Notch signals positively regulate activity of the mTOR pathway in T-cell acute lymphoblastic leukemia

Abstract

Constitutive Notch activation is required for the proliferation of a subgroup of T-cell acute lymphoblastic leukemia (T-ALL). Downstream pathways that transmit pro-oncogenic signals are not well characterized. To identify these pathways, protein microarrays were used to profile the phosphorylation state of 108 epitopes on 82 distinct signaling proteins in a panel of 13 T-cell leukemia cell lines treated with a gamma-secretase inhibitor (GSI) to inhibit Notch signals. The microarray screen detected GSI-induced hypophosphorylation of multiple signaling proteins in the mTOR pathway. This effect was rescued by expression of the intracellular domain of Notch and mimicked by dominant negative MAML1, confirming Notch specificity. Withdrawal of Notch signals prevented stimulation of the mTOR pathway by mitogenic factors. These findings collectively suggest that the mTOR pathway is positively regulated by Notch in T-ALL cells. The effect of GSI on the mTOR pathway was independent of changes in phosphatidylinositol-3 kinase and Akt activity, but was rescued by expression of c-Myc, a direct transcriptional target of Notch, implicating c-Myc as an intermediary between Notch and mTOR. T-ALL cell growth was suppressed in a highly synergistic manner by simultaneous treatment with the mTOR inhibitor rapamycin and GSI, which represents a rational drug combination for treating this aggressive human malignancy.

Figure 1

Reverse phase protein (RPP) microarray profiling of Notch signals in T-ALL cell lines. (A) Six γ-secretase inhibitor (GSI)–sensitive T-ALL cell lines were treated with 1 μM compound E, a potent GSI, or vehicle (DMSO) for 7 days. Cell cycle distribution was determined based on DNA content of propidium iodide–stained populations. Green bars highlight the difference between mock- and GSI-treated cells in the G₀/G₁ fraction. (B) Cell cycle analysis of 7 GSI-resistant cell lines. (C) Lysates derived from GSI- and mock-treated cells were diluted (1:1, 1:3, 1:9) and printed on nitrocellulose-coated slides in 6 replicates. Fluorescent image of a representative RPP microarray probed with an antibody specific for the CDK inhibitor, p27^Kip1, and stained with Alexa Fluor 647 is shown. Each subarray contains lysates derived from a single cell line. For the sole purpose of presentation, brightness and contrast were adjusted equivalently across the entire array to highlight differences in feature intensities. (D) Presentation of 133 phospho-protein or total protein fold change measurements (GSI/DMSO) for each cell line as a scatter plot. Each dot represents an individual measurement. Scatter plots of sensitive cell lines are on the left side and resistant cell lines on the right. See “Materials and methods” for details in the calculation of fold change. All data points are logged in base 2 and median-centered for their corresponding cell line. The blue horizontal lines are arbitrarily placed to assist the comparison of scatter plots.

Figure 2

Identification of changes in phosphorylation and total protein levels that preferentially occur in GSI-sensitive cell lines. (A) Significant list of phospho-epitopes and proteins identified by significance analysis of microarrays (SAM) along with their direction of change and (d)-score rank. See “Materials and methods” for details. When 2 ranks are shown for a particular epitope, 2 distinct antibodies with the same specificity were used. (B) Presentation of fold change measurements as scatter plots for SAM-identified analytes listed in (A). Each dot represents an individual cell line. S denotes sensitive cell lines. R denotes resistant lines. All fold-change data points are logged in base 2 and median-centered for their corresponding cell line. Horizontal line indicates the mean fold change of the group. For cdc2 and p-S6 RP, 2 distinct antibodies were used.

Figure 3

Notch inhibition suppresses phosphorylation of effectors in the mTOR pathway. (A) Five GSI-sensitive cell lines were exposed to either GSI (compound E) or vehicle (DMSO) for 7 days. After the incubation period, whole-cell lysates were prepared and equivalent amounts of total protein were loaded per lane on a polyacrylamide gel for Western blot analysis using the panel of antibodies shown on the left. (B) Similar experiment as in panel A except the cell lines were stably transduced with retroviruses expressing ICN1. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples.

