Puja Sapra

Down-modulation of survivin expression and inhibition of tumor growth in vivo by EZN-3042, a locked nucleic acid antisense oligonucleotide.

Survivin plays an important role in preventing apoptosis and permitting mitosis, and is highly expressed in various human cancers. EZN-3042 is a locked nucleic acid antisense oligonucleotide (LNA-AsODN) against survivin. We report the effects of EZN-3042 in animal models. In a chemical-induced liver regeneration model, treatment with a mouse homolog of EZN-3042 resulted in 80% down-modulation of survivin mRNA. In A549 and Calu-6 lung xenograft models, treatment with EZN-3042 single agent induced 60% down-modulation of survivin mRNA in tumors and 37–45% tumor growth inhibition (TGI). In Calu-6 model, when EZN-3042 was combined with paclitaxel, an 83% TGI was obtained. EZN-3042 is currently being evaluated in a Phase 1 clinical trial as a single agent and in combination with docetaxel.

Abstract C215: EZN‐2208, a novel pegylated SN‐38 drug conjugate, markedly inhibits tumor growth and metastatic spreading in preclinical models of human neuroblastoma

Treatment of neuroblastoma (NB) is successful in less than half of patients with high‐risk disease. Camptothecin and its analog irinotecan (CPT‐11), hold great promise but several limitations suggest that chemical modifications may improve the therapeutic index. EZN‐2208 is a water soluble pegylated SN38 drug conjugate, composed of a four‐arm 40 KDa polyethylene glycol (PEG) linked via glycine residue to SN38. In various preclinical models of solid tumors EZN‐2208 was more efficacious than CPT‐11. In phase I trials in adult tumors, EZN‐2208 was well‐tolerated with neutropenia as the dose limiting toxicity. Here, the anti‐tumor activity of EZN‐2208 was first evaluated in preclinical, pseudometastatic and an orthotopic, models of human NB. Mice were treated every other day for 5 total doses with 10 mg/kg of CPT‐11 or with the SN38 equivalents of EZN‐2208. In the first model, mice treated with EZN‐2208 displayed significant increased life span compared to control mice or those treated with CPT‐11. After 150d post cell implantation, all EZN‐2208‐treated mice were still disease‐free, while control and CPT‐11‐treated animals died with metastatic disease. In the orthotopic model, mice treated with EZN‐2208 showed a dramatic arrest and regression in primary tumor growth compared to control mice. While CPT‐11‐treated mice died with widespread tumor masses within 80 days, long term survival was seen in 100% of EZN‐2208‐treated animals. 21 days after the end of treatment tumors had almost disappeared, as assessed by staining histological sections of the tumors with antibodies recognizing NB cells and the cell proliferation marker, Ki‐67. In a second set of in vivo experiments, MTD doses of both CPT‐11 and EZN‐2208 were compared in immunodeficient (GI‐LI‐N cells) and immunocompetent (NXS2 cells) orthotopic NB animal models. While CPT‐11 at MTD dose led to a partial increased in long term survival, EZN‐2208‐treated, GI‐LI‐N‐bearing mice were 100% cured after 180 days post cells implantation. In the very aggressive syngeneic NB animal model (NXS2 cells), while CPT‐11 did not exert any anti‐tumor effect, EZN‐2208 led to 100% and 40% of long term survivors, in mice challenged with 5x104 and 5x105 tumor cells, respectively. The differences in the anti‐angiogenic activity between CPT‐11 and EZN‐2208 was evaluated by chorioallantoic membrane (CAM) assay. Incubation of the CAMs with EZN‐2208 significantly reduced the number of radiating vessels that invaded the implant compared to either specimens alone or CAMs incubated CPT‐11. In the last set of in vivo experiments, the effects of EZN‐2208 and CPT‐11 were compared in a luciferase‐transfected human NB cells inoculated in the right flank of mice. EZN‐2208 led to a significant tumor regression compared to CPT‐11. Finally, mechanistic experiments showed enhanced TUNEL and Histone H2ax staining in tumors removed from mice treated with EZN‐2208, indicating its effect on tumor cell apoptosis. In conclusion, EZN‐2208 could be considered as a new, promising anti‐NB agent, to be administered alone and/or in combination with traditional chemotherapeutics. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C215.

