Purpose: Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel human telomerase antagonist, imetelstat, on primary human glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo.Experimental Design: Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro. The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels, telomere length, proliferation, clonogenicity, and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts were used to assess the in vivo efficacy of imetelstat.Results: Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC 50 0.45 μmol/L). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation, and eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood-brain barrier in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic administration of imetelstat (30 mg/kg every day for three days), showed ∼70% inhibition of telomerase activity. Chronic systemic treatment produced a marked decrease in the rate of xenograft subcutaneous tumor growth.Conclusion: This preclinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination with standard therapies. Clin Cancer Res; 16(1); 154-63. ©2010 AACR.

Exposing rare but highly malignant tumor cells that migrate from the primary tumor mass into adjacent tissue(s) or circulate in the bloodstream is critical for early detection and effective intervention(s). Here, we report on an aptamer-based strategy directed against epidermal growth factor receptor (EGFR), the most common oncogene in glioblastoma (GBM), to detect these deadly tumor cells. GBMs are characterized by diffuse infiltration into normal brain regions, and the inability to detect GBM cells renders the disease surgically incurable with a median survival of just 14.2 months. To test the sensitivity and specificity of our platform, anti-EGFR RNA aptamers were immobilized on chemically modified glass surfaces. Cells tested included primary human GBM cells expressing high levels of the wild-type EGFR, as well as genetically engineered murine glioma cells overexpressing the most common EGFR mutant (EGFRvIII lacking exons 2-7) in Ink4a/Arf-deficient astrocytes. We found that surfaces functionalized with anti-EGFR aptamers could capture both the human and murine GBM cells with high sensitivity and specificity. Our findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment.

SUMMARY Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify a SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and is capable of driving glioma initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc finger E-box binding homeobox 1 (ZEB1) as potential SOX2 targets, which are frequently co-expressed irrespective of driver mutations. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor suppressor deficient astrocytes into glioma initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using an orthotopic mouse model of primary human glioblastoma (GBM) and a brain metastatic renal tumor of clear cell renal cell carcinoma (CCRCC) histology, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-13C2]glucose. The [3-13C]lactate/[2,3-13C2]lactate ratio was similar for both the GBM and renal tumor and their respective surrounding brains (GBM: 0.197 ± 0.011 and 0.195 ± 0.033 (p=1); CCRCC: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than PPP flux in these tumors, and that PPP flux into the lactate pool was similar in both tissues. Remarkably, 13C-13C coupling was observed in molecules derived from Krebs cycle intermediates in both tumors, denoting glucose oxidation. In the renal tumor, in contrast to GBM and surrounding brain, 13C multiplets of GABA differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. Additionally, the orthotopic renal tumor, the patient’s primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a CCRCC tissue microarray suggesting that GABA synthesis is cell-autonomous in at least a subset of renal tumors. Taken together, these data demonstrate that 13C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.

We evaluated the effect of the pregnane X receptor (PXR) agonist rifampin on metformin pharmacokinetics, organic cation transporter 1 (OCT1) and OCT2 mRNA levels, and glucose levels, using the oral glucose tolerance test (OGTT) in 16 healthy subjects. The glucose-lowering effects of metformin were evaluated by OGTT before and after metformin treatment on days 1 and 2 and again on days 13 and 14 after a 10-day course of rifampin. Rifampin increased the difference in maximum glucose levels (ΔG(max)) by 41.9% (P = 0.024) and the area under the concentration-time curve (AUC) during the first 60 min after glucose ingestion (ΔAUC(gluc60)) by 54.5% (P = 0.020). Renal clearance (CL(R)) of metformin was increased by 16% (P = 0.008), but the systemic exposure was only slightly increased (13%, P = 0.049), possibly because of increased absorption. Rifampin increased OCT1 mRNA levels 4.1-fold in peripheral blood cells (P = 0.001); however, OCT2 mRNA was not detected. Our results suggest that rifampin increases OCT1 expression and hepatic uptake of metformin, leading to enhanced glucose-lowering action.