Figure 4

S6 RP dephosphorylation in G0/G1-gated cells occurs prior to substantial cell cycle arrest. Cells were exposed to GSI (DAPT) or vehicle (DMSO) for the indicated amount of time. At each time point, cells were intracellularly stained with an antibody specific for phospho-S6 RP and their DNA contents measured by propidium iodide staining. Cells in the G₀/G₁ fraction were gated based on DNA content and their level of S6 RP phosphorylation was determined. The fraction of the mean fluorescent intensity (MFI) of GSI-treated cells to the MFI of mock-treated cells was calculated and plotted over time (open circles, left Y-axis). The ratio of the percentage of cells in G₀/G₁ phase in GSI-treated populations to that in mock-treated populations was measured over 8 days to monitor cell cycle arrest (solid circles, right Y-axis; axis is reversed to facilitate comparison between the 2 graphs). Data shown are representative of 3 independent experiments with similar results.

Figure 5

Notch inhibition reduces cell size. (A) The indicated cell lines were treated with GSI (compound E) or vehicle (DMSO) for 7 days. After the incubation period, relative size (forward scatter) of G₀/G₁-gated cells was determined by flow cytometry. Histograms with FSC on the X-axis are shown. (B) The magnitude of size reduction for each of the 13 tested lines was measured after exposure to GSI for 7 days and correlated with its corresponding suppression in p70 S6 kinase (T389) phosphorylation as measured using RPP microarrays. Size of GSI-treated cells is reported as a percentage of mock (DMSO)–treated cells on the X-axis. The level of phospho-p70 S6 kinase (T389) is shown on the Y-axis as the median-centered log₂ ratio of GSI/DMSO-treated cells.

Figure 6

Effects of Notch on the mTOR pathway are independent of changes in Akt activity but dependent on c-Myc. (A) Notch inhibition blocks the induction of S6 RP phosphorylation in response to fetal calf serum (FCS) and insulin-like growth factor I (IGF-I) stimulation. TALL-1 cells were treated with GSI (DAPT) or vehicle (DMSO) for 6 days. The cells were then serum starved for 24 hours and subsequently stimulated with FCS (10%) or IGF-I (20 ng/mL) for an additional 24 hours. Wortmannin (50 nM) was added to the cells during the last hour of incubation. Cell lysates were prepared and equivalent amounts of protein were loaded per lane on a gel for Western blot analysis using the 2 antibodies shown. Expression of Hes-1 mRNA normalized to GAPDH in each sample was measured using quantitative reverse-transcription–polymerase chain reaction (RT-PCR) to confirm inhibition of Notch activity. The values shown indicate the amount of Hes-1 mRNA relative to serum-starved mock-treated cells. GSI-treated (right panel) and DMSO control (left panel) samples were processed on the same blot. (B) Protein microarray ratiometric data for phospho-Akt (Ser473) and phospho-S6 RP in GSI-sensitive cell lines. Ratios (GSI/DMSO) are logged in base 2 and median-centered for each cell line. (C) GSI treatment abolishes S6 RP phosphorylation in the absence of changes in Akt and GSK3β phosphorylation. HPB-ALL and TALL-1 cells were treated with GSI (compound E) or vehicle (DMSO) for 3 days. Whole-cell lysates were prepared and equivalent amounts of proteins were loaded per lane for Western blot analysis using the indicated antibodies on the left. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples. (D) GSI-induced dephosphorylation of S6 ribosomal protein is not dependent on changes in PI3K/Akt activity. HPB-ALL and T-ALL cells were exposed to either DMSO or 1 μM GSI (compound E) for 3 days. At the end of the 3-day period, cells were intracellularly stained with an antibody specific for either phospho-S6 RP or phospho-Akt (Ser 473) and analyzed using flow cytometry. Gates were placed around mock- and GSI-treated cell populations on forward and side scatter dot plots with equivalent levels of phospho-Akt staining. Phospho-S6 RP staining was compared between the 2 groups using the same gated populations. Each bar graph represents background-subtracted mean fluorescent intensity of the indicated phospho-epitope normalized to the corresponding value in mock-treated control cells. Error bars represent standard deviation. (E) Similar experiment as in Figure 3 except the cell lines were stably transduced with retroviruses expressing c-Myc. Blots were probed with the antibodies indicated on the left panel. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples.

Figure 7

Synergistic suppression of T-ALL growth with rapamycin and GSI. GSI-sensitive cell lines (left panel) and -resistant cell lines (right panel) were cultured in the presence of varying concentrations of GSI (compound E), rapamycin, or combination of both drugs at a fixed molar ratio of 1000:1 (compound E/Rapamycin). The number of viable cells in each well was determined using a MTS-based assay at the end of a 5- or 6-day incubation period. See “Materials and methods” for details in the calculation of “fraction affected” and combination index (CI). Error bars represent standard deviation. Lines are placed at 0.25 to facilitate comparisons between graphs.