Abstract C141: In vitro and in vivo characterization of two novel β‐catenin RNA antagonists, EZN‐3889 and 3892

Background: β‐Catenin is an important signaling molecule in the Wnt pathway that plays a key role in tumorgenesis. In the absence of Wnt signaling, the cytoplasmic level of β‐catenin is kept low due to rapid proteasomal‐mediated degradation of GSK3β phosphorylated β‐catenin. Activation of Wnt signaling leads to the inactivation of GSK3β, resulting in stabilization and accumulation of β‐catenin in the cytoplasm. Consequently, β‐catenin translocates into the nucleus, where it binds with members of the T‐cell factor (Tcf)/lymphocyte enhancer‐binding factor family of transcription factors and activates the expression of many target genes important for cancer development. Most colon cancers have activating mutations in the APC tumor suppressor or in β‐catenin itself. Furthermore, activating β‐catenin mutations have been found in a variety of other tumors such as melanomas, hepatocellular carcinomas, skin, breast, and prostate cancer, whereas β‐catenin is not activated in most normal tissues. Therefore, inhibition of β‐catenin is likely to have therapeutic effects in many cancers. We report here the identification of two β‐catenin LNA‐based mRNA‐antagonists, EZN‐3889 and EZN‐3892. Material and Methods:In vitro, the ability of the compounds to inhibit mRNA, cell growth, and reporter gene were evaluated by qRT‐PCR, MTS, and luciferase assays respectively, in multiple cell lines. In vivo, β‐catenin mRNA down‐modulation in liver and human tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the compounds. Results: These molecules were potent down‐modulators of β‐catenin (IC50 = 0.1 to 3 nM) as well as protein (> 80% at 20 nM) of multiple cell lines, when transfected into tumor cells. Growth inhibition appeared to correlate with the status of either APC mutation or activating β‐catenin mutations, suggesting that β‐catenin is the driver of cancer cells. Interestingly, we found that these two antagonists specifically down modulated β‐catenin mRNA and protein effectively in multiple cell lines with a long‐lasting effect in the absence of any tranfection reagent. In mice, both molecules significantly down modulated β‐catenin mRNA in the liver. Conclusions: β‐catenin antagonists potently and specifically inhibited β‐catenin mRNA expression both in vitro and in vivo after intravenous injection. Further studies will examine the antitumor efficacy of the compounds. hese novel agents specifically inhibit a transcription factor that has been difficult to target with conventional agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C141.

Abstract C146: LNA (locked nucleic acid) oligonucleotide based GLI2 RNA antagonist EZN‐4482 effectively inhibited tumor growth and increased survival of animals with liver metastasis

Background: The Hedgehog (Hh) pathway has been implicated in the growth of a number of human malignancies and cancer stem cells. In the presence of Hh ligands, PTCH1 inhibition of a membrane protein, Smoothened (SMO), is relieved. SMO activation triggers a cascade of downstream events that ultimately activate the zinc finger transcription factors GLI2 and GLI3 and induce transcription of their target genes such as GLI1, PTCH1, Cyclin D, and BCL2. Emerging evidence suggests that GLI1 and GLI2 represent the main activators of Hh pathway due to amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway. Therefore, specific down regulation of GLIs with RNA antagonists such as siRNA and antisense molecules may offer an effective therapeutic approach for cancer treatment. We reveal here the biological activities of LNA‐based GLI2 mRNA antagonist, EZN‐4482 and 4496. Material and Methods:In vitro, the ability of the antagonists to down modulate mRNA and cell growth was evaluated by qRT‐PCR and MTS respectively. In vivo, GLI2 mRNA down‐modulation in tumors, which were grown on the flank of nude mice, was evaluated after intravenous administration of the antagonists. The effect of antagonists on tumor growth and survival of mice were evaluated in PC3 prostate tumor grown on the flank and in A549 lung carcinoma cells that had metastasized to the liver respectively. Results: Both antagonists, when transfected into tumor cells, were potent down‐modulators of GLI2 mRNA (IC50 ≤ 2 nM), resulting caspase ativation and growth inhibition. Interestingly, we found that without any transfection reagent, 1–10 micromolar concentrations of these antagonists were able to down modulate GLI2 mRNA and inhibit the growth of multiple cell lines while their scrambled control oligonucleotides showed no effect, suggesting that the effect was specific. In mice, EZN‐4482 (3 mg/kg given q3d x4) down modulated GLI2 mRNA in PC3 prostate tumors. Moreover, using murine specific probes, we found EZN‐4482 also inhibited GLI1/2 and PTCH1 in the same tumors. Moderate tumor growth inhibition was found with EZN‐4482 in the PC3 xenograft model. Additionally, we show that EZN‐4482 prolonged the survival of animals with liver metastasis derived from A549 cells. Conclusions: GLI2 antagonists potently and specifically inhibited GLI2 mRNA expression and tumor growth in two tumor models. The possible mechanisms of efficacy include inhibition of mRNA levels of GLI1/2 and PTCH1 in mouse stromal cells. While small molecule inhibitors of SMO are being evaluated clinically, this approach is limited to tumors where pathway activation is upstream of GLIs or tumors that are resistant to such inhibitors. Therefore, a GLI2 RNA antagonist may be an effective therapy to treat a broad spectrum of cancers including ones that fail treatment with SMO inhibitor therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C146.

