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...

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.

In Linux, real‐time tasks are supported by separating real‐time task priorities from non‐real‐time task priorities. However, this separation of priority ranges may not be effective when real‐time tasks make the system calls that are taken care of by the kernel threads. Thus, Linux is considered a soft real‐time system. Moreover, kernel threads are configured to have static priorities for throughputs. The static assignment of priorities to kernel threads causes trouble for real‐time tasks when real‐time tasks require kernel threads to be invoked to handle the system calls because kernel threads do not discriminate between real‐time and non‐real‐time tasks. We present a dynamic kernel thread scheduling mechanism with weighted average priority inheritance protocol (PIP), a variation of the PIP. The scheduling algorithm assigns proper priorities to kernel threads at runtime by monitoring the activities of user‐level real‐time tasks. Experimental results show that the algorithms can greatly improve the unexpected execution latency of real‐time tasks.