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Supplementary Data from The Telomerase Antagonist, Imetelstat, Efficiently Targets Glioblastoma Tumor-Initiating Cells Leading to Decreased Proliferation and Tumor Growth

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 (IC50 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

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 (IC50 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

While screening for plant homologs of telomeric-complex proteins, we isolated a cDNA for the Terminal acidic SANT 1 (Tacs1) gene of maize, encoding a 45-kDa protein with a C-terminal Myb/SANT-like domain. Gene expression and protein modeling data indicate that the TACS1 protein may function in chromatin remodeling within shoot primordia or other meristem-containing tissues.

The influence of photoperiod and temperature on the seasonal (fall to winter) cold acclimation and accu- mulation of a 25 kDa dehydrin in Rhododendron 'Chionoidesʼ was studied by exposing two groups of plants each in the greenhouse or outdoors to either a natural photoperiod (or short days) or an extended photoperiod (or long days) regime. Results suggest that the shortening daylength alone is suffi cient to trigger both the fi rst stage of cold acclima- tion and concomitant 25 kDa dehydrin induction. Exposure of the plants to natural photoperiod and temperatures induced the greatest cold hardiness and 25 kDa accumulation, while exposure to extended photoperiods (long days) and warmer temperatures (in the greenhouse) failed to induce any signifi cant freezing tolerance in leaves. Whereas short days trigger the cold acclimation process initially, low inductive temperatures can eventually replace the photoperiod stimulus. Seasonal accumulation of 25 kDa dehydrin, on the other hand, appears to be predominantly effected by short photoperiods. Data indicated that the leaf water content of outdoor plants maintained under natural photoperiod was lower than that of plants grown under extended photoperiod. This was also true for the greenhouse plants at the fi rst (September) and the last (January) sampling. It is hypothesized that early 25 kDa dehydrin accumulation may be due to short-day-induced cellular dehydration. Accumulation of two other dehydrins of 26 kDa and 32 kDa molecular masses does not appear to be associated with short day (SD)-induced fi rst stage of cold acclimation. Results show that their accumulation may be regulated by low, subfreezing temperatures and may be associated with the second and/or third stage of cold acclimation of 'Chionoidesʼ rhododendron leaves. Freezing temperatures constitute one of the major environmen- tal constraints limiting growth, development, and distribution of plants. Most tropical and subtropical plant species and herbaceous annuals lack the ability to adapt to cold winters and are typically injured or killed by slightly subfreezing temperatures. In contrast

We screened maize (Zea mays) cDNAs for sequences similar to the single myb-like DNA-binding domain of known telomeric complex proteins. We identified, cloned, and sequenced five full-length cDNAs representing a novel gene family, and we describe the analysis of one of them, the gene Single myb histone 1 (Smh1). The Smh1 gene encodes a small, basic protein with a unique triple motif structure of (a) an N-terminal SANT/myb-like domain of the homeodomain-like superfamily of 3-helical-bundle-fold proteins, (b) a central region with homology to the conserved H1 globular domain found in the linker histones H1/H5, and (c) a coiled-coil domain near the C terminus. The Smh-type genes are plant specific and include a gene family in Arabidopsis and the PcMYB1 gene of parsley (Petroselinum crispum) but are distinct from those (AtTRP1, AtTBP1, and OsRTBP1) recently shown to encode in vitro telomere-repeat DNA-binding activity. The Smh1 gene is expressed in leaf tissue and maps to chromosome 8 (bin 8.05), with a duplicate locus on chromosome 3 (bin 3.09). A recombinant full-length SMH1, rSMH1, was found by band-shift assays to bind double-stranded oligonucleotide probes with at least two internal tandem copies of the maize telomere repeat, TTTAGGG. Point mutations in the telomere repeat residues reduced or abolished the binding, whereas rSMH1 bound nonspecifically to single-stranded DNA probes. The two DNA-binding motifs in SMH proteins may provide a link between sequence recognition and chromatin dynamics and may function at telomeres or other sites in the nucleus.