Abstract C148: LNA (locked nucleic acid)‐based RNA antagonists mediate target mRNA silencing without transfection by simple addition to cell culture media

Background: Since oligonucleotides do not penetrate cells well, most RNA‐based therapies, such as antisense molecules and siRNAs use transfection conditions in tissue culture and delivery technologies in animals to facilitate entry of oligonucleotides into target cells. However, such vehicles are known to substantially alter cellular function and thereby significantly confound analyses. We have been developing LNA‐based RNA antisense molecules to key cancer targets including survivin, HIF‐1α, androgen receptor, GLI2, β‐catenin, HER3, p110 alpha, and HSP27. LNA‐based oligonucleotides were choosen since these molecules are highly stabile in plasma, have extraordinary high affinity to mRNA, and potently silence mRNA in cells under transfection conditions (IC50 in the high picomolar ‐ low nanomolar range). We now report that LNA based RNA antagonist, when used in the high nanomolar ‐ low micromolar range readily down modulate mRNA and protein of interests in multiple cell lines without any delivery means. Material and Methods: In vitro, the ability of the antagonists to down modulate mRNA and cell growth was evaluated by qRT‐PCR and MTS respectively. Cellular localization of the antagonists was evaluated by examining a FAM‐labeled HER3 antagonist EZN‐3920 under fluorescent microscope. In vivo, HER3 mRNA down‐modulation and protein levels in tumors, which were grown on the flank of nude mice, were evaluated after intravenous administration of the antagonists. Results: The down‐modulation of mRNA in tissue culture‐treated cells was quick, robust, long lasting, and specific followed by potent protein inhibition. Importantly, we show that mechanisms underlying the down‐modulation correlated with nuclear localization of the LNA‐ASO, suggesting that RNas H was the primary mechanism involved in target inhibition. Using this simple and effective approach, we were able to demonstrate that HER3, GLI2, and β‐catenin were likely crucial growth and survival factors for certain cancer cells. Conclusions: LNA‐based RNA antagonists down‐modulate target mRNA and protein in the absence of any agent to facilitate cell penetration. Such antagonists represent an attractive option for rapid, simple, and specific determination of gene function, target validation, and identification of drug‐sensitive tumors for further in vivo evaluation. Beyond this, these results helps validate the use of intravenously administered LNA oligonucleotides simply prepared in saline, as is being done in on‐going clinical trials with LNA‐based RNA antagonists to HIF‐1α and survivin. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C148.

Abstract C144: A locked nucleic acid antisense oligonucleotode against androgen receptor, down‐modulates target mRNA and causes antitumor effects in xenograft models of prostate cancer

Background: Androgen‐deprivation therapies remain the first line treatment for prostate cancer. Despite an excellent initial response, most of these patients will succumb to the castration resistant form of the disease. Interestingly, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional androgen receptor (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)‐based antisense oligonucleotide (ASO), EZN‐4176, that silence AR and is associated with tumor growth inhibition. Methods: Target mRNA and protein knockdown, growth inhibition, and apoptosis induction effects of EZN‐4176 were evaluated by qRT‐PCR, western blot analysis, ELISA, MTS assay, and caspase3/7 activity assay, respectively, in AR‐positive cancer cell lines (LNCaP, 22RV1) or AR‐negative cell line (PC3) in the presence or absence of lipofection (transfection reagent). In vivo, therapeutic efficacy of EZN‐4176 was evaluated in AR‐positive CWR22 (androgen dependent) and 22Rv1 (castration refractory) tumor models. Results: EZN‐4176 resulted in potent down‐modulation of AR mRNA or protein (IC50 Conclusions: Our data suggest that EZN‐4176 is a potent LNA‐based ASO of AR that causes target down‐modulation and TGI effects in vivo and hence may provide a novel strategy to treat advanced prostate cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C144.