Yixian Zhang

ErbB3 Ablation Impairs PI3K/Akt-Dependent Mammary Tumorigenesis

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

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.

EZN-2208 (PEG-SN38) Overcomes ABCG2-Mediated Topotecan Resistance in BRCA1-Deficient Mouse Mammary Tumors

BRCA1 dysfunction in hereditary breast cancer causes defective homology-directed DNA repair and sensitivity towards DNA damaging agents like the clinically used topoisomerase I inhibitors topotecan and irinotecan. Using our conditional K14cre;Brca1F/F;p53F/F mouse model, we showed previously that BRCA1;p53-deficient mammary tumors initially respond to topotecan, but frequently acquire resistance by overexpression of the efflux transporter ABCG2. Here, we tested the pegylated SN38 compound EZN-2208 as a novel approach to treat BRCA1-mutated tumors that express ABCG2. We found that EZN-2208 therapy resulted in more pronounced and durable responses of ABCG2-positive tumors than topotecan or irinotecan therapy. We also evaluated tumor-specific ABCG2 inhibition by Ko143 in Abcg2−/− host animals that carried tumors with topotecan-induced ABCG2 expression. Addition of Ko143 moderately increased overall survival of these animals, but did not yield tumor responses like those seen after EZN-2208 therapy. Our results suggest that pegylation of Top1 inhibitors may be a useful strategy to circumvent efflux transporter-mediated resistance and to improve their efficacy in the clinic.

Abstract PR12: Selective targeting of MYC mRNA by chemically stabilized antisense oligonucleotides

Despite its well-characterized properties as a proto-oncogene in numerous cancers, direct potent and selective inhibition of MYC or its protein product c-Myc with cell-permeable synthetic agents remains a significant challenge. Systemic inhibition of c-Myc activity in mice by expression of dominant negative MYC transgenes has been shown to dramatically decrease tumor burden with apparently tolerable side effects (Soucek et al, Nature 2008). In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC at the transcriptional, translational, and post-translational level. Here we describe the application of synthetic antisense oligonucleotides designed to target specific sequences of the MYC mRNA (MYCASOs). We have incorporated several chemical features into MYCASOs, including the addition of locked nucleic acid (LNA) bases at the 59 and 39 ends, phosphorothioate linkers, and internal DNA bases in order to increase mRNA target affinity and cleavage, cellular permeability, stability, and systemic distribution in vivo. Treatment of MYC¬-expressing cancer cells with MYCASOs leads to a potent decrease in MYC mRNA and c-Myc protein levels. Cleaved MYC mRNA in MYCASO-treated cells is detected with a sensitive 59 rapid amplification of cDNA ends (RACE) assay. MYCASO-treatment of cancer cell lines leads to significant inhibition of cellular proliferation and induces apoptosis in c-Myc-dependent myeloma and leukemia cell lines while decreasing c-Myc-mediated gene expression. In vivo administration of MYCASOs simply dissolved in saline was performed in mice by tail vein injection. MYCASOs are well tolerated up to 25 mg per kg on a twice-weekly dosing regimen. In a MYC-induced model of hepatocellular carcinoma, MYCASO treatment leads to detectable cleavage of the MYC transcript, decreases c-Myc levels within tumors, and significantly decreases tumor burden. MYCASOs represent a new chemical tool for in vitro and in vivo modulation of c-Myc activity, and promising therapeutic agents for MYC-driven tumors. Citation Format: Christopher J. Ott, Raj Bandaru, Taylor Gill, Junyan Tao, Xin Chen, Yixian Zhang, James E. Bradner. Selective targeting of MYC mRNA by chemically stabilized antisense oligonucleotides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR12.

