Gain-of-Function NOTCH1 Mutations Occur Frequently in Human T Cell Acute Lymphoblastic Leukemia.

Abstract

NOTCH1 was discovered originally through its involvement in a rare (7;9) translocation found in human T cell acute lymphoblastic leukemia (T-ALL). Here, we report that >50% of human T-ALLs have activating NOTCH1 mutations, occurring as amino acid substitutions in an extracellular heterodimerization (HD) domain and/or as frameshift and stop codon mutations that result in the deletion of a C-terminal PEST destruction box. Normal pro-NOTCH1 is processed into a heterodimer consisting of an extracellular subunit and a transmembrane subunit, which associate non-covalently through the HD domain. NOTCH1 activation is triggered by binding of Serrate or Delta-like ligands to the extracellular subunit, which induces successive proteolytic cleavages in the transmembrane subunit that are dependent on i) metalloproteases and ii) gamma-secretase. The γ-secretase cleavage releases intracellular NOTCH1 (ICN1), which translocates to the nucleus and forms a transcriptional activation complex with the transcription factor CSL and co-activators of the Mastermind family. Normal turnover of ICN1 is regulated by the C-terminal PEST sequence. Data pointing to the existence of frequent abnormalities of NOTCH1 in T-ALL stemmed from a functional screen of 30 T-ALL cell lines. This identified five T-ALL cell lines that underwent growth arrest in response to i) treatment with an inhibitor γ-secretase, and ii) retroviral transduction of dominant negative Mastermind-like-1. Sequencing of of cDNAs from 4 of these 5 cell lines demonstrated both a missense mutation in the HD domain and a frameshift mutation in the PEST domain lying in cis in the same NOTCH1 allele. Subsequent sequencing of genomic DNA obtained from bone marrow lymphoblasts of 96 children and adolescents with T-ALL demonstrated identical or similar mutations in NOTCH1 in 53 samples (55.2%). Mutations in the HD domain alone were observed in 26 cases (27.1%), in the PEST domain alone in 11 cases (11.4%), and in both the HD and PEST domains in 16 cases (16.7%). Mutations were observed in tumors associated with expression of HOX11 (2/3), HOX11L2 (10/13; 77%), TAL1 (12/31; 39%), LYL1 (9/14; 64%), MLL-ENL (1/3) or CALM-AF10 (1/2), which span the major molecular T-ALL subtypes. In contrast, NOTCH1 mutations were not observed in genomic DNAs samples obtained from B-ALL lymphoblasts (N=89), or from T-ALL patients with NOTCH1-associated disease at the time of clinical remission (N=4). Reporter gene assays conducted with plasmids expressing normal and mutated forms of NOTCH1 showed that a PEST deletion or various HD mutations alone caused ~1.5-fold and 3–9-fold stimulations of reporter gene activity, respectively, whereas normal NOTCH1 lacked intrinsic signaling activity. More strikingly, the combination of various HD mutations and a PEST deletion in cis caused synergistic 20–40-fold stimulations of reporter gene activity that were completely abrogated by a γ-secretase inhibitor, indicating that signaling depends on proteolysis. These results suggest a model in which HD domain mutations promote ICN1 production, and PEST domain mutations enhance ICN1 stability. Our findings greatly expand the role of NOTCH1 in the pathogenesis of human T-ALL, and provide a rationale for targeted therapies that interfere with NOTCH signaling.

Notch Antagonists: Potential Modulators of Cancer and Inflammatory Diseases

“…Following encouraging results obtained using human cancer cell lines in vitro together with xenograft experimental models,[118][119][120][121] ongoing clinical trials are now investigating the safety and potential efficacy of GSIs, alone or in combination with other drugs, in various types and stages of cancer or inflammation. During the development and evaluation of GSIs for AD, undesirable side-effects were observed which were attributed to the poor selectivity of these GSIs and their interference with the processing of AD-unrelated protein substrates by γ-secretase, in particular the components of the Notch signaling pathway.…”

The eye of Drosophila as a model system for studying intracellular signaling in ontogenesis and pathogenesis

“…This translocation, as it turned out, leads to formation of a truncated Notch1 gene, expression of which leads to constitutive, ligand independent activation of the Notch cascade [35]. Subsequent studies have revealed frequent point mutations in the gene for Notch1, especially in areas that encode its heterodimerization domain and PEST domain, which controls degradation of NICD [36]. Activating mutations in the Notch1 heterodimeriza tion domain stimulate ligand independent S2 receptor cleavage, which is accompanied by S3 cleavage and acti vation of Notch, whereas mutations in the PEST domain improve the stability of the liberated through the S3 cleavage intracellular Notch (NICD) domain [22,37].…”

Chemische Kontrolle des Schicksals und Entwicklungspotenzials von Stammzellen

“…Eine Eigenschaft, die CSCs und normale Stammzellen gemeinsam haben, ist die Fähigkeit zur Selbsterneuerung. [252] Für Signalwege wie PI3K/Akt, [74] Wnt/b-Catenin, [253] Notch [254] und Hh [255] wurde festgestellt, dass sie in humanen Tumoren gegenüber normalen Stammzellen verändert waren. [256] Mehr und mehr Untersuchungen deuten darauf hin, dass diese Signalwege in normalen Zellen anders ablaufen als in Krebszellen und dass man dieses unterschiedliche Verhalten nutzen kann.…”