Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling - PubMed (original) (raw)

Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling

Andrew P Weng et al. Mol Cell Biol. 2003 Jan.

Abstract

Constitutive NOTCH signaling in lymphoid progenitors promotes the development of immature T-cell lymphoblastic neoplasms (T-ALLs). Although it is clear that Notch signaling can initiate leukemogenesis, it has not previously been established whether continued NOTCH signaling is required to maintain T-ALL growth. We demonstrate here that the blockade of Notch signaling at two independent steps suppresses the growth and survival of NOTCH1-transformed T-ALL cells. First, inhibitors of presenilin specifically induce growth suppression and apoptosis of a murine T-ALL cell line that requires presenilin-dependent proteolysis of the Notch receptor in order for its intracellular domain to translocate to the nucleus. Second, a 62-amino-acid peptide derived from a NOTCH coactivator, Mastermind-like-1 (MAML1), forms a transcriptionally inert nuclear complex with NOTCH1 and CSL and specifically inhibits the growth of both murine and human NOTCH1-transformed T-ALLs. These studies show that continued growth and survival of NOTCH1-transformed lymphoid cell lines require nuclear access and transcriptional coactivator recruitment by NOTCH1 and identify at least two steps in the Notch signaling pathway as potential targets for chemotherapeutic intervention.

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Figures

FIG. 1.

FIG. 1.

(a) Schematic representation of various forms of NOTCH referred to in this study. The normal mature heterodimeric receptor is produced by cleavage at S1. Cleavages at S2 and S3 are normally regulated by ligand binding to NEC but occur constitutively in the case of the ΔE form. Cleavage of ΔE at S2 by a metalloprotease yields the ΔE* form. Cleavage of ΔE* at S3 by presenilins releases ICN from the membrane. ICN1 constructs utilized in T6E/DFP-AA rescue experiments are indicated (ICN, ΔRAMΔP, and ΔTADΔP). P, PEST domain. (b) Schematic representation of functional domains of MAML1. Amino acid residue positions are indicated; 1016 represents full-length MAML1. ICN, Notch-binding domain; p300, p300 recruitment domain according to Fryer et al. (20); CoA*, recruitment domain for unidentified transcriptional coactivator(s).

FIG. 2.

FIG. 2.

Potency of presenilin inhibitor compounds in suppression of transcriptional stimulation by ΔE. U2OS cells were transiently transfected with a ΔE expression construct along with a CSL-luciferase reporter. Average normalized luciferase reporter activity (± standard deviations) from triplicate samples is expressed as a percentage of that observed in mock-treated cells.

FIG. 3.

FIG. 3.

Suppression of T6E cell growth by the presenilin inhibitor compound DFP-AA. T6E (left panel) and I22 (right panel) cells were cultured in the presence of the indicated concentrations of DFP-AA or carrier (mock) and were counted daily. Extrapolated cell counts were calculated as described in Materials and Methods.

FIG. 4.

FIG. 4.

Presenilin inhibitor treatment alters cell cycle progression and induces apoptosis in T6E, but not I22, cells. (a and b) Cell cycle analysis. T6E and I22 cells were treated with the indicated concentrations of DFP-AA for 3 and 8 days, respectively. DNA content was measured by flow cytometry after staining with propidium iodide. (a) Representative propidium iodide (PI) fluorescence histograms with superimposed cell cycle analysis models (including background correction). (b) Cell cycle fractions determined from histograms as for panel a. (c and d) Apoptosis analysis. (c) Sub-G1 fractions determined by using histograms as for panel a. (d) T6E and I22 cells treated with DFP-AA for 8 days were dual-stained with Annexin-V (for apoptotic cells) and 7-ADD (to exclude dead cells) and were analyzed by flow cytometry. Percentages of apoptotic cells (Annexin-V+/7-AAD−) are indicated after excluding cell debris by forward/side scatter gating.

FIG. 5.

FIG. 5.

