Marlene Balys

Cloning and Expression of a Novel, Tissue Specifically Expressed Member of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controllingO-linked glycosylation.

CHARACTERIZATION OF A UDP-GALNAC:POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE THAT DISPLAYS GLYCOPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE ACTIVITY

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests thatO-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

Background: Eradication of primary human leukemia cells represents a major challenge. Therapies have not substantially changed in over 30 years. Results: Using normal versus leukemia specimens enriched for primitive cells, we document aberrant regulation of glutathione metabolism. Conclusion: Aberrant glutathione metabolism is an intrinsic property of human leukemia cells. Significance: Interventions based on modulation of glutathione metabolism represent a powerful means to improve therapy. The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

Chemical genomic screening reveals synergism between parthenolide and inhibitors of the PI-3 kinase and mTOR pathways

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However, despite the activity of this agent, PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus, we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal, we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment, exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together, our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells.

Lpa2 Is a Negative Regulator of Both Dendritic Cell Activation and Murine Models of Allergic Lung Inflammation

Negative regulation of innate immune responses is essential to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration, and activation and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G protein-coupled receptors, whose function in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. In this article, we show that DC from lpa2−/− mice are hyperactive compared with their wild-type counterparts and are less susceptible to inhibition by different LPA species. In transient-transfection assays, we found that lpa2 overexpression inhibits NF-κB–driven gene transcription. Using an adoptive-transfer approach, we found that allergen-pulsed lpa2−/− DC induced substantially more lung inflammation than did wild-type DC after inhaled allergen challenge. Finally, lpa2−/− mice develop greater allergen-driven lung inflammation than do their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits DC activation and allergic airway inflammation.

Selective Activity of the Histone Deacetylase Inhibitor AR-42 against Leukemia Stem Cells: A Novel Potential Strategy in Acute Myelogenous Leukemia

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus, it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression–based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide, we identified AR-42 (OSU-HDAC42), a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate, but with improved activity at submicromolar concentrations. Here, we report that AR-42 induces NF-κB inhibition, disrupts the ability of Hsp90 to stabilize its oncogenic clients, and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide, the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2–driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials, we expect that our results can be extended to the clinic. Mol Cancer Ther; 13(8); 1979–90. ©2014 AACR.

Perinatal methanol exposure in the rat. I. Blood methanol concentration and neural cell adhesion molecules.

Although the acute toxicity of methanol is well documented, few studies have addressed the consequences of perinatal exposures to the low concentrations that are expected to arise from its proposed use as a component of automobile fuel. This report describes the general research design of a series of studies, the effects of methanol exposures on blood concentrations in dams and neonates, and indices of brain development. Four cohorts of Long-Evans pregnant rats, each cohort consisting of an exposure (n = 12) and a control (n = 12) group, were exposed whole-body to 4500 ppm methanol vapor or air for 6 hr daily beginning on Gestation Day 6. Both dams and pups were then exposed through Postnatal Day 21 (PND 21). Blood methanol concentrations determined by gas chromatography from samples obtained immediately following a 6-hr exposure reached approximately 500-800 micrograms/ml in the dams during gestation and lactation. Average concentrations for pups attained levels about twice those of the dams. Selected offspring from Cohort 4 were exposed for one additional 6-hr session at ages that extended out to PND 52. Regression analyses showed that the blood methanol concentrations of the pups declined until about PND 48, at which time their levels approximated those of their dams. Such pharmacokinetic differences might increase the risks posed to developing organisms. Light-microscopic analysis showed no significant abnormalities in the brains of the methanol-treated animals. However, assays of neural cell adhesion molecules (NCAMs) in brains of pups sacrificed on PND 4 showed staining for both the 140 and the 180 kDa isoforms to be less intense in the cerebellum of exposed animals. NCAM differences were not apparent in animals sacrificed 15 months after their final exposure.

Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Index case of acute myeloid leukemia in a family harboring a novel CEBPA germ line mutation

The persistence of a CEBPA mutation at the time of complete remission warrants germ line analysis.Not all patients harboring germ line CEBPA mutations have a family history of AML.