Very rare cases of human T cell acute lymphoblastic leukemia (T-ALL) harbor chromosomal translocations that involve NOTCH1, a gene encoding a transmembrane receptor that regulates normal T cell development. Here, we report that more than 50% of human T-ALLs, including tumors from all major molecular oncogenic subtypes, have activating mutations that involve the extracellular heterodimerization domain and/or the C-terminal PEST domain of NOTCH1. These findings greatly expand the role of activated NOTCH1 in the molecular pathogenesis of human T-ALL and provide a strong rationale for targeted therapies that interfere with NOTCH signaling.T-ALL is an aggressive cancer that preferentially affects children and adolescents. It is commonly associated with acquired chromososomal translocations and other genetic or epigenetic abnormalities, which lead to aberrant expression of a select group of transcription factors (1). NOTCH1 was discovered as a partner gene in a (7;9) chromosomal translocation found in G1% of T-ALLs (2). It encodes a transmembrane receptor that is required for the commitment of pluripotent progenitors to T cell fate (3) and the subsequent assembly of pre-T cell receptor complexes in immature thymocytes (4).Cleavage of pro-NOTCH1 by a furinlike protease during transit to the cell surface (5) produces a NOTCH1 heterodimer comprised of noncovalently associated extracellular (NEC) and transmembrane (NTM) subunits (6). The heterodimerization domain (HD) responsible for stable subunit association consists of a 103 amino acid region of NEC (HD-N) and a 65 amino acid region in NTM (HD-C) (7). Physiologic activation of NOTCH receptors occurs when ligands of the Delta-SerrateLag2 (DSL) family bind to the NEC subunit and initiate a cascade of proteolytic cleavages in the NTM subunit. The final cleavage, catalyzed by ,-secretase (8, 9), generates intracellular NOTCH (ICN), which translocates to the nucleus and forms a large transcriptional activation complex that includes proteins of the Mastermind family (10-12).Prior work has shown that enforced NOTCH1 signaling is a potent inducer of T-ALL in the mouse (13-15) and is required to sustain the growth of a human t(7;9)-positive T-ALL cell line (16). To investigate the possibility of a more general role for NOTCH signaling in human T-ALL, we tested T-ALL cell lines lacking the t(7;9) for NOTCH dependency by treating these cells with a ,-secretase inhibitor (17). Of 30 human T-ALL cell lines tested, 5 showed a G 0 /G 1 cell-cycle arrest that equaled or exceeded that of T6E, a reference NOTCH1-dependent murine T-ALL cell line (Fig. 1A). This drug-induced growth suppression was abrogated by retroviral expression of ICN1 (Fig. 1B) and reproduced ( fig. S1) by retroviral expression of dominant negative Mastermindlike-1 (16). These results indicated that the growth of these five cell lines depends on NOTCHtransduced signals.Because physical dissociation of the NOTCH extracellular domain has been linked to receptor activation (6, 18), we reasoned that the HD domain of NOTC...

Barrier structures (e.g. epithelia around tissues, plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometers within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Due to their fast diffusion and versatile biological activities, reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. We probed the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor1. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100−200 μm into the tail fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by Dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.