Leukemogenic forms of ICN1 rescue T6E cells from presenilin inhibitor-mediated growth suppression. (a) Growth advantage of ICN1-transduced cells under conditions of presenilin inhibitor treatment. T6E cells were transduced with retroviruses encoding various constitutively nuclear forms of NOTCH1 (shown in Fig. 1a) and GFP on a bicistronic mRNA or GFP alone and then were treated with 1 μM DFP-AA or carrier (mock) beginning at day 3 posttransduction and continuing for the duration of the experiment. The percentage of GFP+ cells in each of the cultures was determined daily by flow cytometry, gating for live cells by forward/side scatter criteria. All cultures were repeated in duplicate; a single representative experiment is shown. (b) Cell cycle analysis. T6E cells from the experiment depicted in panel a were harvested after 9 days of exposure to 1 μM DFP-AA or carrier (mock) and were stained with DRAQ5 dye. DNA content was measured by DRAQ5 fluorescence in GFP− and GFP+ subpopulations by flow cytometry.

FIG. 6.

FIG. 6.

Presenilin inhibitor treatment blocks NOTCH1 signal transduction in T6E, but not I22, cells. (a) Effects on NOTCH1 polypeptides. T6E and I22 cells were treated with 1 μM DFP-AA for the indicated periods of time. NOTCH1 polypeptides were then immunoprecipitated from whole-cell extracts with an antibody directed against the intracellular domain of NOTCH1 (α-TC) and were analyzed on a Western blot stained with α-TC. ΔE, ΔE*, and ICN are NOTCH1-derived polypeptides diagrammed in Fig. 1. (b) Effects on HES1 transcript level. After T6E and I22 cells were treated with 1 μM DFP-AA for the indicated periods of time, total RNA was collected and analyzed on a Northern blot with probes for HES1 and GAPDH.

FIG. 7.

FIG. 7.

Mapping and functional characterization of dominant-negative MAML1 peptides. (a) Mapping MAML1 residues needed for ternary complex formation. Highly purified MAML1 peptides incubated with recombinant ICN1 RAM-ANK and immunopurified CSL were scored for complex formation in an EMSA. MAML1 peptides were included at increasing concentrations of up to a fivefold molar excess over the concentration of RAM-ANK. (b) Dominant-negative activities of MAML1 peptides. Each well of a 24-well plate containing U2OS cells was transiently transfected with pcDNA3 expression constructs for ICN1 (10 ng), various MAML1 peptides (50 ng), a CSL-luciferase reporter (125 ng), and a Renilla luciferase internal control reporter (2.5 ng). Mean normalized luciferase activity (± standard deviations) from triplicate wells is expressed relative to that observed in cells transfected with reporter constructs plus empty pcDNA3 (60 ng).

FIG. 8.

FIG. 8.

Dominant-negative MAML1 peptides specifically suppress growth of human and murine NOTCH1-transformed T-cell lines. (a) Growth suppression of cells expressing the dominant-negative MAML1(13-74)-GFP fusion protein. Each cell line was transduced with MAML1(13-74)-GFP or GFP retrovirus at titers such that only a subpopulation of cells (∼40 to 70%) were transduced. The percentage of GFP+ cells was determined daily by flow cytometry, gating for live cells by forward/side scatter criteria. The percentage of GFP+ cells remaining at each day is expressed as a fraction of the initial (day 3 posttransduction) GFP+ percentage. All cell lines except BJAB were used in three independent experiments; a single representative experiment is shown. (b) Absolute growth rates of SUPT-T1 and BW5147 cultures in which >95% of cells had been transduced by the indicated retroviruses. Cell counts were performed daily starting at day 2 postretroviral transduction, and extrapolated cell counts were calculated as described in Materials and Methods. (c) Cell cycle effects of MAML1(13-74)-GFP. DNA content was measured from the SUP-T1 and BW5147 cultures depicted in panel b on day 7 posttransduction (corresponding to day 5 in panel b). Cells were stained with propidium iodide and were analyzed by flow cytometry. MamGFP, MAML1(13-74)-GFP; GFP, GFP only.

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