The STAT3 transcription factor is an important regulator of stem cell self-renewal, cancer cell survival, and inflammation. In the pancreas, STAT3 is dispensable for normal development whereas the majority of pancreatic ductal adenocarcinomas (PDAC) show constitutive activation of STAT3, suggesting its potential as a therapeutic target in this cancer. Here, we sought to define the mechanisms of STAT3 activation and its functional importance in PDAC pathogenesis. Large-scale screening of cancer cell lines with a JAK2 inhibitor that blocks STAT3 function revealed a >30-fold range in sensitivity in PDAC, and showed a close correlation of sensitivity with levels of tyrosine-phosphorylated STAT3 and of the gp130 receptor, an upstream signaling component. Correspondingly, upregulation of the IL6/LIF-gp130 pathway accounted for the strong STAT3 activation in PDAC subsets. To define functions of STAT3 in vivo, we developed mouse models that test the impact of conditional inactivation of STAT3 in KRAS-driven PDAC. We showed that STAT3 is required for the development of the earliest pre-malignant pancreatic lesions, acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Moreover, acute STAT3 inactivation blocked PDAC initiation in a second in vivo model. Our results demonstrate that STAT3 has critical roles throughout the course of PDAC pathogenesis, supporting the development of therapeutic approaches targeting this pathway. Moreover, our work suggests that gp130 and phospho-STAT3 expression may be effective biomarkers for predicting response to JAK2 inhibitors.

Nutritional excess and/or obesity represent well-known predisposition factors for the development of noninsulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y 632 MPM motif that is implicated in the binding of p85␣/p110␣ PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.mTOR (mammalian target of rapamycin) is a Ser/Thr kinase that belongs to the phosphatidylinositol (PI) kinase-related protein kinase family (17). In the budding yeast Saccharomyces cerevisiae, TOR functions as a nutrient-dependent mediator of cell autonomous growth (17). In metazoans, TOR participates in both nutrient-and hormone-dependent signaling pathways (17). mTOR partitions between two scaffold proteins, Raptor (14, 27) and Rictor (25, 33, 52). The rapamycin-sensitive Raptor/mTOR complex (TORC1) regulates growth via S6K1 and 4EBP1/PHAS (14, 27). The rapamycin-insensitive Rictor/ mTOR (TORC2) complex regulates cellular proliferation via Akt (51) and cytoskeleton organization via protein kinase C␣ (52) and small GTPases Rho and Rac (25).AMP-activated protein kinase (AMPK) acts as an intracellular energy sensor that controls glucose and lipid metabolism in peripheral tissues (6). Its activation leads to a homeostatic response by down-regulation of anabolic pathways and upregulation of catabolic pathways (6). AMPK-and PI3-kinase/ Akt-dependent signaling pathways converge at the level of tuberus sclerosis complex 2 (TSC2 or tuberin) which functions as a GTPase-activating protein for the small GTPase Rheb (20)(21)(22). Genetic studies in Drosophila melanogaster place Rheb downstream of insulin/insulin-like growth factor I and nutrient-dependent signaling pathways (53). Rheb is negatively regulated by AMPK/TSC (20, 22) and functions as an upstream activator of mTOR (20,34,53). Normal cell growth and homeostasis rely on a proper balance between nutrient-and hormone-dependent regulation of the AMPK/mTOR axis, while its deregulation could lead to metabolic and growth-related diseases (11,20,22,67).Several in vitro and in vivo studies show a beneficial metabolic effect of AMPK activation on glucose homeostasis and peripheral insul...

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.The metabolic control systems in HSCs are poorly understood although these cells have been shown to be highly sensitive to energetic and oxidative stress and must be able to shift Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2011 March 1. Lkb1 mutant animals displayed progressive pancytopenia as well as rapid loss of bone marrow myeloid, B lymphoid and erythroid cells (Fig. 1c, d and Supplementary Fig. 2a), and markedly decreased cellularity of the thymus and spleen ( Supplementary Fig. 2c, d and data not shown). Notably, in the bone marrow and thymus, immature lymphoid cells declined at a faster rate than the more differentiated cells ( Supplementary Fig. 2b, c). The Lkb1 mutants also exhibited a pronounced loss of HSC and multipotent progenitor populations at day 5 after pIpC treatment (Fig. 1e, f and Supplementary Fig. 2f). Furthermore, Lkb1 mutant bone marrow cells formed fewer and smaller colonies in in vitro colony forming assays ( Fig. 1g and Supplementary Fig. 2e). Comparable in vivo and in vitro phenotypes were seen using a second model system in which Lkb1 was deleted using the tamoxifen-inducible Rosa26-creERt2 strain ( Supplementary Fig. 2g, h, and not shown). These results show that Lkb1 is critically required for haematopoiesis and for the maintenance of HSCs and progenitor cells. Lkb1 function in bone marrow is cell intrinsicBoth theMx1-cre and Rosa26-creERt2 systems induce Cre recombinase activity in many cell types; thus bone marrow transplants were used to determine whether Lkb1 has a cellautonomous role in haematopoiesis. We performed non-competitive transplants of CD45.1 whole bone marrow from contr...

KDM6A, an X chromosome-encoded histone demethylase and member of the COMPASS-like complex, is frequently mutated in a broad spectrum of malignancies and contributes to oncogenesis with poorly characterized mechanisms. We found that KDM6A loss induced squamous-like, metastatic pancreatic cancer selectively in females through deregulation of the COMPASS-like complex and aberrant activation of super-enhancers regulating ΔNp63, MYC, and RUNX3 oncogenes. This subtype of tumor developed in males had concomitant loss of UTY and KDM6A, suggesting overlapping roles, and points to largely demethylase independent tumor suppressor functions. We also demonstrate that KDM6A-deficient pancreatic cancer is selectively sensitive to BET inhibitors, which reversed squamous differentiation and restrained tumor growth in vivo, highlighting a therapeutic niche for patient tailored therapies.