BACKGROUND The majority of established human prostate cancer cell lines are derived from metastatic lesions and are already tumorigenic in vivo, therefore immortalized normal prostate cell lines may provide a more relevant model to unveil the mechanisms associated with cancer progression and metastasis. METHODS PZ-HPV-7, an immortalized human prostate epithelial cell line was used to generate xenograft tumors in mice. A subline designated HPV-PZ-7T was subsequently derived from the subrenal capsule xenograft of a nude mouse. These cells were further characterized using karyotyping, immunofluorescence, qRT-PCR, Western blotting, and three-dimensional cultures in Matrigel. RESULTS The PZ-HPV-7 cell line possesses a typical epithelial morphology, expresses basal cell markers, and is capable of forming web-like structures with evidence of budding on Matrigel. PZ-HPV-7 is non-tumorigenic in immunocompromised mice by either subcutaneous injection or subrenal grafting. In contrast, the PZ-HPV-7T cells, derived from a xenograft tumor induced by co-inoculation with matrigel using subrenal grafting, possess a mesenchymal phenotype as well as luminal cell markers and are highly tumorigenic and metastatic in nude mice. Functionally and biochemically, the PZ-HPV-7T subline appears to have undergone an epithelial-to-mesenchymal transition (EMT) from the parental PZ-HPV-7 line. CONCLUSION We have developed a novel EMT model using an immortalized normal prostate epithelial cell line and generated a new prostate cancer cell line, PZ-HPV-7T, which may represent an excellent system to study mechanisms associated with prostate cancer progression and metastasis. Prostate 71:626–636, 2011. © 2010 Wiley-Liss, Inc.

Prostate cancer is the most common malignancy in men, and patients with metastatic disease have poor outcome even with the most advanced therapeutic approaches. Most cancer therapies target the bulk tumor cells, but may leave intact a small population of tumor-initiating cells (TICs), which are believed to be responsible for the subsequent relapse and metastasis. Using specific surface markers (CD44, integrin alpha(2)beta(1) and CD133), Hoechst 33342 dye exclusion, and holoclone formation, we isolated TICs from a panel of prostate cancer cell lines (DU145, C4-2 and LNCaP). We have found that prostate TICs have significant telomerase activity which is inhibited by imetelstat sodium (GRN163L), a new telomerase antagonist that is currently in Phase I/II clinical trials for several hematological and solid tumor malignancies. Prostate TICs telomeres were of similar average length to the telomeres of the main population of cells and significant telomere shortening was detected in prostate TICs as a result of imetelstat treatment. These findings suggest that telomerase inhibition therapy may be able to efficiently target the prostate TICs in addition to the bulk tumor cells, providing new opportunities for combination therapies.

Chemotherapeutic resistance is one of the major factors contributing to cancer mortality. Although evidence supports a mechanistic link between metabolic enzyme polymorphisms and drug sensitivity, little is known about the metabolic changes tumor cells undergo when treated with a chemotherapeutic agent. Moreover, the metabolic consequences of exposure to these agents may provide essential adaptive functions contributing to chemotherapeutic resistance. Here we used metabolic flux analysis to identify metabolic changes accompanying and potentially accounting for docetaxel resistance in DU145 prostate cancer cells. We focused on the utilization of glucose and glutamine, the two most abundant extracellular nutrients, which together account for the bulk of carbon and nitrogen metabolism in tumor cells. Docetaxel resistant (DOCETR) cells consumed glutamine more avidly and secreted glutamate and ammonia at a greater rate than sensitive parental cells. Surprisingly, contribution of glutamine carbon into the TCA cycle was unchanged in DOCETR cells despite the higher glutamine utilization. Transfer of glutamine carbon into both the intracellular and extracellular glutamate pools, on the other hand, was higher in DOCETR cells, indicating that the DOCETR cells had an increased ability to convert glutamine to glutamate and then to secrete the glutamate. Accordingly, these cells also had increased expression and activity of glutaminase, the major enzyme responsible for glutamine deamidation. Because glutaminase activity does not yield energy unless the resulting glutamate is further oxidized, we hypothesized that this metabolic transition served a non-bioenergetic role, possibly supporting enhanced rates of glutathione synthesis for modification and secretion of docetaxel. Consistent with this idea, DOCETR cells displayed an increase in de novo synthesis of glycine, which is required for the production of glutathione. Importantly, a glutaminase inhibitor re-sensitized DOCETR cells to docetaxel, strongly suggesting that their enhanced glutamine metabolism was involved in resistance to the drug. We speculate that alterations in glutamine metabolism provide a general and targetable mechanism for xenobiotic resistance in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2011-1248

Conventional therapies for prostate cancer, especially in its androgen-independent form, may result in the survival of small populations of resistant cells with tumor-initiating potential. These “cancer stem cells” are believed to be responsible for cancer relapse, and therapeutic strategies targeting these cells are of great importance. Telomerase is a ribonucleoprotein enzyme responsible for telomere elongation and is activated in the majority of malignancies, including prostate cancer, but is absent in most normal cells. Putative tumor-initiating cells have significant levels of telomerase, indicating that they are an excellent target for telomerase inhibition therapy. In this review, we present some evidence for the hypothesis that conventional therapies (standard chemotherapy and/or radiation therapy) in combination with telomerase inhibitors may result in effective and more durable responses.