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IL-7 supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. STAT5 is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7R, JAK and FLT3. To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant pre-leukemia (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R; Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1*6 mutant. Expression of STAT5B1*6 in T-cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with Pimozide induced differentiation and loss of LSCs, whilst enhancing response to chemotherapy. Furthermore, Pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL.

Cancer cell growth is dependent upon the sustainability of proliferative signaling and resisting cell death. Macroautophagy/autophagy promotes cancer cell growth by providing nutrients to cells and preventing cell death. This is in contrast to autophagy promoting cell death under some conditions. The mechanism regulating autophagy-mediated cancer cell growth remains unclear. Herein, we demonstrate that TSSC4 (tumor suppressing subtransferable candidate 4) is a novel tumor suppressor that suppresses cancer cell growth and tumor growth and prevents cell death induction during excessive growth by inhibiting autophagy. The oncogenic proteins ERBB2 (erb-b2 receptor tyrosine kinase 2) and the activation EGFR mutant (EGFRvIII, epidermal growth factor receptor variant III) promote cell growth and TSSC4 expression in breast cancer and glioblastoma multiforme (GBM) cells, respectively. In EGFRvIII-expressing GBM cells, TSSC4 knockout shifted the function of autophagy from a pro-cell survival role to a pro-cell death role during prolonged cell growth. Furthermore, the interaction of TSSC4 with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) via its conserved LC3-interacting region (LIR) contributes to its inhibition of autophagy. Finally, TSSC4 suppresses tumorsphere formation and tumor growth by inhibiting autophagy and maintaining cell survival in tumorspheres. Taken together, sustainable cancer cell growth can be achieved by autophagy inhibition via TSSC4 expression. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; CQ: chloroquine; EGFRvIII: epidermal growth factor receptor variant III; ERBB2: erb-b2 receptor tyrosine kinase 2; GBM: glioblastoma multiforme; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule Associated protein 1 light chain 3; TSSC4: tumor suppressing subtransferable candidate 4

The gene encoding the transcription factor, Grainyhead-like 3 (Grhl3), plays critical roles in mammalian development and homeostasis, and these have been uncovered through analysis of loss-of-function models. Grhl3-null embryos exhibit a range of gross phenotypes including a shortened longitudinal axis, thoraco-lumbo-sacral spina bifida and soft-tissue syndactyly. Additional studies reveal that these embryos also exhibit a proliferation/differentiation imbalance in the epidermis. This manifests as a failure in skin barrier formation resulting in peri-natal lethality, and defective wound repair. Conditional inactivation of Grhl3 in the squamous epithelium of adult skin, head and neck tissues, and oesophagus reproduces this proliferation/differentiation imbalance and leads to squamous cell carcinomas. These observations establish GRHL3 as a critical tumour suppressor. Despite these extensive analyses of Grhl3 loss-of-function models, the consequences of gain-of-function of this gene have been difficult to achieve. We have redressed this issue through the generation of a novel mouse model that expresses Grhl3 from a transgene integrated in the Rosa26 locus on an endogenous Grhl3-null background. Expression of the transgene rescues both the neurulation and skin barrier defects of the knockout mice, allowing survival into adulthood. Despite this, the mice are not normal, exhibiting a range of phenotypes attributable to dysregulated Grhl3 expression. In mice homozygous for the transgene, we observe a severe Shaker-Waltzer phenotype associated with hearing impairment. Micro-CT scanning of the cochleae and the vestibular apparatus revealed profound structural alterations underlying these phenotypes. In addition, these mice exhibit other developmental anomalies including hair loss, digit defects and epidermal dysmorphogenesis. These findings indicate that diverse developmental processes display low tolerance to dysregulation of Grhl3.

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., This study shows that the closely related transcription factors ZEB1 and ZEB2 cooperate to restrain myeloid and lymphoid differentiation programs in hematopoietic stem and progenitor cells, ensuring fidelity of differentiation in multiple lineages.

ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

International audience; 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 Kras G12D-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 Kras G12D-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 Kras G12D 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.

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.

Aberrant expression of the SNAIL & ZEB families of Epithelial to Mesenchymal Transition (EMT) modulators is an emerging theme in acute leukemia biology. EMT regulation has long been implicated in epithelial tumor pathogenesis, however a role in hematopoietic malignancy has only recently been explored. In this study, we describe a novel mechanism of AML pathogenesis involving SNAI1 induced epigenetic dysregulation via corruption of the histone demethylase LSD1. SNAI1 is expressed ∼10-fold higher in AML blasts compared to normal hematopoietic cells, suggesting a functional role for SNAI1 in AML. Indeed, SNAI1 knockdown induces differentiation in human AML cells, while Snai1 deletion delays tumour onset in mouse AML models. Furthermore, transgenic Snai1 expression perturbs myeloid development, resulting in accumulation of immature myeloid cells with enhanced self-renewal capacity - ultimately predisposing mice to AML. Intriguingly, impaired myeloid development by Snai1 absolutely requires its interaction with Lsd1, a H3K4 histone demethylase that has emerged as a novel therapeutic target in AML. Mechanistically, this interaction results in corruption of Lsd1 function via aberrant recruitment to Snai1 gene targets, and sequestration away from its own targets. Ultimately, this leads to changes in H3K4me1/2 methylation at specific promoters and enhancers, and subsequently altered gene expression. This model provides insight into the mechanisms by which LSD1 can be perturbed in AML, and offers a possible explanation for why LSD1 inhibition/loss has somewhat dichotomous effects on normal and malignant hematopoietic stem cells. Furthermore, as other members of the SNAIL and ZEB families are known to interact with LSD1, it is likely that this model will be more broadly applicable to other EMT modulators in AML, representing a possible new focus for future therapeutic studies.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. ispartof: Blood vol:117 issue:21 pages:5620-5630 ispartof: location:United States status: published

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.

Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.

Relatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A–mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A–independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell–derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

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.

There is growing evidence that the p53 tumour suppressor downregulates vascular endothelial growth factor (VEGF) expression, although the underlying mechanisms remain unclear and controversial. Here we provide insights from in vitro experiments and in vivo xenotransplantation assays that highlight a dual role for p53 in regulating VEGF during hypoxia. Unexpectedly, and for the first time, we demonstrate that p53 rapidly induces VEGF transcription upon hypoxia exposure by binding, in an HIF-1α-dependent manner, to a highly conserved and functional p53-binding site within the VEGF promoter. However, during sustained hypoxia, p53 indirectly downregulates VEGF expression via the retinoblastoma (Rb) pathway in a p21-dependent manner, which is distinct from its role in cell-cycle regulation. Our findings have important implications for cancer therapy, especially for tumours that harbour wild-type TP53 and a dysfunctional Rb pathway.

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.

Introduction: Regulation of the epithelial to mesenchymal transition (EMT) is an emerging theme in acute leukemia biology (1, 2). EMT is required for many aspects of development, including gastrulation and mesoderm formation. It has also been associated with malignant processes such as epithelial tumor cell invasion and metastasis, and the development, function and chemo-resistance of cancer stem cells. Master regulators of EMT belong to the Snail (Snai1/Snai2/Snai3) and Zeb (Zeb1/Zeb2) families of transcription factors. While they have primarily been studied in relation to solid organ development and embryogenesis, recent work has begun to uncover novel roles for these proteins in both normal and malignant hematopoiesis. A mounting body of evidence implicates the founding member of the Snail family, SNAI1, in Acute Myeloid Leukaemia (AML) development. Transgenic Snai1 expression induces myeloid leukaemia in mice (3), and human AML cells show increased SNAI1 expression compared to haematopoietic stem and progenitor cells (HSPCs). We have also found that shRNA knockdown of SNAI1 in AML cells induces differentiation, as measured by increased CD11b expression. In this paper we elucidate the mechanisms by which elevated levels of Snai1 impair normal hematopoiesis and promote leukemogenesis. Results: We employed murine retroviral and transgenic models to drive high levels of Snai1 in hematopoietic cells in vivo. Homozygous expression of a Snai1 transgene, using the endothelial and hematopoietic restricted Tie2-Cre, resulted in embryonic lethality at E15.5-17.5. Embryos were highly anemic, exhibiting normal primitive erythropoiesis, but severely impaired definitive erythropoiesis. Retroviral mediated overexpression of Snai1 during adult hematopoiesis resulted in a significant expansion of the Granulocyte-Macrophage progenitor (GMP) compartment, with a concomitant reduction in Megakaryocyte-Erythroid progenitor (MEP) numbers. Downstream myeloid development was also perturbed, with a strong differentiation bias towards macrophage production, at the expense of granulocyte development. Mice (n=4) have also begun to develop an aggressive AML beginning at 12 months of age, consistent with the previously published transgenic model (3). The effects of Snai1 expression on myeloid development are reminiscent of those observed in mice lacking Gfi1 or Gfi1b, suggesting possible competition for, or altered function of, their common co-repressor: the chromatin modulator Lsd1. To test this hypothesis, we generated a mutant form of Snai1 that does not bind Lsd1 and retrovirally expressed it in HSPCs. Unlike wild-type Snai1, the mutant form was unable to induce hematopoietic defects or AML development in mice. We next conducted gene expression profiling on the HPC7 hematopoietic progenitor cell line, with or without ectopic Snai1 expression. We observed a significant up-regulation of GMP-associated genes and more mature monocyte/macrophage related genes. These data, along with morphological analysis, suggested that Snai1 expressing HPC7 cells take on a macrophage-biased GMP-like phenotype, but do not fully differentiate into mature myeloid cells. Moreover, there was a significant correlation to gene expression programs from Gfi1/1b knockout HSCs, further implicating loss of Gfi1/1b activity in driving these myeloid phenotypes. Strikingly, we also observed evidence of inflammatory cytokine production and activation of key downstream signaling pathways that contribute to AML cell survival and proliferation. Whether this effect is due to altered regulation of Lsd1 targets, or instead represents a distinct Lsd1-independent mechanism, remains to be determined. Conclusion: We have shown that Snai1 expression perturbs myeloid cell development and induces AML in an Lsd1-dependent manner. Snai1 expression likely contributes to increased survival and proliferation of leukemic cells via induction of key inflammatory signaling pathways that support tumor cell growth. These data confirm the importance of Snai1 in AML development and pathogenesis. References: 1. Goossens S, et al. (2015).Nature communications 6:5794. 2. Stavropoulou V, et al. (2016). Cancer cell 30(1):43-58. 3. Perez-Mancera PA, et al. (2005). Human molecular genetics 14(22):3449-3461. Disclosures No relevant conflicts of interest to declare.

To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.

Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or Eμ-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy. ispartof: Molecular and Cellular Biology vol:30 issue:22 pages:5394-405 ispartof: location:United States status: published

SUMMARY Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/ loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest—for example, to enhance lineagedirected differentiation—with the use of a trapcoupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.

To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system.