Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a γ-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with γ-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.

Direct communication between arteries and veins without intervening capillary beds is the primary pathology of arteriovenous malformations (AVMs). Although Notch signaling is implicated in embryonic arteriovenous (AV) differentiation, its function in the adult mammalian vasculature has not been established due to the embryonic lethality that often occurs in both gain- and loss-of-function mutants. We expressed a constitutively active Notch4, int3, in the adult mouse endothelium by using the tetracycline-repressible system to suppress int3 during embryogenesis. int3 caused profound blood vessel enlargement and AV shunting, which are hallmarks of AVM, and led to lethality within weeks of its expression. Vessel enlargement, a manifestation of AVM, occurred in an apparently tissue-specific fashion; the liver, uterus, and skin were affected. int3-mediated vascular defects were accompanied by arterialization, including ectopic venous expression of ephrinB2, increased smooth muscle cells, and up-regulation of endogenous Notch signaling. Remarkably, the defective vessels and illness were reversed upon repression of int3 expression. Finally, endothelial expression of a constitutively active Notch1 induced similar hepatic vascular lesions. Our results provide gain-of-function evidence that Notch signaling in the adult endothelium is sufficient to render arterial characteristics and lead to AVMs

The acquisition of metastatic ability by tumor cells is considered a late event in the evolution of malignant tumors. We report that untransformed mouse mammary cells that have been engineered to express the inducible oncogenic transgenes MYC and Kras D12 , or polyoma middle T, and introduced into the systemic circulation of a mouse can bypass transformation at the primary site and develop into metastatic pulmonary lesions upon immediate or delayed oncogene induction. Therefore, previously untransformed mammary cells may establish residence in the lung once they have entered the bloodstream and may assume malignant growth upon oncogene activation. Mammary cells lacking oncogenic transgenes displayed a similar capacity for long-term residence in the lungs but did not form ectopic tumors.Metastatic dissemination of cancer cells, the major cause of cancer mortality, is traditionally viewed as a late-stage event (1), although mammary epithelial cells have been shown to disseminate systemically from early neoplastic lesions in transgenic mice and from ductal carcinoma in situ in women (2). There is ample evidence that the ability of fully transformed tumor cells to metastasize depends on the regulation of developmental programs and external environmental cues (3-11), but to what extent the seeding or growth of tumor cells at the ectopic site is dependent on the initiating transforming event(s) is a subject of debate (12). We have developed a system that separates the process of seeding cells in the lung from the process of malignant growth at an ectopic site by using animals engineered to express potent oncogenes in a doxycycline-dependent mammary-specific manner. After intravenous (IV) injection of marked mammary cells that have different genetic potentials [no oncogenes, or oncogenes that will be expressed only after cells have taken up residence in an ectopic site (the lungs)], normal mammary cells can lodge in the lungs, grow slowly, and become frank metastatic malignancies once potent oncogenes are turned on.We recently described tri-transgenic TetO-MYC;TetO-Kras D12 ;MMTV-rtTA (TOM;TOR; MTB) mice that coordinately express MYC and mutant Kras oncogenes in mammary epithelial cells when fed doxycycline (13). Doxycycline-naïve animals do not express the transgenic oncogenes and have morphologically and functionally normal mammary glands, but they develop diffuse autochthonous tumors within 3 to 4 weeks after doxycycline exposure. Tumors that form because of the expression of these oncogenes display malignant characteristics, such as transplantability and metastasis ( fig. S1) To investigate whether mammary cells from these mice can be induced to form metastasis in the absence of transformation at the primary site, we modified the traditional experimental metastasis assay (14). In the modified approach, instead of IV delivery of tumor cells from doxycycline-treated animals into new recipients, we injected dissociated morphologically normal mammary cells from mature TOM;TOR;MTB animals never exposed to doxycy...