Maoliang Wang

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.

Reduced Expression of the Androgen Receptor by Third Generation of Antisense Shows Antitumor Activity in Models of Prostate Cancer

The androgen receptor (AR) is a member of a unique class of transcription factors because it contains a ligand-binding domain that, when activated, results in nuclear translocation and the transcriptional activation of genes associated with prostate cancer development. Although androgen deprivation therapies are effective initially for the treatment of prostate cancer, the disease eventually relapses and progresses to castration-resistant prostate cancer (CRPC). Nonetheless, the AR still plays a critical role because late-stage investigational agents that deplete testosterone (abiraterone) or block ligand binding (MDV3100) can still control tumor growth in patients with CRPC. These findings indicate that downmodulation of AR expression may provide a complementary strategy for treating CRPC. In this article, we describe a novel, locked, nucleic acid–based antisense oligonucleotide, designated EZN-4176. When administered as a single agent, EZN-4176 specifically downmodulated AR mRNA and protein, and this was coordinated with inhibition of the growth of both androgen-sensitive and CRPC tumors in vitro as well as in animal models. The effect was specific because no effect on growth was observed with a control antisense oligonucleotide that does not recognize AR mRNA, nor on tumors derived from the PC3, AR-negative, tumor cell line. In addition, EZN-4176 reduced AR luciferase reporter activity in a CRPC model derived from C4-2b cells that were implanted intratibially, indicating that the molecule may control prostate cancer that has metastasized to the bone. These data, together with the continued dependency of CRPC on the AR signaling pathway, justify the ongoing phase I evaluation of EZN-4176 in patients with CRPC. Mol Cancer Ther; 10(12); 2309–19. ©2011 AACR.

Downregulation of HER3 by a Novel Antisense Oligonucleotide, EZN-3920, Improves the Antitumor Activity of EGFR and HER2 Tyrosine Kinase Inhibitors in Animal Models

Among the four human EGF receptor (HER) family members (EGFR, HER2, HER3, HER4), HER3 is of particular interest as it interacts with HER2 and EGFR via heterodimerization and is a key link to the phosphoinositide 3-kinase (PI3K)/AKT signal transduction axis. Recent studies indicate that HER3 plays a critical role in mediating resistance to agents that target EGFR or HER2. As HER3 lacks significant kinase activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors of HER3 have been sought as cancer therapeutics. We describe here a locked nucleic acid (LNA)-based HER3 antisense oligonucleotide, EZN-3920, that specifically downmodulated the expression of HER3, which was associated with growth inhibition. EZN-3920 effectively downmodulated HER3 expression, HER3-driven PI3K/AKT signaling pathway, and growth in tumors derived from BT474M1 breast and HCC827 lung carcinoma cell lines, which overexpress HER2 and EGFR, respectively. Furthermore, when EZN-3920 was coadministered with gefitinib or lapatinib in xenograft tumor models, enhanced antitumor activity compared with the effect of monotherapy was found. The effect was associated with a blockade of induced HER3 mRNA expression caused by lapatinib or gefitinib treatment. Finally, EZN-3920 sustained its antiproliferative effect in trastuzumab-resistant cells and three independently derived gefitinib-resistant cells. Our findings show that downmodulation of HER3 by EZN-3920 leads to the suppression of tumor growth in vitro and in vivo, suggesting that HER3 can be an effective target for the treatment of various cancers that have been activated by HER3 alone or where HER3 activation is associated with EGFR or HER2 expression. Mol Cancer Ther; 12(4); 427–37. ©2013 AACR.

Abstract A147: EZN-3920, a HER3 antagonist, enhances the antitumor effect of gefitinib and lapatinib in xenograft models.

Background: The HER family consists of four tyrosine kinase receptors designated as EGFR, HER2, HER3 and HER4. Numerous new studies now indicated that HER3 plays a critical role in cancer development since it 1) heterodimerizes with EGFR or HER2, 2) is a critical link to the PI3K/AKT axis, and 3) can mediate resistance to HER1/2-targeted therapeutics. Resistance to the tyrosine kinase inhibitors (TKIs), such as gefitinib and lapatinib, are associated with overexpression of HER3 ultimately leading to activation of the PI3K/AKT axis. Thus, it is a logical approach to combine agents capable of down modulating HER3 with TKIs. To this end, we have used an RNA antagonist of HER3, EZN-3920, to explore its ability to inhibit tumor growth and enhance the activity of HER1/2-directed therapeutics. Methods: Tissue culture cells were treated with EZN-3920, a locked nucleic acid (LNA)-based oligonucleotide complementary to HER3. EZN-3920 was either added to tissue culture media (i.e. no transfection) in vitro or prepared in saline and given IV in vivo. Endpoints were measured by qRT-PCR, MTT, Western Blot analysis, Immunohistochemistry, and tumor size. Results: EZN-3920 down modulated HER3 mRNA, protein expression, PI3K/AKT signaling, and inhibited tumor cell proliferation. In vivo systemic administration of EZN-3920 resulted in specific down-modulation of HER3 mRNA and protein expression, as well as blockade in PI3K/AKT signaling pathways in tumors derived from HCC827 lung carcinoma cells that was associated with tumor growth inhibition. Interestingly, the combination of EZN-3920 at 30 mg/kg (biweekly for 4 weeks, i.v.) and gefitinib at 15 mg/kg (5 times a week for two weeks, p.o) completely shrank established HCC827 tumors while either treatment alone only delayed tumor growth by 40–60%. Similarly, in BT-474-M1 breast carcinoma xenograft models, EZN-3920 in combination with lapatinib resulted in superior antitumor effect than either agent alone. Conclusions: The studies suggest that the antitumor effects of gefitinib or lapatinib can be enhanced by HER3 down modulation in NSCLC and breast tumors, respectively. On-going studies will determine if tumors that are resistant to HER1/2 therapeutics will still respond to EZN-3920 alone or its combination with standard HER1/2 therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A147.

