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...

Acute lymphoblastic leukemias carrying a chromosomal translocation involving the mixed-lineage leukemia gene (MLL, ALL1, HRX) have a particularly poor prognosis. Here we show that they have a characteristic, highly distinct gene expression profile that is consistent with an early hematopoietic progenitor expressing select multilineage markers and individual HOX genes. Clustering algorithms reveal that lymphoblastic leukemias with MLL translocations can clearly be separated from conventional acute lymphoblastic and acute myelogenous leukemias. We propose that they constitute a distinct disease, denoted here as MLL, and show that the differences in gene expression are robust enough to classify leukemias correctly as MLL, acute lymphoblastic leukemia or acute myelogenous leukemia. Establishing that MLL is a unique entity is critical, as it mandates the examination of selectively expressed genes for urgently needed molecular targets.