Abstract 5647: Marked sensitivity to mRNA targeting by LNA antisense in tumor cells without a delivery system: Lessons learned and potential translation to the clinic

Background: Unlike siRNAs, single-stranded locked nucleic acid-based antisense oligonucleotides (LNA-ONs) have shown the ability to down regulate mRNAs in vitro and in vivo without any delivery systems such as transfection reagents or liposomes. Hence, LNA-ONs may have significant advantages as a therapeutic compared to siRNAs. Investigation of LNA-ONs that target HIF-1α, survivin, or androgen receptor in cancer patients is ongoing. EZN-3920, a LNA-ON to HER3, down regulates HER3 mRNA over a large concentration range (IC50 = 0.1-10 μM) when multiple cancer cell lines are evaluated (Zhang et al, 2011 Gene Therapy. 18:326). We assess here 1) the generality of down-regulation efficiency using two additional LNA-ONs in multiple cell lines and in cells prepared directly from patient tumors, and 2) the correlation of target down-regulation, growth inhibition, and LNA-ON concentration observed in vitro with in vivo xenograft models. Methods: LNA-ONs were 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, and tumor size. Gene Expression Profiling was performed by Asuragen, Inc. Concentration of LNA-ONs in tissues was measured by LC/MS/MS. Results: LNA-ONs targeting PI3KCA p110α (EZN-4150) or β-catenin (EZN-3892) potently down regulated the target mRNA in 50% of tumor cell lines, thereby confirming the observations made with EZN-3920. Diverse sensitivity of the HER3-directed LNA-ON was also observed in cells derived from over 40 primary tumors. Cell lines that were highly sensitive to LNA-ONs in proliferation assay were also responsive in xenograft tumor models. When anti-tumor efficacy of the LNA-ONs was achieved, the concentration of the drug within the tumor matched the concentration required to effectively down regulate the target in vitro. To understand the mechanisms underlying the resistance to LNA-ONs-mediated down-modulation, several cell lines were made highly resistant to LNA-ONs in vitro by chronic exposure to the agent over several months. Tumors from such cell lines were also resistant to LNA-ONs in xenograft models and can be cross-resistant to multiple LNA-ONs. We are currently identifying common genetic differences in the sensitive and resistant cell. Conclusion: We have shown that LNA-ONs, given without any delivery systems, have broad utility in many cancer cell lines including primary tumor cells in which the intended target is a driver for growth. Furthermore, those cell lines sensitive to LNA-ONs in vitro or ex vivo may help identify tumors in animal models that will be responsive to LNA-ONs. Lastly, mechanistic studies on resistant cells may provide an understanding of the diverse response of tumors to LNA-ONs that could ultimately be used to select patients who might preferentially respond to therapy with LNA-ONs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5647. doi:1538-7445.AM2012-5647

Abstract A74: Targeting the β-catenin oncogene in multiple myeloma disease using locked nucleic acid technology.

