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The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (m1DH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the m1DH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of m1DH1 impaired the growth of IDH1-mutant—but not IDH1-wild-type—glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that m1DH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

A Role for iNOS in Fasting Hyperglycemia and Impaired Insulin Signaling in the Liver of Obese Diabetic Mice Masaki Fujimoto 1 , Nobuyuki Shimizu 1 , Kaiko Kunii 1 , J.A. Jeevendra Martyn 1 , Kohjiro Ueki 2 and Masao Kaneki 1 1 Department of Anesthesia and Critical Care, Massachusetts General Hospital, Shriners Hospital for Children, Harvard Medical School, Boston, Massachusetts 2 Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan Address correspondence and reprint requests to Masao Kaneki, Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, 149 13th St., Rm. 6604, Charlestown, MA 02129. E-mail: mkaneki{at}partners.org Abstract Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat diet–induced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, l -NIL, in obese diabetic ( ob/ob ) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5- and 2-fold, respectively, and enhanced IRS-1–and IRS-2–mediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice. Br-cGMP, 8-bromoguanosine-3′,5′-cyclic monophosphorothioate GAPDH, glyceraldehyde-3-phosphate dehydrogenase GSNO, S-nitrosoglutathione IKK, inhibitor κΒ kinase iNOS, inducible NO synthase IR, insulin receptor IRS, IR substrate LPS, lipopolysaccharide NF-κB, nuclear factor-κB PI3, phosphatidylinositol-3 SREBP, sterol regulatory element–binding protein Footnotes Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org . Accepted February 15, 2005. Received December 9, 2004. DIABETES

A small-molecule activator specific for PKM2 binds to a site distinct from the endogenous activator fructose-1,6-bisphosphate, promoting tetramerization and constitutive activation of PKM2, to inhibit xenograft tumor growth in mice. Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor α-chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions including asthma. STAT6 binds poly(ADP-ribose) polymerase (PARP)14, an ADP-ribosyl monotransferase. Elimination of PARP14 by gene targeting led to attenuation of OVA-specific allergic lung inflammation. However, PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation, and it is not clear whether inhibition of the catalytic function has any biological consequence. Using BALB/c mice sensitized to the allergen Alternaria alternata, we show that peroral administration of RBN012759, a highly selective inhibitor of ADP-ribosylation by PARP14 with negligible impact on other members of the PARP gene family, achieved biologically active plasma concentrations and altered several responses to the Ag. Specifically, the pharmaceutical compound decreased mucus after allergen challenge, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that PARP14 catalytic activity can contribute to pathogenesis in allergic or atopic processes and propose that other biological endpoints dependent on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

Nat. Chem. Biol. 8, 839–847 (2012); published online 26 August 2012; corrected after print 11 October 2012 In the version of this article initially published, the accession codes to the Protein Data Bank were omitted. The error has been corrected in the HTML and PDF versions of the article.

Adaptive resistance limits immune checkpoint blockade therapy (ICBT) response duration and magnitude. Interferon γ (IFNγ), a critical cytokine that promotes cellular immunity, also induces adaptive resistance to ICBT. Using syngeneic mouse tumour models, we confirmed that chronic IFNγ exposure confers resistance to anti-Programmed cell death protein 1 (α-PD-1) therapy. We identified consistent upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in both chronic IFNγ-treated cancer cells and patient melanoma with elevated IFNG expression. Knockdown or pharmacological inhibition of PARP14 increased effector T cell infiltration into tumours derived from cells pre-treated with IFNγ and decreased the presence of regulatory T cells, leading to restoration of α-PD-1 sensitivity. Finally, we determined that tumours which spontaneously relapsed following α-PD-1 therapy could be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.Competing Interest StatementK.K., N.R.P., and M.N are all employees and shareholders of Ribon Therapeutics at the time of data collection. P.E.R. served as a consultant to Ribon Therapeutics. A.H. received research sponsorship from Ribon Therapeutics. All other authors declare no competing interests.Footnotes* All authors have been updated with their corresponding ORCIDs.

Higher-density inoculation of fibroblasts into a three-dimensional scaffold should accelerate wound healing after skin implantation. This study attempted to develop tissue-engineered skin with a higher density of fibroblasts. We first attempted to fabricate three-dimensional high-cell-density aggregates (spheroids) of normal human fibroblasts for application to tissue-engineered skin. Our method consisted of rotational shaking with nontreated dishes, decreasing fibroblast-meterial interactions, and augmenting cell-cell interaction. To prompt aggregate formation, the medium was supplemented with insulin, dexamethasone, ascorbic acid, and basic fibroblast growth factors that potentiate secretion of extracellular matrices. Under such improved conditions, fibroblasts were able to form spheroidal aggregates within 24 to 36h of rotational culture. Although the formed aggregates were irregular in shape and were composed of only several cells after 12h, they became almost spheroidal after 24h. The aggregates grew even more round after 36h, and their surfaces became smooth. After 36h of rotational culture, the fibroblast aggregates were collected and reinoculated onto a biodegradable mesh composed of polyglycolic acid coated with collagen. The aggregates were trapped in the material and became attached after 24h. Finally, because transforming growth factor-β^sub 3^ (TGF-β^sub 3^) is known to accelerate wound healing, we conducted a semiquantitative analysis of TGF-β^sub 3^ mRNA in both the fibroblast monolayers (two-dimensional culture) and the aggregates (three-dimensional culture). Analysis of TGF-β^sub 3^ mRNA expression showed that mRNA expression was greater in the fibroblasts of aggregates than in a monolayer. Therefore, our newly developed dermal graft is expected to accelerate wound healing faster than conventional grafts.[PUBLICATION ABSTRACT]