The TCF-1 and LEF-1 transcription factors have cooperative and opposing roles in T cell development and malignancy

Abstract

The TCF-1 and LEF-1 transcription factors are known to play critical roles in normal thymocyte development. Unexpectedly, we found that TCF-1-deficient (Tcf7(-/-)) mice developed aggressive T cell malignancy, resembling human T cell acute lymphoblastic leukemia (T-ALL). LEF-1 was aberrantly upregulated in premalignant Tcf7(-/-) early thymocytes and lymphoma cells. We further demonstrated that TCF-1 directly repressed LEF-1 expression in early thymocytes and that conditional inactivation of Lef1 greatly delayed or prevented T cell malignancy in Tcf7(-/-) mice. In human T-ALLs, an early thymic progenitor (ETP) subtype was associated with diminished TCF7 expression, and two of the ETP-ALL cases harbored TCF7 gene deletions. We also showed that TCF-1 and LEF-1 were dispensable for T cell lineage commitment but instead were required for early thymocytes to mature beyond the CD4(-)CD8(-) stage. TCF-1 thus has dual roles, i.e., acting cooperatively with LEF-1 to promote thymocyte maturation while restraining LEF-1 expression to prevent malignant transformation of developing thymocytes.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Tcf7−/− mice develop T-cell lymphoblastic lymphomas

(A) Kaplan-Meier survival curve of Tcf7−/− mice. A cohort of Tcf7−/− mice (n = 24) along with WT and Tcf7+/− littermates (each >10) were monitored for 40 weeks. None of the control mice developed lymphomas (not depicted). (B) Histological analysis of thymi and spleens from WT or Tcf7−/− mice that developed lymphomas. Representative images are shown (black bar = 100 μm). All Tcf7−/− mice with lymphomas exhibited similar diffuse infiltration and effacement of normal tissue structure by neoplastic cells. Insets in right panels show neoplastic cells on higher magnification (yellow bar = 20 μm). (C and D) Immunophenotypic analysis of Tcf7−/− lymphomas. Lymphoma cells were harvested and surface-stained. Initial gating was on lymphoma blasts based on their increased forward and side scatters, and surface markers were analyzed on the blasts. Representative contour plots for 31 DN lymphomas (C) or 8 DP lymphomas (D) are shown. Marked in red on top of the contour plots is the frequency of each immunophenotype observed.

Figure 2. Aberrant upregulation of Id2 and LEF-1 in Tcf7−/− thymocytes and T-cell lymphomas

(A) Assessment of TCF-1-repressed genes in Tcf7−/− lymphomas by GSEA. The gene set of TCF-1-repressed genes was analyzed by GSEA against rank-ordered data set of Tcf7−/− lymphomas vs. WT control (Ctrl) and Tcf7−/− DN3 cells. The enrichment plot is shown, with genes at the leading edge highlighted in a red rectangle. (B) Heatmap of the 63 genes in the leading edge as in (A). Id2, Lef1, and Dtx1 are marked by green arrows. For functional annotation of these genes, see Figure S2A. (C) Validation of increased Id2 and Lef1 transcripts in Tcf7−/− DN3 thymocytes and lymphomas. Data are means ± s.d. (n ≥ 4 for control and Tcf7−/−, and n =12 for lymphomas). *, p<0.05; ***, p<0.001 compared with control DN3 cells. (D) Aberrant expression of Id2 and LEF-1 proteins in Tcf7−/− T-cell lymphomas. Cell lysates were isolated from control DN thymocytes, independent lines of lymphomas from Tcf7−/− or Tcf7−/−Id2−/− mice. The lysates were western-blotted for Id2 and LEF-1 with β-actin as an equal loading control.

Figure 3. Molecular resemblance of Tcf7−/− lymphomas to human ETP-ALLs

(A) and (B) GSEA on downregulated genes in Tcf7−/− lymphomas. The downregulated gene set was run against the gene expression profiles of 12 ETP-ALLs and 40 non-ETP T-ALLs. The enrichment plot is shown in (A), with the red rectangle marking negatively enriched genes in ETP-ALLs. (B) shows heatmap of the top 10 most differentially regulated genes between ETP-ALLs and non-ETP T-ALLs. (C) Identification of monoallelic TCF7 deletion in ETP-ALL cases. Genomic DNA from 15 ETP-ALL samples was hybridized to the Affymetrix SNP6 GeneChip Human Mapping Arrays. The signal strength was manually curated for the TCF7 and flanking gene loci on chromosome 5q and shown in the heatmap. The color-coded relative signal strength is displayed below the heatmap. The TCF7 locus was highlighted in green rectangle. The green vertical arrows mark the 2 ETP-ALL cases that harbor monoallelic loss of TCF7. The SNP6 microarray data were deposited in the dbGaP database, accession number phs000340.v1.p1.

