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. 2009 May;100(5):920-6.
doi: 10.1111/j.1349-7006.2009.01130.x.

Gain-of-function mutations and copy number increases of Notch2 in diffuse large B-cell lymphoma

Suk-young Lee  1 , Keiki Kumano, Kumi Nakazaki, Masashi Sanada, Akihiko Matsumoto, Go Yamamoto, Yasuhito Nannya, Ritsuro Suzuki, Satoshi Ota, Yasunori Ota, Koji Izutsu, Mamiko Sakata-Yanagimoto, Akira Hangaishi, Hideo Yagita, Masashi Fukayama, Masao Seto, Mineo Kurokawa, Seishi Ogawa, Shigeru Chiba
Affiliations

Gain-of-function mutations and copy number increases of Notch2 in diffuse large B-cell lymphoma

Suk-young Lee et al. Cancer Sci. 2009 May.

Abstract

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T-cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T-cell acute lymphoblastic leukemias. Notch2, another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B-cell generation. Here, we report that 5 of 63 (approximately 8%) diffuse large B-cell lymphomas, a subtype of mature B-cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain, or a single amino acid substitution at the C-terminus of Notch2 protein. Furthermore, high-density oligonucleotide microarray analysis revealed that some diffuse large B-cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation-sensitive luciferase reporter assay in vitro, mutant Notch2 receptors show increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers.

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Figures

Figure 1
Figure 1
Mutations of the Notch2 gene in diffuse large B‐cell lymphomas. Polymerase chain reaction–single‐stranded conformational polymorphism and sequence analyses for samples having the nonsense mutation at 7454, C/T (W109539, W121672 and L8). Arrowheads indicate shifted bands. The shifted bands in (a) and (c) are obviously dominant against the normal band, suggesting the small amount of normal tissue contamination and unbalanced ratio of mutant and normal alleles. Those in (b) and (e) are minor compared with the normal band, suggesting the contamination of normal tissues, and those in (c) are comparable with the normal band. The shifted bands were excised from the gel and the extracted DNA was sequenced for samples W121672 and W117336. (f) Sequence of DNA prepared from the bone marrow cells obtained from the patient W109539.
Figure 2
Figure 2
Immunohistochemical staining of lymphoma specimens for CD10, BCL6, and MUM‐1. Antibodies used were anti‐CD10 monoclonal antibody (mAb) (56C6; Novocastra, Norwell, MA, USA), anti‐BCL6 mAb (P1F6; Novocastra), and antihuman MUM‐1 mAb (MUM1p; Dako, Glostrup, Denmark). The detection of antibody binding was visualized by the avidin–biotin complex method using diaminobenzidine as the chromogen. An Elipse 80i microscope was used (Nikon, Tokyo, Japan); original magnification, × 200. Camera, Dxm1200F (Nikon). Acquisition software, Act‐1 (Nikon).
Figure 3
Figure 3
Copy number increases of mutated Notch2 allele in diffuse large B‐cell lymphomas. (a) High‐density oligonucleotide microarray analysis using the CNAG program (CREST, Japan Science and Technology Agency, Tokyo, Japan) for samples W109539 and W121672. The copy number of the Notch2‐encompassing allele is greatly increased in W109539 and mildly increased in W121672. Red arrow, centromere. hetero, heterozygous; SNP, single nucleotide polymorphism. (b) Fluorescence in situ hybridization analysis for sample W109539 using probes corresponding to Notch2 (green signals) and a reference sequence on 1q23.3 (red signals). (c) Copy number evaluation of the Notch2 gene by quantitative real‐time polymerase chain reaction for samples L8 and W121672. The quantity of genomic DNA, extracted from samples L8 and W121672, MKN45 [a stomach cancer cell line having a copy number loss at the Notch2 (1p13) locus], and normal peripheral blood mononuclear cells (PBMNC), was normalized by real‐time reverse transcription–polymerase chain reaction for the control locus (2q35). Statistical analysis (Student's t‐test) showed that the Notch2 gene dose was unchanged in sample L8, and significantly increased in sample W121782, relative to the Notch2 gene dose in the PBMNC, whose mean level was adjusted to two copies. The number of samples was 24 in each arm. *P < 0.0001; **P = 0.79.
Figure 4
Figure 4
Functional analysis of human full‐length Notch2 cDNA (wtN2), and Notch2 with the nonsense mutation (nsmN2), single‐base deletion mutation (delstN2), or R2453Q mutation (rqN2). (a) Flow cytometric analysis of CHO(r) clones expressing wtN2, nsmN2, delstN2, and rqN2 at similar expression levels. Each clone (cl1 and cl2 represented by green and red lines, respectively) of wtN2/CHO(r), nsmN2/CHO(r), delstN2/CHO(r), and rqN2/CHO(r) was analyzed by flow cytometry using the antihuman Notch2 antibody MHN2‐25. Purple curves represent isotype control. (b) Western blot analysis of CHO(r) clones expressing wtN2, nsmN2, delstN2, and rqN2 using an antibody recognizing the intracellular domain of Notch2. Asterisks indicate the transmembrane species of each Notch2 protein. MW, molecular weight. (c) Reporter gene transactivation by wtN2, nsmN2, delstN2, and rqN2. Each clone (cl1 and cl2) was cultured in a dish coated with human Delta1‐Fc (D1‐Fc) or control IgG. Data are means of quadricate experiments. Error bars represent standard deviations. A representative experiment from repeated experiments is shown. sRAU, relative arbitrary units standardized by β‐galctosidase activity. (d) Inhibition of luciferase activity by N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor. Bulk CHO(r) cells transfected with wtN2 or nsmN2 were stimulated with D1‐Fc or control IgG with graded concentrations of DAPT. RAU, relative arbitrary units.
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