β-catenin is an oncogenic transcription factor that lies down stream of the Wnt signaling pathway. Typical of many transcription factors, it has been exceeding difficult to identify small molecule inhibitors of β-catenin. Therefore, we have applied an alternative technology using locked nucleic acid (LNA)-based short antisense oligonucletides (ON) to specifically destroy β-catenin mRNA. Such LNA-ONs to the androgen receptor, HIF-1α, and survivin are currently being evaluated in cancer patients. Previously, we established anti-tumor activity of a β-catenin specific LNA-ON (EZN-3892) in colorectal and lung cancer xenograft models (Dumble, M et al , Poster, EORTC 2010, Berlin.). In the current studies, we have explored the LNA-ONs in experimental models of multiple myeloma (MM) since uncontrolled clonal expansion of plasma cells within the bone marrow, which typifies MM, is highly dependent on stromal secretion of various cytokines (e.g. IL6) and notably several Wnt ligands. Wnt signaling can promote hematopoietic stem cell proliferation and self renewal as well as trigger the proliferation of tumor cells via the activation of -catenin. In addition, -catenin has been shown to be stabilized and in the nucleus of primary patient MM samples as well as many MM cell lines (Sukhdeo, K et al , 2007, PNAS (104), 7516–7521). Finally, several groups have shown that inhibition of β-catenin by siRNA silencing or a natural product small molecule results in decreased MM cell viability and growth, although further development of these molecules has not occurred. The study reported herein describes a novel 16-mer LNA-ON antagonist of β-catenin, EZN-3892, and its anti-tumor effect on a panel of eight MM cell lines. Robust mRNA decrease is measured in all MM cell lines treated with a dose titration of EZN-3892 in 3-day culture without the aid of transfection agents (“gymnotic culture”). As expected, this mRNA decrease results in an inhibition of β-catenin protein. Surprisingly however, inhibition of the β-catenin protein is not sufficient to result in an anti-proliferative effect in all eight cell lines with only 2/8 lines having 25–35% inhibition of growth when treated with 5 M of EZN-3892. Despite moderate anti-proliferative effect in vitro, the anti-tumor effect is much greater when tested in vivo in tumor xenograft experiments. Treatment of mice bearing OPM-2 MM tumor xenografts with a well tolerated dose of EZN-3892 (50 mg/kg EZN-3892, Q3D×4), results in 85% tumor growth inhibition after 10 days. Tumor growth inhibition is associated with an inhibition of β-catenin in the tumor environment (stroma) and tumor cell death. Interestingly, tumor cells that showed no growth inhibition in 2-D culture likewise show no tumor growth inhibition in a xenograft model suggesting the 2-D proliferation assay in vitro is predictive of tumor response in vivo. Similar observations have been made with EZN-3892 and mouse xenograft models derived from human colorectal and lung cancer cells with activated β-catenin signaling. This work raises the possibility that targeted inhibition of β-catenin within the tumors, as well as the stroma surrounding the tumor are critically important in the control of MM. 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 A74.

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 5394: Dual inhibition of the androgen receptor by ligand blockade and antisense-mediated downregulation is associated with synergistic antitumor activity model of prostate cancer

Background: Although new agents to treat prostate cancer have recently been approved, and new investigational drugs (Abiraterone and MDV3100) that mediate hormone deprivation show promising data from late-stage clinical trials, these therapies typically extend overall survival up to 4 months in patients with advanced disease who have failed conventional androgen deprivation therapy. Hence, there continues to be great medical need. EZN-4176 is a novel LNA-based antisense oligonucleotide (EZN-4176) that down regulates androgen receptor (AR) expression. Previously, antitumor activity of EZN-4176 in multiple xenograft models including both androgen-sensitive and castrate-resistant tumors was shown. More importantly, a synergistic effect was also found when EZN-4176 was combined with MDV3100. Further exploration of the mechanistic underpinning of the effect was explored here. Methods: The mRNA, growth, luciferase activity, protein, and prostate specific antigen (PSA) were evaluated by qRT-PCR, MTT, SteadyGlo, western blot analysis, and ELISA assay, respectively. The effect of EZN-4176 on AR transcriptional activity was evaluated in cells stably express luciferase gene regulated by AR. In vivo, the effect of EZN-4176 on AR transcriptional activity was evaluated in a bone tumor model developed by intratibia injection of C4-2b-AR-luc cells. Results: Combination of EZN-4176 with MDV3100 resulted in a much improved inhibitory effect in colony formation assay in vitro. This effect was not associated with enhanced efficiency of AR mRNA down-modulation as treatment with EZN-4176 with or without MDV3100 showed similar effects on the level of AR mRNA. However, when AR transcriptional activity was examined, we found that the combined effect was greater than each agent alone. In C4-2b-AR-luc bone tumor model, EZN-4176 potently and specifically down-modulated AR-luciferase reporter activity. Further examinations of the anti-tumor effect in this model will help understand the potential of the combinatorial effect in a highly relevant disease model. Conclusions: Our preliminary data suggest that the synergistic anti-tumor effect of EZN-4176 in combination with MDV3100 is in part associated with much reduced transcriptional activity of AR. EZN-4176 alone or in combination with anti-androgens offers great potential in treating patients with advanced prostate cancer. 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 5394. doi:10.1158/1538-7445.AM2011-5394