The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3 0 -P5 0 thiophosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first-(GRN163) and secondgeneration (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC 50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipidconjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Differential regulation of telomerase activity in normal and tumor cells provides a rationale for the design of new classes of telomerase inhibitors. The telomerase enzyme complex presents multiple potential sites for the development of inhibitors. GRN163L, a telomerase enzyme antagonist, is a lipid-modified 13-mer oligonucleotide N3V ! P5V -thiophosphoramidate, complementary to the template region of telomerase RNA (hTR). We evaluated both the in vitro and in vivo effects of GRN163L using A549-luciferase (A549-Luc) human lung cancer cells expressing a luciferase reporter. GRN163L (1 Mmol/L) effectively inhibits telomerase activity of A549-Luc cells, resulting in progressive telomere shortening. GRN163L treatment also reduces colony formation in soft agar assays. Surprisingly, after only 1 week of treatment with GRN163L, A549-Luc cells were unable to form robust colonies in the clonal efficiency assay, whereas the mismatch control compound had no effect. Finally, we show that in vivo treatment with GRN163L is effective in preventing lung metastases in xenograft animal models. These in vitro and in vivo data support the development of GRN163L as a therapeutic for the treatment of cancer. (Cancer Res 2005; 65(17): 7866-73)

Telomerase activity is undetectable in most normal tissues but the vast majorities of cancers express active telomerase. Therefore, telomerase serves as an attractive target for the treatment of cancers. GRN163L is a lipid-modified oligonucleotide N3'-->P5' thio-phosphoramidate complementary to the RNA template region of human telomerase. The anti-telomerase activity of GRN163L was evaluated using MDA-MB-231 and MDA-MB-435 human breast adenocarcinoma cell lines. Twice weekly administration of GRN163L resulted in the inhibition of telomerase activity and progressive telomere shortening. Cells treated with GRN163L did not demonstrate decreased cell proliferation for up to 2 weeks. However, after additional treatment, cell proliferation gradually decreased in GRN163L-treated cells compared to untreated or mismatch control oligoncleotide treated cells. Furthermore, anti-tumorigenic effects were seen in cells treated with GRN163L, as cells lose their ability to form colonies in soft agar and were unable to form colonies in the clonal efficiency assay upon incubation with GRN163L. Moreover, breast cancer cells that were treated with GRN163L for only 1 week prior to plating in invasion chambers, and when bulk telomere are still long, exhibit significantly diminished invasive potential. These results reveal critical information regarding the effectiveness of GRN163L as a potential therapeutic agent for the treatment of human breast cancer.

Peloruside A is a microtubule-stabilizing agent isolated from a New Zealand marine sponge. Peloruside prevents growth of a panel of cancer cell lines at low nanomolar concentrations, including cell lines that are resistant to paclitaxel. Three xenograft studies in athymic nu/nu mice were performed to assess the efficacy of peloruside compared with standard anticancer agents such as paclitaxel, docetaxel, and doxorubicin. The first study examined the effect of 5 and 10 mg/kg peloruside (QDÂ5) on the growth of H460 non-small cell lung cancer xenografts. Peloruside caused tumor growth inhibition (%TGI) of 84% and 95%, respectively, whereas standard treatments with paclitaxel (8 mg/kg, QDÂ5) and docetaxel (6.3 mg/kg, Q2DÂ3) were much less effective (%TGI of 50% and 18%, respectively). In a second xenograft study using A549 lung cancer cells and varied schedules of dosing, activity of peloruside was again superior compared with the taxanes with inhibitions ranging from 51% to 74%, compared with 44% and 50% for the two taxanes. A third xenograft study in a P-glycoprotein-overexpressing NCI/ADR-RES breast tumor model showed that peloruside was better tolerated than either doxorubicin or paclitaxel. We conclude that peloruside is highly effective in preventing the growth of lung and P-glycoprotein-overexpressing breast tumors in vivo and that further therapeutic development is warranted.

HIF-1α plays a major role in activating gene transcription and is important for maintaining homeostasis under hypoxic conditions. Since tumors are often in a hypoxic state, HIF-1α is a potential target for the development of novel cancer therapeutics. This study was performed to determine the antitumoral efficacy of an antisense HIF-1α inhibitor, RX-0047 on different human cancer cell lines (MDA-MB 231, HME50-T, PC-3, Panc-1 and A549) in vitro. A549 lung cancer and PC-3 prostate cancer cells containing a luciferase gene reporter were used for in vivo xenograft animal models. Progressive tumor development was quantified using live animal BLI (bioluminescence imaging) in addition to ex vivo imaging and histology. All cell lines tested were sensitive to inhibition of cell growth with 10 nM and higher ranges of RX-0047, additionally RX-0047 sensitizes cells to ionizing radiation treatments. Finally, RX-0047 (30 mg/kg) inhibited the formation of human lung metastasis in xenograft mouse models and reduced tumor size in flank models. Keywordshypoxia; HIF-1α; RX-0047; cancer; bioluminescence imagingThe hypoxia-inducible factors (HIFs) are key transcriptional regulators of the hypoxic response in both adult and embryonic organisms. HIF-1 regulates cellular adaption to oxygen deficiency by regulating genes involved in erythropoiesis, iron metabolism, glycolysis, angiogenesis, inhibition of apoptosis, tissue matrix metabolism, cell proliferation and survival, which are key factors for tumor growth [Semenza, 2002;Bracken et al., 2003;Goda et al., 2003]. Loss of HIF-1α activity dramatically decreases tumor growth, vascularization, and energy metabolism, whereas overexpression of HIF-1α increases HIF-1 transcription factor activity and promotes tumor growth [Powis and Kirkpatrick, 2004].HIF-1 is a heterodimer consisting of the oxygen sensitive HIF-1α (120 kDa) and the constitutively expressed HIF-1β subunit (80 kDa) which is also known as aryl hydro-carbon receptor nuclear translocator, ARNT . Both subunits contain a basic-helix-loop-helix motif and a Per Arnt Sim (PAS) protein-protein interaction domain . Recent studies demonstrated that hydroxylation of HIF-1α at two NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript specific proline residues (Pro-402 and Pro-564) is catalyzed by prolyl-4-hydroxylase (PHD) using molecular oxygen, Fe +2 and oxoglutarate as substrates [Ivan et al., 2001]. Under normoxic conditions, the tumor suppressor von Hippel-Lindau (VHL) protein specifically interacts with hydroxylated HIF-1α and mediates the assembly of a complex that activates an ubiquitin-dependent proteasome system, therefore HIF-1α subunit is kept low due to massive ubiquitination and subsequent proteosomal degradation [Salceda and Caro, 1997;Maxwell et al., 1999]. However, during hypoxia, proline is not hydroxylated, and ubiquitination is inhibited, causing accumulation of the HIF-1α protein [Salceda and Caro, 1997;Maxwell et al., 1999;Bruick, 2003]. Stabilized HIF-1α protein translocates into the nu...