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Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a KrasG12D-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of KrasG12D-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout KrasG12D mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus–based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

Epithelial homeostasis within the epidermis is maintained by means of multiple cell–cell adhesion complexes such as adherens junctions, tight junctions, gap junctions, and desmosomes. These complexes co-operate in the formation and the regulation of the epidermal barrier. Disruption of the epidermal barrier through the deregulation of the above complexes is the cause behind a number of skin disorders such as psoriasis, dermatitis, keratosis, and others. During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their adhesive capacities and gain mesenchymal properties. ZEB transcription factors are key inducers of EMT. In order to gain a better understanding of the functional role of ZEB2 in epidermal homeostasis, we generated a mouse model with conditional overexpression of Zeb2 in the epidermis. Our analysis revealed that Zeb2 expression in the epidermis leads to hyperproliferation due to the combined downregulation of different tight junction proteins compromising the epidermal barrier. Using two epidermis-specific in vivo models and in vitro promoter assays, we identified occludin as a new Zeb2 target gene. Immunohistological analysis performed on human skin biopsies covering various pathogeneses revealed ZEB2 expression in the epidermis of pemphigus vulgaris. Collectively, our data support the notion for a potential role of ZEB2 in intracellular signaling of this disease.

The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state.

ETP-ALL (Early T cell Progenitor Acute Lymphoblastic Leukemia) represents a high-risk subtype of T cell acute lymphocytic leukemia (T-ALL). Therapeutically, ETP-ALL patients frequently relapse after conventional chemotherapy highlighting the need for alternative therapeutic approaches. Using our ZEB2Tg ETP-ALL mouse model we previously documented the potential utility of the catalytic LSD1 inhibitor (GSK2879552) for treating mouse/human ETP-ALL. However, this approach proved to be inefficient, especially in killing human LOUCY cell ETP-ALL xenografts in vivo. Here we have revealed the novel involvement of ZEB2/LSD1 complexes in repressing the intrinsic apoptosis pathway by inhibiting the expression of several pro-apoptotic proteins such as BIM (BCL2L11) as a major driver for ETP-ALL survival. Treatment with LSD1i (particularly with the steric inhibitor SP2509) restored the expression of ZEB2/LSD1 pro-apoptotic BIM (BCL2L11) target. In combination with a JAK/STAT pathway inhibitor (JAKi, Ruxolitinib) or with a direct inhibitor of the anti-apoptotic BCL2 protein (BCL2i, ABT-199) resistance of human and mouse ETP-ALL to LSD1i was reversed. This new combination approach efficiently inhibited the growth of human and mouse ETP-ALL cells in vivo by enhancing their differentiation and triggering an apoptotic response. These results set the stage for novel combination therapies to be used in clinical trials to treat ETP-ALL patients.

Vascular endothelial growth factor (VEGF) and β‐catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone‐related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over‐expression in osteo‐chondroprogenitors and their progeny largely pheno‐copied constitutive β‐catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2‐ and phosphatidyl inositol 3‐kinase‐mediated pathway inducing β‐catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3‐β phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate β‐catenin activity may have widespread physiological and clinical ramifications.

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2 -driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R . This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2 -driven mouse model. Driver mutations in early T-cell precursor leukaemia (ETP-ALL) are poorly characterized. Here the authors show that Zeb2 overexpression is often found in ETP-ALL, can recapitulate the disease in transgenic mice and confers survival advantage by upregulating IL-7 signalling.

The ubiquitin ligase RACO-1 is found to be a co-factor for the AP-1 transcription factor c-Jun, and its depletion reduces AP-1 target gene expression, proliferation and tumour formation. RACO-1 is stabilised by growth factors through the MEK/ERK pathway, which promotes K63 linked ubiquitylation, opposing the K48-linked proteolytic auto-ubiquitylation of RACO-1. The AP-1 transcription factor c-Jun is essential for cellular proliferation in many cell types, but the molecular link between growth factors and c-Jun activation has been enigmatic. In this study we identify a previously uncharacterized RING-domain-containing protein, RACO-1 (RING domain AP-1 co-activator-1), as a c-Jun co-activator that is regulated by growth factor signalling. RACO-1 interacted with c-Jun independently of amino-terminal phosphorylation, and was both necessary and sufficient for c-Jun/AP-1 activation. Growth factor-mediated stimulation of AP-1 was attributable to MEK/ERK-dependent stabilization of RACO-1 protein. Stimulation of the MEK/ERK pathway strongly promoted Lys 63-linked ubiquitylation of RACO-1, which antagonized Lys 48-linked degradative auto-ubiquitylation of the same Lys residues. RACO-1 depletion reduced cellular proliferation and decreased expression of several growth-associated AP-1 target genes, such as cdc2 , cyclinD1 and hb-egf . Moreover, transgenic overexpression of RACO-1 augmented intestinal tumour formation triggered by aberrant Wnt signalling and cooperated with oncogenic Ras in colonic hyperproliferation. Thus RACO-1 is a co-activator that links c-Jun to growth factor signalling and is essential for AP-1 function in proliferation.

Vascular endothelial growth factor (VEGF) and b-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive b-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing b-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-b phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate b-catenin activity may have widespread physiological and clinical ramifications.