NOTCH1 was discovered through analysis of a (7;9) chromosomal translocation found in <1% of T-ALLs,[unreadable] which creates a truncated NOTCH1 gene encoding constitutively active ICN1-like polypeptides. Prior work[unreadable] (from us and others) has shown that constitutively active forms of NOTCH 1 are potent inducers of T cell[unreadable] acute lymphoblastic leukemia/lymphoma (T-ALL) in mice. Recently, we identified frequent gain of function[unreadable] NOTCH1 mutations in human T-ALL cell lines and primary tumors. Two different types of mutations occur:[unreadable] point mutations involving a heterodimerization domain lying just external to the transmembrane domain; and[unreadable] frameshift or stop codon mutations that result in deletion of the cytoplasmic tail of NOTCH 1. These mutations[unreadable] activate NOTCH1 signaling synergistically, probably by increasing both the production and stability of ICN1.[unreadable] Beyond providing important pathogenic insights, these findings have exciting therapeutic implications;[unreadable] because NOTCH1 receptors bearing these mutations depend on the protease gamma-secretase for[unreadable] function, these molecular lesions can be targeted with gamma-secretase inhibitors.[unreadable] Based on these new insights, we propose the following three specific aims:[unreadable] 1. To determine how mutations in the heterodimerization domain of NOTCH1 increase signaling[unreadable] 2. To determine how mutations that delete the C-terminal sequences of NOTCH1 increase signaling[unreadable] 3. To determine the effects of leukemia-associated NOTCH1 mutations on human hematopoietic stem cell[unreadable] development and transformation[unreadable] Molecular models for how NOTCH1 mutations produce increases in downstream signaling will be tested,[unreadable] initially in cell-based assays and subsequently in a robust murine leukemia model. Because human cells[unreadable] may have different requirements for transformation than murine cells, we will explore the effects of various[unreadable] mutated forms of NOTCH1 on primary human hematopoietic progenitors. Together, these studies will yield[unreadable] important new insights into how NOTCH1 contributes to human acute leukemias. Because the NOTCH[unreadable] pathway is a tractable therapeutic target, these insights are likely to yield new therapeutic opportunities in[unreadable] acute leukemia and other cancers associated with abnormalities in NOTCH signaling.