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. 2008 Nov 24;205(12):2851-61.
doi: 10.1084/jem.20081561. Epub 2008 Nov 3.

Oncogenesis of T-ALL and nonmalignant consequences of overexpressing intracellular NOTCH1

Xiaoyu Li  1 , Fotini Gounari, Alexei Protopopov, Khashayarsha Khazaie, Harald von Boehmer
Affiliations

Oncogenesis of T-ALL and nonmalignant consequences of overexpressing intracellular NOTCH1

Xiaoyu Li et al. J Exp Med. .

Abstract

Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL). We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells. Early consequences are the generation of polyclonal nontumorigenic CD4(+)8(+) T cell receptor (TCR)-alphabeta(+) cells that do not qualify as tumor precursors despite the observation that they overexpress Notch 1 and c-Myc and degrade the tumor suppressor E2A by posttranslational modification. The first tumorigenic cells are detected among more immature CD4(-)8(+)TCR-alphabeta(-) cells that give rise to monoclonal tumors with a single, unique TCR-beta chain and diverse TCR-alpha chains, pinpointing malignant transformation to a stage after pre-TCR signaling and before completion of TCR-alpha rearrangement. In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level. Even though the tumors consist of phenotypically heterogeneous cells, no evidence for tumor stem cells was found. As judged by array-based comparative genomic hybridization (array CGH) and spectral karyotype (SKY) analysis, none of the tumors arise because of genomic instability.

