Gain-of-function NOTCH1 mutations are found in 50%-70% of human T cell acute lymphoblastic leukemia/ lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show "addiction" to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.

NOTCH2 mutations in splenic marginal zone lymphoma are associated with poor prognosis.

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo β-selection, during which cells expressing functionally rearranged TCRβ proliferate and differentiate into CD4+CD8+ progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-β-selected thymocytes. Following successful β-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-β-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.

Point mutations that trigger ligand-independent proteolysis of the Notch1 ectodomain occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL) but are rare in murine T-ALL, suggesting that other mechanisms account for Notch1 activation in murine tumors. Here we show that most murine T-ALLs harbor Notch1 deletions that fall into 2 types, both leading to ligand-independent Notch1 activation. Type 1 deletions remove exon 1 and the proximal promoter, appear to be RAG-mediated, and are associated with mRNA transcripts that initiate from 3′ regions of Notch1. In line with the RAG dependency of these rearrangements, RAG2 binds to the 5′ end of Notch1 in normal thymocytes near the deletion breakpoints. Type 2 deletions remove sequences between exon 1 and exons 26 to 28 of Notch1, appear to be RAG-independent, and are associated with transcripts in which exon 1 is spliced out of frame to 3′ Notch1 exons. Translation of both types of transcripts initiates at a conserved methionine residue, M1727, which lies within the Notch1 transmembrane domain. Polypeptides initiating at M1727 insert into membranes and are subject to constitutive cleavage by γ-secretase. Thus, like human T-ALL, murine T-ALL is often associated with acquired mutations that cause ligand-independent Notch1 activation.