The histone H3 demethylase Not dead yet-1 (Ndy1/KDM2B) is a physiological inhibitor of senescence. Here, we show that Ndy1 is down-regulated during senescence in mouse embryonic fibroblasts (MEFs) and that it represses the Ink4a/Arf locus. Ndy1 counteracts the senescence-associated down-regulation of Ezh2, a component of polycomb-repressive complex (PRC) 2, via a JmjC domain-dependent process leading to the global and Ink4a/Arf locus-specific up-regulation of histone H3K27 trimethylation. The latter promotes the Ink4a/ Arf locus-specific binding of Bmi1, a component of PRC1. Ndy1, which interacts with Ezh2, also binds the Ink4a/Arf locus and demethylates the locus-associated histone H3K36me2 and histone H3K4me3. The combination of histone modifications driven by Ndy1 interferes with the binding of RNA Polymerase II, resulting in the transcriptional silencing of the Ink4a/Arf locus and contributing to the Ndy1 immortalization phenotype. Other studies show that, in addition to inhibiting replicative senescence, Ndy1 inhibits Ras oncogene-induced senescence via a similar molecular mechanism.cancer ͉ chromatin ͉ epigenetics ͉ Fbxl10 ͉ JHDM1B

BACKGROUND & AIMS The Hippo signaling pathway is a context-dependent regulator of cell proliferation, differentiation, and apoptosis in species ranging from Drosophila to humans. In this study, we investigated the role of the core Hippo kinases—Mst1 and Mst2—in pancreatic development and homeostasis. METHODS We used a Cre/LoxP system to create mice with pancreas-specific disruptions in Mst1 and Mst2 (Pdx1-Cre;Mst1−/−;Mst2fl/fl mice), the mammalian orthologs of Drosophila Hippo. We used a transgenic approach to overexpress Yap, the downstream mediator of Hippo signaling, in the developing pancreas of mice. RESULTS Contrary to expectations, the pancreatic mass of Pdx1-Cre;Mst1−/−;Mst2fl/fl mice was reduced compared with wild-type mice, largely because of postnatal de-differentiation of acinar cells into duct-like cells. Development of this phenotype coincided with postnatal reactivation of YAP expression. Ectopic expression of YAP during the secondary transition (a stage at which YAP is normally absent) blocked differentiation of the endocrine and exocrine compartments, whereas loss of a single Yap allele reduced acinar de-differentiation. The phenotype of Pdx1-Cre;Mst1−/−;Mst2fl/fl mice recapitulated cellular and molecular changes observed during chemical-induced pancreatitis in mice. CONCLUSIONS The mammalian Hippo kinases, and YAP, maintain postnatal pancreatic acinar differentiation in mice.

A common integration site, cloned from MoMuLV-induced rat T cell lymphomas, was mapped immediately upstream of Not dead yet-1 (Ndy1)/KDM2B, a gene expressed primarily in testis, spleen, and thymus, that is also known as FBXL10 or JHDM1B. Ndy1 encodes a nuclear, chromatin-associated protein that harbors Jumonji C (JmjC), CXXC, PHD, proline-rich, F-box, and leucine-rich repeat domains. Ndy1 and its homolog Ndy2/KDM2A (FBXL11 or JHDM1A), which is also a target of provirus integration in retrovirus-induced lymphomas, encode proteins that were recently shown to possess Jumonji C-dependent histone H3 K36 dimethyldemethylase or histone H3 K4 trimethyl-demethylase activities. Here, we show that mouse embryo fibroblasts engineered to express Ndy1 or Ndy2 undergo immortalization in the absence of replicative senescence via a JmjC domain-dependent process that targets the Rb and p53 pathways. Knockdown of endogenous Ndy1 or expression of JmjC domain mutants of Ndy1 promote senescence, suggesting that Ndy1 is a physiological inhibitor of senescence in dividing cells and that inhibition of senescence depends on histone H3 demethylation.cancer ͉ histone demethylase ͉ immortalization ͉ senescence ͉ insertional mutagenesis

Epigenetic mechanisms mediate heritable control of cell identity in normal cells and cancer. We sought to identify epigenetic regulators driving the pathogenesis of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal human cancers. We found that KDM2B (also known as Ndy1, FBXL10, and JHDM1B), an H3K36 histone demethylase implicated in bypass of cellular senescence and somatic cell reprogramming, is markedly overexpressed in human PDAC, with levels increasing with disease grade and stage, and highest expression in metastases. KDM2B silencing abrogated tumorigenicity of PDAC cell lines exhibiting loss of epithelial differentiation, whereas KDM2B overexpression cooperated with Kras G12D to promote PDAC formation in mouse models. Gain-and loss-of-function experiments coupled to genome-wide gene expression and ChIP studies revealed that KDM2B drives tumorigenicity through 2 different transcriptional mechanisms. KDM2B repressed developmental genes through cobinding with Polycomb group (PcG) proteins at transcriptional start sites, whereas it activated a module of metabolic genes, including mediators of protein synthesis and mitochondrial function, cobound by the MYC oncogene and the histone demethylase KDM5A. These results defined epigenetic programs through which KDM2B subverts cellular differentiation and drives the pathogenesis of an aggressive subset of PDAC.