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Figures

Figure 1.
Figure 1.
Phenotypic analysis of GFP+ cells in various tissues after transplantation of ICN1-overexpressing BM cells. (a) Single-cell suspensions were prepared from blood and BM at 2 wk and from blood, lung, and liver at 8 wk after reconstitution with retrovirally transduced BM. GFP+ and GFP− cells were stained with CD4 and CD8 antibodies. Infiltration by GFP+CD4+8+ and GFP+CD4−8+ malignant cells was analyzed in nonlymphoid organs such as lung and liver. Numbers indicate the percentage of cells within each quadrant. Data are representative of three independent experiments. (b) BrdU–labeling kinetics: continuous (top) and pulse (bottom) BrdU labeling of lymphocytes subsets. BrdU was administered to control mice and mice at 2 or 8 wk after BM transplantation with ICN1-overexpressing cells. The percentage of BrdU-labeled cells was analyzed by flow cytometry as previously described (13). (c) CFSE labeling: normal CD4+8+ thymocytes and ICN1-overexpressing CD4+8+ splenocytes were labeled with CFSE and injected into nu/nu mice. Dilution of CFSE label was analyzed by FACS analysis. (d) BrdU–labeling kinetics: continuous (top) and pulse (bottom) BrdU labeling of ICN1-overexpressing TCR-αβ− CD4−8+ splenocytes versus control normal CD4−8+ thymocytes. (e) TCR-β expression by various lymphocyte subsets in ICN1-transplanted mice at 2 wk after BMT, as well as control mice. TCR-β staining was performed with the pan–TCR-β antibody H57.
Figure 2.
Figure 2.
TCR expression by malignant and nonmalignant cells. (a) TCR-β rearrangement in various cell types. Analysis of TCR-β rearrangement by Southern blot analysis in GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) thymocytes derived from retroviral GFP-empty vector–transplanted mice, as well as GFP+CD4+8+(GFP+/DP) and GFP+CD4−8+(GFP+/CD8+) splenocytes derived from retroviral GFP-ICN1–transplanted mice at 2 and 8 wk after BMT. Molecular markers are shown on the left. (b) TCR-β and TCRV-α2 expression by various lymphocyte subsets. (left) Staining with the pan TCR-β antibody H57. (right) Staining with Vα2 TCR antibody.
Figure 3.
Figure 3.
Kinetic analysis of tumor development. Various cell numbers of ICN1-overexpressing cells with a distinct surface phenotype (as indicated) were injected into nu/nu mice and the kinetics of tumor development were monitored.
Figure 4.
Figure 4.
Overexpressed ICN1 inhibits E2A activity by promoting protein degradation at different stages of disease development. (a) E2A E-box motif binding activities were analyzed by electrophoretic mobility shift assay (EMSA) using nuclear extracts of GFP+CD4+8+ (GFP+/DP) cells derived from various mice. (b) E2A and phosphorylated ERK1/2 protein expression levels were analyzed by Western blot in whole-cell lysates from various cells. (c) Quantitative analysis of E47 mRNA expression in various cell types. GAPDH mRNA was used as control. **, P < 0.01, compared with E2A+/− group. Data are reported as mean ± SEM for triplicate experiments. (d) Ubiquitination assay: whole-cell lysates were immunoprecipitated by E2A antibody. Immunoprecipitates were blotted with antiubiquitin antibody. (e) ICN1-induced degradation and phosphorylation of E2A by ERK1/2 MAP kinases. Whole-cell lysates were from various cells that have incubated in the absence or presence of 50 μM PD98059 for 90 min; PD98059 is a specific inhibitor of MEK1 and MEK2. Western blot analysis was performed to evaluate E2A protein expression levels. β-Actin is shown as loading control.
Figure 5.
Figure 5.
Nondegradable mutant E47 is overexpressed in ICN1-DsRed tumor cells. Western blot analysis of mutant E47 protein expression in whole-cell lysates from ICN1 tumor cells transduced with the empty retroviral vector MSCV-IRES-GFP or retroviral construct MSCV-IRES-GFP-MmE47. β-Actin is shown as loading sample.
Figure 6.
Figure 6.
Essential role of ICN1 and c-Myc, but not E47, down-regulation in tumor growth. Tumor development and growth dependent on c-Myc and ICN1, respectively. The empty GFP-IRES retroviral vector (a), the GFP-IRES retroviral vector containing mutant E47 (b), or the GFP-IRES retroviral vector containing DNMAML1 (c) were introduced into tumors expressing a DsRed-ICN1 retrovirus. Double-fluorescent tumor cells were sorted and 106 cells were injected into nu/nu recipient mice. Tumor cells were analyzed when mice had developed palpable tumors. Nude mice injected with tumors containing empty retroviral vectors died of lymphoma 3 wk of after transfer. Mice injected with cells containing MmE47 or DN-MAML1 developed tumors 7 wk after injection. Double-fluorescent cells did not form tumors in c. (d) Analysis of GFP+ and GFP− cells in blood of Rag2−/−, γc−/− mice that were γ irradiated and reconstituted with retrovirally transduced BM cells from either floxed c-Myc, CD4-cre, or CD4-cre mice. CD4-cre mice were killed at 8.5 wk when they exhibited large lymphoblastic tumors. In contrast, CD4-cre c-MycFL/FL mice developed no tumors up to 15 wk.
Figure 7.
Figure 7.
Specific gene expression. (a) NF-κB DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA) using nuclear extracts of GFP+/CD4+8+ DP cells derived from various mice. (b) c-Myc, cyclin D1, and cyclin D3 protein levels in various cells were analyzed by Western blot in whole-cell lysates. β-Actin is shown as loading control. (c) Total RNA isolated from various cells was analyzed by quantitative real-time PCR using specific primers for c-Myc. GAPDH mRNA was used as control. *, P < 0.05; **, P < 0.01. Data are shown as mean ± SEM for triplicate experiments. (d) The c-Myc-p53 axis: Western blot analysis in whole-cell lysates from various cells was performed. β-Actin is shown as loading sample. (e) Quantitative analysis of p19Arf, Mdm2, p53, and Bmi-1 mRNA expression in various cells. GAPDH mRNA was used as control. **, P < 0.01; ***, P < 0.001. Data are shown as mean ± SEM for triplicate experiments. N, CTRL GFP+/DP thymus; NT, ICN1, GFP+/DP spleen 2 wk; TU, ICN1 GFP+/DP spleen 8 wk.
Figure 8.
Figure 8.
Genomic analysis. (left) SKY analysis of ICN1-induced tumor cells. SKY table of stained ICN1-induced tumor chromosomes using SKY paint shows the spectral, classified, and banded-inverted chromosomes. (right) Array CGH. Genomic DNAs extracted from ICN1-induced malignant tumor GFP+CD4+8+ (Tumor XLT7), abnormal nonmalignant ICN1-overexpressing cells GFP+CD4+8+, and control GFP+CD4+8+ thymocytes were subjected to high-resolution oligo array–based comparative genomic hybridization. Tail DNA from the same mouse was used as reference for the hybridization. Malignant tumor GFP+CD4+8+ cells from four independent tumor mice were analyzed by Array CGH.
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