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.

Summary KRIT1 (Krev/Rap1 Interaction Trapped-1) mutations are observed in ~40% of autosomal dominant cerebral cavernous malformations (CCM), a disease occurring in up to 0.5% of the population. We show that KRIT1 functions as a switch for β1 integrin activation by antagonizing ICAP1 (Integrin Cytoplasmic Associated Protein-1)-mediated modulation of “inside-out” activation. We present co-crystal structures of KRIT1 with ICAP1 and ICAP1 with integrin β1 cytoplasmic tail to 2.54 Å and 3.0 Å resolution (the resolutions at which I/σI = 2 are 2.75 Å and 3.0 Å, respectively). We find that KRIT1 binds ICAP1 by a bidentate surface, KRIT1 directly competes with integrin β1 to bind ICAP1, and that KRIT1 antagonizes ICAP1-modulated integrin activation using this site. We also find that KRIT1 contains an N-terminal Nudix domain, in a region previously designated as unstructured. We therefore provide new insights to integrin regulation and CCM-associated KRIT1 function.

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with ␥-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-endstage Tal1/Ink4a/Arf ϩ/Ϫ leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSItreated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is associated with the misexpression of the basic helix-loop-helix protein TAL1/SCL and LIM-domain only proteins LMO1 and LMO2. [1][2][3][4][5][6][7][8] These oncogenes are found misexpressed in greater than 60% of human T-ALL patients. 3,9 Mouse models of T-ALL recapitulate the disease through ectopic expression of Tal1 in the thymus. [10][11][12] These mice develop respiratory distress due to the presence of large thymic masses and have detectable T-cell blasts in peripheral blood lymphocytes (PBLs), spleen, liver, and kidney. 10 Misexpression of Tal1 results in perturbed thymocyte development by interfering with the basic-helix-loop-helix (bHLH) heterodimer E47/HEB that regulates the expression of genes critical for thymocyte differentiation including Rag1/2, CD4, CD3, and TCR␣/␤. 13,14 Consistent with this finding, loss of the E2A gene that encodes the E47 and E12 bHLH protein is associated with human B-progenitor ALL. 15 Mutations in the Notch1 receptor have been frequently detected in mouse T-ALL models [16][17][18][19] and importantly in 54% of T-ALL patients. 20 These mutations cluster in the heterodimerization domain (HD) 20 and the juxtamembrane (JME) region, 21 or result in truncation of PEST regulatory sequences. 18,20 Mutations in the HD domain result in increased susceptibility to cleavage by the gamma-secretase complex; JME mutations may facilitate metalloprotease cleavage, whereas deletion of PEST regulatory sequences is thought to result in increased Notch1 stability. [21][22][23] Treatment of mouse Tal1 leukemic cell lines in vitro with ␥-secretase inhibitors (GSIs) results in cell cycle arrest and apoptosis, revealing that Notch1 signaling is required for leukemic growth/survival. 18Notch1-mediated leukemic growth in mouse and ...

Loss-of-function mutations in genes encoding KRIT1 (also known as CCM1), CCM2 (also known as OSM and malcavernin) or PDCD10 (also known as CCM3) cause cerebral cavernous malformations (CCMs). These abnormalities are characterized by dilated leaky blood vessels, especially in the neurovasculature, that result in increased risk of stroke, focal neurological defects and seizures. The three CCM proteins can exist in a trimeric complex, and each of these essential multi-domain adaptor proteins also interacts with a range of signaling, cytoskeletal and adaptor proteins, presumably accounting for their roles in a range of basic cellular processes including cell adhesion, migration, polarity and apoptosis. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of current models of CCM protein function focusing on how known protein-protein interactions might contribute to cellular phenotypes and highlighting gaps in our current understanding.

The INK4A/ARF tumor suppressor locus is frequently inactivated in hepatocellular carcinoma (HCC), yet the consequences of this remain unknown. We recently described a HCC mouse model in which loss of the Ink4a/Arf locus accelerates the development of metastasis and enhances tumor cell migration and invasion in cell culture assays. We show here that knockdown of p19Arf in an HCC cell line increases invasion in cell culture assays. Furthermore, reintroduction of p19Arf into HCC cell lines lacking Ink4a/Arf inhibits tumor cell invasion, without affecting cell proliferation, or cell transformation as measured by soft agar colony formation. Inhibition of cell invasion by p19Arf was dependent on its C-terminal binding protein (CtBP) interaction domain but independent of Mdm2 binding and nucleolar localization. Indeed, RNA interference-mediated knockdown of CtBP1 or CtBP2 decreased cell invasion, and ectopic expression of CtBP2 enhanced tumor cell migration and invasion. Thus, our data indicate a novel role for the Arf tumor suppressor protein in regulating phenotypes associated with tumor progression and metastasis in HCC cells. [Cancer Res 2008;68(2):476-82]