Myeloid-derived suppressor cells (MDSCs) regulate T cell immunity, and this population is a new therapeutic target for immune regulation. A previous study showed that transforming growth factor-β (TGF-β) is involved in controlling MDSC differentiation and immunoregulatory function in vivo. However, the direct effect of TGF-β on MDSCs with various cytokines has not previously been tested. Thus, we examined the effect of various cytokine combinations with TGF-β on MDSCs derived from bone marrow cells. The data show that different cytokine combinations affect the differentiation and immunosuppressive functions of MDSCs in different ways. In the presence of TGF-β, interleukin-6 (IL-6) was the most potent enhancer of MDSC function, whereas granulocyte colony-stimulating factors (G-CSF) was the most potent in the absence of TGF-β. In addition, IL-4 maintained MDSCs in an immature state with an increased expression of arginase 1 (Arg1). However, regardless of the cytokine combinations, TGF-β increased expansion of the monocytic MDSC (Mo-MDSC) population, expression of immunosuppressive molecules by MDSCs, and the ability of MDSCs to suppress CD4+ T cell proliferation. Thus, although different cytokine combinations affected the MDSCs in different ways, TGF-β directly affects monocytic-MDSCs (Mo-MDSCs) expansion and MDSCs functions.

TM accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement the defects in growth and protein N-glycosylation, and to correct the accumulation of Dol-P-P in the cwh8⌬ yeast mutant. Northern blot analyses demonstrate a wide distribution of the DOLPP1 mRNA in mouse tissues. Overexpression of Dolpp1p in yeast, COS, and Sf9 cells produces substantial increases in Dol-P-P phosphatase activity but not in dolichyl monophosphate or phosphatidic acid phosphatase activities in microsomal fractions. Subcellular fractionation and immunofluorescence studies localize the enzyme encoded by DOLPP1 to the endoplasmic reticulum of COS cells. The results of protease sensitivity studies with microsomal vesicles from the lpp1⌬/dpp1⌬ yeast mutant expressing DOLPP1 are consistent with Dolpp1p having a luminally oriented active site. The sequence of the DOLPP1 cDNA predicts a polypeptide with 238 amino acids, and a new polypeptide corresponding to 27 kDa is observed when DOLPP1 is expressed in yeast, COS, and Sf9 cells. This study is the first identification and characterization of a cDNA clone encoding an essential component of a mammalian lipid pyrophosphate phosphatase that is highly specific for Dol-P-P. The specificity, subcellular location, and topological orientation of the active site described in the current study strongly support a role for Dolpp1p in the recycling of Dol-P-P discharged during protein N-glycosylation reactions on the luminal leaflet of the endoplasmic reticulum in mammalian cells.The multisubunit complex, oligosaccharyltransferase (1), catalyzes the transfer of Glc 3 Man 9 GlcNAc 2 from dolichyl pyrophosphate (Dol-P-P) 1 to appropriate asparagine residues during the co-translational N-glycosylation of nascent polypeptides in yeast and mammalian cells (2-4). During the primary N-glycosylation reaction, Dol-P-P is released on the luminal surface of the endoplasmic reticulum (ER). In order for Dol-P-P to be re-utilized as a glycosyl carrier lipid for additional rounds of lipid intermediate biosynthesis, it must be converted to dolichyl phosphate (Dol-P) and translocated to the cytoplasmic leaflet of the ER (4). Although it cannot yet be excluded that Dol-P-P, or perhaps Dol-P, diffuses transversely from the luminal leaflet to the cytoplasmic face by a protein-mediated mechanism, it is more likely that it is dephosphorylated on the luminal surface to form free dolichol that could more readily diffuse back to the cytoplasmic leaflet. Cytoplasmically oriented dolichol would then be re-phosphorylated by dolichol kinase (5, 6). Recent studies (7,8) have shown that the CWH8 gene in Saccharomyces cerevisiae encodes a Dol-P-P phosphatase that actively converts Dol-P-P to Dol-P and is also capable of dephosphorylating Dol-P at a slower rate. Moreover, the yeast Dol-P-P phosphatase is recovered in crude microsomal fractions, but its subcellular location has not been established.Although there have been numerous reports (4, 7-13) that crude microsomal fractions from...

Recent molecular evidence suggests an association with a new herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), and primary effusion lymphomas (PELs). PELs have a characteristic morphology, phenotype, and clinical presentation, with malignant effusions in the absence of a contiguous solid tumor mass. We have established a cell line (KS-1) from a KSHV-positive human immunodeficiency virus (HIV)-negative patient with pleural cavity-based lymphoma that was passaged into triple-immunodeficient BNX mice. In contrast to cell lines from body cavity-based lymphomas derived from HIV-positive individuals that contain both KSHV and Epstein Barr viral genome, these cells contain only KSHV, allowing for uncontaminated virologic studies. Ultrastructural examination identified malignant cells with features of late differentiating B cells (immunoblasts). Cells with viral cytopathic effect contained typical 110-nm intranuclear herpesvirus nucleocapsids and complete cytoplasmic virions, confirming the association of PEL with KSHV.