4851

NOTCH1

TAN1|hN1;notch 1;NOTCH1;notch 1

Notch homolog 1, translocation-associated|neurogenic locus notch homolog protein 1|translocation-associated notch protein TAN-1

9q34.3

protein-coding

Notch signaling is of crucial importance in normal T-cell development and Notch 1 is frequently mutated in T-cell acute lymphoblastic leukemias (T-ALL), leading to aberrantly high Notch signaling. In this report, we determine whether T-ALL mutations occur not only in Notch1 but also in the F-box protein hCdc4 (Sel-10, Ago, or Fbxw7), a negative regulator of Notch1. We show that the hCDC4 gene is mutated in leukemic cells from more than 30% of patients with pediatric T-ALL and derived cell lines. Most hCDC4 mutations found were missense substitutions at critical arginine residues (Arg(465), Arg(479), and Arg(505)) localized in the substrate-binding region of hCdc4. Cells inactivated for hCdc4 and T-ALL cells containing hCDC4 mutations exhibited an increased Notch1 protein half-life, consistent with the proposed role of hCdc4 in ubiquitin-dependent proteolysis ofNotch1. Furthermore, restoration of wild-type but not mutant hCdc4 in HCT 116 hCDC4-negative cells led to an increased Notch1 ubiquitylation and decreased Notch1 signaling. These results show that hCdc4 mutations interfere with normal Notch1 regulation in vivo. Finally, we found that mutations in hCDC4 and NOTCH1 can occur in the same cancers and that patients carrying hCDC4 and/or NOTCH1 mutations have a favorable overall survival. Collectively, these data show that mutation of hCDC4 is a frequent event in T-ALL and suggest that hCDC4 mutations and gain-of-function mutations in NOTCH1 might synergize in contributing to the development of pediatric T-ALL leukemogenesis.

PURPOSE: The molecular pathogenesis of pediatric ependymoma remains unclear. Ourstudy was designed to identify genetic changes implicated in ependymoma progression. PATIENTS AND METHODS: We characterized 59 ependymoma samples (33 atdiagnosis and 26 at relapse) using array-comparative genomic hybridization (aCGH). Specific chromosomal imbalances were confirmed by fluorescent in situ hybridization, and candidate genes were assessed by real-time quantitative polymerase chain reaction (qPCR), immunohistochemistry, sequencing, and in vitrofunctional studies. RESULTS: aCGH analysis revealed a significant increase in genomic imbalances on relapse compared with diagnosis, such as gain of 9qter and1q (54% v 21% and 12% v 0%, respectively) and loss of 6q (27% v 6%). Supervised tumor classification showed that gain of 9qter was associated with tumor recurrence, age older than 3 years, and posterior fossa location. Using a candidate-gene strategy, we found an overexpression of two potential oncogenes at the locus 9qter: Tenascin-C and Notch1. Moreover, Notch pathway analysis (qPCR) revealed overexpression of Notch ligands, receptors, and target genes (Hes-1, Hey2, and c-Myc), and downregulation of Notch repressor Fbxw7. We confirmed by immunohistochemistry the overexpression of Tenascin-C and Hes-1. We detected Notch1 missense mutations in 8.3% of the tumors (only in the posterior fossa location and in case of 9q33-34 gain). Furthermore, inhibition of Notch pathway with a gamma-secretase inhibitor impaired the growth of ependymoma stem cell cultures. CONCLUSION: The activation of the Notch pathway and Tenascin-C seem tobe important events in ependymoma progression and may represent future targets for therapy. We report, to our knowledge for the first time, recurrent oncogenicmutations in pediatric posterior fossa ependymomas.