Notch1 promotes survival of E2A-deficient T cell lymphomas through pre-T cell receptor-dependent and -independent mechanisms

Abstract

Loss of E2A transcription factor activity or activation of the intracellular form of Notch1 (ICN) leads to the development of leukemia or lymphoma in humans or mice, respectively. Current models propose that ICN functions by suppressing E2A through a pre-T cell receptor (TCR)-dependent mechanism. Here we show that lymphomas arising in E2A(-/-) mice require the activation of Notch1 for their survival and have accumulated mutations in, or near, the Notch1 PEST domain, resulting in increased stability and signaling. In contrast, lymphomas arising in p53(-/-) mice show the activation of Notch1, but no mutations were identified in ICN. The requirement for Notch1 signaling in E2A(-/-) lymphomas cannot be overcome by ectopic expression of pTalpha; however, pTalpha is required for optimal survival and expansion of these cells. Our findings indicate that the activation of Notch1 is an important "second hit" for the transformation of E2A(-/-) T cell lymphomas and that Notch1 promotes survival through pre-TCR-dependent and -independent mechanisms.

Figure 1.

Expression of Notch1 target genes in E2A^–/– and p53^–/– lymphomas. (A) Northern blot of total RNA isolated from the indicated lymphomas probed with Notch1 or actin cDNA. (B) Western blot of protein isolated from the indicated lymphomas 24 hours after treatment with GSI (+) or DMSO (–) using anti–activated Notch1 (V1744) antibody. The position of molecular mass markers, in kilodaltons, is shown to the left of the blot. (C) Northern blot of total RNA isolated from the indicated lymphomas probed with Deltex1, Hes1, Notch3, nRARP, pTα, and actin cDNA. 0531 and 1.F9 are E2A^–/–, and 16610D9, K052FA2, K052FA2C1, and K052DA20 are p53^–/– lymphoma lines. E2A^–/– and p53^–/– lymphomas are primary tumors isolated directly from mice.

Figure 2.

Inhibition of Notch1 signaling inhibits survival of E2A^–/– lymphomas. (A) E2A^–/– lymphoma lines 0531, 1.F9, and 0714 and the Notch1-deficient p53^–/– lymphoma 16610D9 were treated with GSI or vehicle (DMSO), and the number of viable cells was determined every 24 hours. Relative viability is the number of viable cells (GSI)/number of viable cells (DMSO) × 100. (B) Relative viability of Notch1-expressing p53^–/– lymphoma lines treated with GSI or DMSO. (C) Lymphomas were treated with GSI or vehicle (DMSO) starting 24 hours after infection with MigR1 () or MigR1-ICN (▪) virus. Number of viable cells was determined 48 hours later. Infection efficiency was greater than 90% for all populations. (D) 16610D9 (♦), 0531 (•), 1.F9 (▴), and K052FA2C1 (▪) lines were infected with MigR1 (open symbols) or MigR1-DNMAML (filled symbols), and the percentage of cells expressing GFP was determined every 24 hours by FACS. Relative GFP is the percentage GFP⁺ divided by the percentage GFP⁺ at 24 hours after infection. Infection efficiency for each line was 60% to 80%. (E) MigR1-infected cells () and MigR1-DN MAML–infected cells (▪) were incubated with DHE for 30 minutes starting 48 hours after infection. The percentage of GFP⁺ cells demonstrating increased fluorescence staining with DHE is shown.

Figure 3.

Inhibition of Notch1 affects the expression of Notch1 target genes in E2A^–/– lymphomas. (A) Northern blot of total RNA isolated from lymphomas treated with DMSO or GSI for 48 hours or sorted GFP⁺ cells from DN-MAML–expressing cells harvested 48 hours after infection. The blot was probed sequentially with the indicated cDNA probes. (B) Flow cytometric analysis of cell surface TCR-β on E2A^–/– lymphomas treated with DMSO (black histogram) or GSI (gray histogram) for 24 hours. Broken line represents staining with irrelevant fluorescently labeled antibody.

Figure 4.

pTα is insufficient to promote the survival of GSI-treated E2A^–/– lymphomas. (A) FACS analysis of E2A^–/– lymphoma (1.F9) infected with control (black histogram) or pTα (gray histogram) producing virus and stained with anti-hCD25 and anti–TCR-β antibody (solid lines) or control IgG (stippled lines). Anti–TCR-β staining is shown on hCD25⁺ cells. (B) Total cell numbers 48 hours after treatment of control (▪) or pTα () virus–infected 16610D9, 1.F9, or 0531 lymphomas treated with DMSO (–) or GSI (+).

Figure 5.

pTα is required for optimal expansion of E2A^–/– lymphomas. (A) FACS analysis of E2A^–/– lymphoma (1.F9) with control (black histogram) or pTα siRNA (gray histogram) virus and stained with anti–TCR-β antibody (solid line) or control IgG (broken line). Anti–TCR-β staining is shown on GFP⁺ cells. (B) Northern blot of RNA isolated from GFP⁺ lymphomas 48 hours after infection with control or pTα siRNA virus. The blot was probed with pTα or actin cDNA, as indicated. (C) The 70Z/3 pre–B cell line (♦), E2A^–/– lymphomas 0531 (•) and 1.F9 (▴), and the Notch3-transformed cell line N3T (▪) were infected with control (open symbols) or pTα siRNA (filled symbols) virus, and the percentage of GFP⁺ cells was determined every 24 hours by flow cytometry. Infection efficiency for each cell line was 40% to 60%. (D) Control () and pTα siRNA virus–infected (▪) cells were incubated with DHE for 30 minutes starting 48 hours after infection. The percentage of GFP⁺ cells showing increased fluorescence with DHE is shown. (E) Western blot of whole cell extracts made from lymphoma lines treated for 48 hours with DMSO (–) or GSI (+) or 48 hours after infection with control virus (B) or pTα siRNA–producing virus (S). The blots were probed sequentially with antibodies detecting p21 or p27. Total protein loading was visualized before hybridization using Amido Black and mirrored the intensity of the nonspecific band present on the p21 blot.