Murine Pten(-/-) T-ALL requires non-redundant PI3K/mTOR and DLL4/Notch1 signals for maintenance and γc/TCR signals for thymic exit.

T cell acute lymphoblastic leukemias (T-ALLs) commonly display constitutively active PI3K/mTOR and Notch signaling. However, controversy surrounds whether these pathways have independent functions and whether Pten loss is sufficient to generate resistance to Notch inhibition. Here we report that Pten(-/-) T-ALL is sensitive to either PI3K/mTOR or Notch inhibition alone, each pathway controlling distinct downstream signaling events that cannot be rescued by activation of the other pathway, consistent with independent, non-redundant functions.

Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2.

Background: Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.

Structural basis for autoinhibition of Notch.

Notch receptors transmit signals between adjacent cells. Signaling is initiated when ligand binding induces metalloprotease cleavage of Notch within an extracellular negative regulatory region (NRR). We present here the X-ray structure of the human NOTCH2 NRR, which adopts an autoinhibited conformation.

Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes.

The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability.

Notch subunit heterodimerization and prevention of ligand-independent proteolytic activation depend, respectively, on a novel domain and the LNR repeats.

Notch proteins are transmembrane receptors that participate in a highly conserved signaling pathway that regulates morphogenesis in metazoans. Newly synthesized Notch receptors are proteolytically cleaved during transit to the cell surface, creating heterodimeric mature receptors comprising noncovalently associated extracellular (N(EC)) and transmembrane (N) subunits. Ligand binding activates Notch by inducing two successive proteolytic cleavages, catalyzed by metalloproteases and gamma-secretase, respectively, that permit the intracellular portion of N to translocate to the nucleus and activate transcription of target genes.

Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia.

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.

Nuclear magnetic resonance structure of a prototype Lin12-Notch repeat module from human Notch1.

Notch1 is a member of a conserved family of large modular heterodimeric type 1 transmembrane receptors that control differentiation in multicellular animals. Receptor maturation is accompanied by a furin-dependent cleavage that converts the Notch1 precursor polypeptide into a heterodimer consisting of an extracellular ligand-binding subunit (NEC) and a transmembrane signaling subunit (NTM). Binding of a physiologic ligand to NEC induces signaling by triggering additional proteolytic cleavages in NTM, which allow its intracellular region to translocate to the nucleus where it participates in a transcriptional activation complex.