Abstract 232: Targeting HER3 mRNA by a locked nucleic antisense molecule enhances the antitumor activity of gefitinib in vivo

Background: The HER family consists of four tyrosine kinase receptors designated as EGFR, HER2, HER3 and HER4. HER3 is a unique member that plays a critical role in tumor growth since it 1) can heterodimerize with EGFR or HER2 and is the main link to the downstream PI3K/Akt signaling axis, 2) can mediate resistance to HER1/2-targeted therapeutics, and 3) unlike EGFR or HER2, is not typically overexpressed but still hyperphosphorylated in a variety of tumors. Resistance to the EGFR-tyrosine kinase inhibitors, such as gefitinib, can be associated with EGFR mutations and/or activation of HER3 ultimately leading to activation of the PI3K/AKT axis. We have used an RNA antagonist of HER3, designated EZN-3920, to explore the ability of this compound to inhibit tumor growth and overcome resistance to HER1/2 therapeutics. Methods: Tissue culture cells were treated with EZN-3920, a locked nucleic acid (LNA)-based oligonucleotide complementary to HER3. Compound was either added to tissue culture media (i.e. no transfection) in vitro or prepared in saline and given IV in vivo. Endpoints were measured by qRT-PCR, MTT, Western Blot analysis, Immunohistochemistry, and tumor size. Results: EZN-3920 down modulated HER3 mRNA, protein expression, PI3K/AKT signaling, and inhibited tumor cell proliferation. In vivo, systemic administration of EZN-3920, prepared in saline, resulted in specific down- modulation of HER3 mRNA and protein expression, as well as blockade in PI3K/AKT signaling pathways in NSCLC HCC827 associated with tumor growth inhibition. Similar results were also shown in tumor derived from BT-474-M1 breast carcinoma xenograft models. Interestingly, in the HCC827 tumor model, EZN-3920 at 30 mg/kg (biweekly for 4 weeks, i.v.) and gefitinib at 15 mg/kg (5 times a week for two weeks) completely shrank established tumors while either treatment alone only delayed tumor growth by 40-60%. Conclusions: The studies suggest that down regulation of HER3 by antisense molecule EZN-3920 inhibits human tumor growth in mice. Beyond this, antitumor effects of gefitinib can be enhanced by HER3 down modulation in gefitinib-sensitive tumors. On-going studies will determine if tumors that are resistant to HER1/2 therapeutics will either have enhanced sensitivity to EZN-3920 and/or the compound can restore sensitivity to such agents. 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 232. doi:10.1158/1538-7445.AM2011-232

Abstract 600: Targeting the hedgehog pathway by LNA (locked nucleic acid) oligonucleotide-based GLI2 RNA antagonists, EZN-4482 and EZN-4496, in vitro and in vivo

Background: Aberrent activation of hedgehog (Hh) signaling has been implicated in the initiation and growth of a number of human malignancies, including those derived from skin, lung, liver, prostate and breast. The GLI family proteins are zinc finger transcription factors and the downstream effectors of Hh pathway, controling expression of the target genes such as GLI1, Ptch1, Cyclin D, and BCL2. Since amplification, overexpression of Hh ligand, and mutations of components along the Hh pathway are found in cancer and cancer stem cells, specific down regulation of GLIs with antisense molecules may offer a novel and effective therapeutic approach for cancer treatment. We report here the biological activities of LNA-based GLI2 mRNA antagonist, EZN-4482 and EZN-4496. Methods: In vitro, the ability of the antagonists to down modulate their direct target, GLI2 mRNA, and to inhibit cell growth was evaluated by qRT-PCR and MTS, respectively, in multiple cell lines of various origins. To identify and investigate the molecular events associated with targeted downmodulation of GLI2, expression of Hh pathway components and downstream effectors were analyzed by gene expression profiling and immnomoblotting analysis. In vivo, GLI2 mRNA down-modulation in PC3 xenograft tumor 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 are specific and potent down-modulators of GLI2 mRNA (by transfection, IC 50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 600.

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.