Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation1–8. RIPK1 is implicated in inflammatory and cell death signalling9–13 and its kinase activity is believed to drive RIPK3-mediated necroptosis14,15. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.

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.

The serine/threonine kinase RIPK1 is recruited to the TNF receptor 1 to mediate pro-inflammatory signalling and to regulate TNF-induced cell death. A RIPK1 deficiency results in perinatal lethality, impaired NFκB and MAPK signalling and sensitivity to TNF-induced apoptosis. Chemical inhibitor and in vitro reconstitution studies suggested RIPK1 displays distinct kinase activity dependent and independent functions. To determine the contribution of RIPK1 kinase to inflammation in vivo, we generated knock-in mice endogenously expressing catalytically inactive RIPK1 D138N. Unlike Ripk1−/− mice, which die shortly after birth, RIPK1D138N/D138N mice are viable. Cells expressing RIPK1 D138N are resistant to TNF- and poly (I:C)-induced necroptosis in vitro and RIPK1D138N/D138N mice are protected from TNF-induced shock in vivo. Moreover, RIPK1D138N/D138N mice fail to control Vaccinia virus replication in vivo. This study provides genetic evidence that the kinase activity of RIPK1 is not required for survival but is essential for TNF-, TRIF- and viral-initiated necroptosis.

Whole genome doubling (WGD) occurs early in tumorigenesis and generates geneticallyunstable tetraploid cells that fuel tumor development. Cells that undergo WGD (WGD + ) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from ~10,000 primary human cancer samples and essentiality data from ~600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD + cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGDcells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD + cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGDcells, its loss induces dramatic mitotic errors in WGD + cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD + cancer cells while sparing the normal, nontransformed WGDcells that comprise human tissue..

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 ...