proteins were blotted with antibodies against peroxiredoxin 5 (PRDX5) and NDUFA2. Tubulin was used as loading control. The densitometric ratios of TALL-1 cells treated with DMSO (grey bar) or GSI (black bar) were calculated. Mice were treated with either vehicle (0.5% methyl cellulose) or GSI at 75 and 100 mg·kg−1 three times a week for 3 weeks (right panel). Mice were killed, tumours were harvested 4 h after the final dose, and superoxide dismutase 2 (SOD2) and NDUFA2 were measured by immunoblot analysis. β-Actin was used for loading control.
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