Figure 4. TCF-1-mediated repression of Id2 and LEF-1 in early thymocytes

(A) Inhibition of GSK-3β repressed Id2 and LEF-1 expression. Sorted DN3 thymocytes were treated with MetBIO or BIO for 6 hrs, and indicated transcripts were quantitatively determined. (B) Activation of β-catenin repressed Id2 and LEF-1. Lin− DN thymocytes were cultured on monolayer of OP9-DL1 cells overnight and then transduced with retrovirus expressing GFP only (pMIG), WT or mutant form of β-catenin. After 24 hrs, GFP+DN3 thymocytes were sorted and measured for expression of indicated genes. For (A) and (B), data are means ± s.d. from 3 independent experiments. **, p<0.01; ***, p<0.001. (C) Schematic showing conserved TCF-1 binding motifs in the Lef1 locus. Shown on the top is the cross-species conservation of the Lef1 regulatory sequences at −10 to +5 kb region based on the UCSC genome browser. Conserved TCF-1 binding motifs and their relative locations are marked. Also see Figure S4C for sequence alignments at the −4.4 kb cluster (TBC). (D) Direct binding of TCF-1 to the Lef1 locus. ChIP was performed on DN3 thymocytes, and all conserved motifs in (C) were assessed for enriched TCF-1 binding. The Axin2 T2-T3 regulatory region was used as a positive control, and the Gapdh gene body as a negative control. Data are pooled results from 2 independent experiments with each motif measured in triplicates. (E) Schematics showing the retroviral reporter constructs. The retrovirus is self-inactivating due to mutations in its long terminal repeats (SIN). PGK, phosphoglycerate kinase promoter. LefPro, murine Lef1 promoter. Arrows denote the transcription initiation sites and orientations. (F) The TCF-1 binding cluster confers LEF-1 repression. Lin− DN thymocytes were infected with the reporter retroviruses as in (B). The expression of Thy1.1 reporter, in terms of frequency and MFI, was determined on GFP+ DN3 thymocytes. Similar results were obtained for other DN subsets (not shown). Gating of Thy1.1+ cells was based on background staining in corresponding GFP− subset. Data are representative of 2 independent experiments with similar results (n = 4).

Figure 5. Elimination of Id2 or LEF-1 delayed or prevented malignant transformation of Tcf7−/− thymocytes

(A) Elimination of Id2 delayed onset of lymphomas in Tcf7−/− mice. Shown are Kaplan-Meier survival curves for Tcf7−/−Id2−/− and Tcf7−/− littermates. (B) Effective elimination of LEF-1 protein using Vav1-Cre. Cell lysates from bone marrow (BM) cells and thymocytes (Thy) of Lef1−/− or control mice were western-blotted for LEF-1 expression. (C) Elimination of Lef1 transcripts in DN3 thymocytes. Lef1 transcripts were measured by quantitative RT-PCR in sorted DN3 thymocytes and normalized to Hprt1. N.D., not detectable. Similar results were obtained for DN4 cells (not shown). (D) Elimination of Lef1 transcripts in HSCs. Bone marrow cells were harvested and sorted for lineage-negative Sca1+c-Kit+Flt3− subset, which contains both long-term and short-term HSCs, followed by measurements of Lef1 transcripts as in (C). Data in (C) and (D) are means ± s.d. (n = 4 from 2 independent experiments). (E) Total thymic cellularity. Data are means ± s.d. (n = 5). (F) DN3 thymocyte numbers. Data are means ± s.d. (n ≥ 7). ***, pTcf7−/− mice from T-cell lymphomas. Shown are Kaplan-Meier survival curves for Lef1−/−Tcf7−/− and Tcf7−/− littermate controls.

Figure 6. Notch1 in Tcf7−/− thymocytes and T-cell lymphomas

(A) Notch signaling was moderately enhanced in Tcf7−/− DN3 thymocytes but was not sustained after transformation. Sorted DN3 thymocytes were measured for the expression of Notch1 and its target genes, in direct comparison with Tcf7−/− lymphomas. Data are means ± s.d. from 3 independent experiments (n = 4~8). (B) Inhibition of γ-secretase did not repress Lef1 expression in DN3 thymocytes. Lin− DN thymocytes were cultured on OP9-DL1 stromal cells in the presence of DMSO or GSI for 24 hrs. DN3 thymocytes were sorted and measured for indicated transcripts. Data are means ± s.d. from 2 independent experiments (n = 4). (C) Forced expression of ICN or DN-MAML did not detectably affect Lef1 expression. Lin− DN thymocytes were infected with pMIG retrovirus or that expressing either ICN or DN-MAML. The GFP+ DN3 cells were sorted 24 hours post-infection and measured for indicated transcripts. Data are means ± s.d. from 3 independent experiments (n = 6). For (A)–(C), *, p<0.05; ***, p<0.001; N.S., not statistically significant. (D) Cleaved Notch1 was expressed in Tcf7−/− but not Lef1−/−Tcf7−/− T-cell lymphomas. Cell lysates from the lymphomas or total thymocytes were western-blotted to detect cleaved Notch1, with β-actin as a loading control. (E) Notch1 was mutated in the PEST domain in Tcf7−/− T-cell lymphomas. Notch1 cDNA was amplified from thymocytes or lymphomas and sequenced for marked regions. For Notch1, the first nucleotide in its open reading frame was designated as 1, and the coding sequence in exon 34 is within 6151 to 7597. Mutation sites were determined based on this numbering.

Figure 7. A cooperative function between TCF-1 and LEF-1 is required for β-selection at the DN3 stage

(A) TCF-1 and LEF-1 double deficiency arrested T-cell development at the DN stage. Lin− thymocytes were fractioned based on CD4 and CD8 expression. (B) Ablation of LEF-1 did not further exacerbate T-cell developmental blocks caused by TCF-1 deficiency within the DN stage. Lin−CD4−CD8− thymocytes were fractionated based on CD25 and CD44 expression. Data in (A) and (B) are representative of at least 3 independent experiments with similar results. The percentage of each subset is marked. (C) TCF-1 and LEF-1 are required for efficient β-selection. DN3 and DN4 thymocytes were intracellularly stained for TCRβ expression, and the percentage of TCRβ+ population is marked in representative histograms. (D) Cumulative frequency of intracellular TCRβ+ DN3 and DN4 thymocytes. Data are means ± s.d. (n = 3). *, p<0.05; **, p<0.01; and ***, p<0.001.