Lee M. Greenberger

Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785).

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (IC50 = 370+/-120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 approximately 5 nM), inhibited cell proliferation (IC50 = 31-125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.

Targeting vascular and avascular compartments of tumors with C. novyi-NT and anti-microtubule agents.

Current approaches for treating cancer are limited, in part, by the inability of drugs to affect the poorly vascularized regions of tumors. We have found that C. novyi-NT in combination with anti-microtubule agents can cause the destruction of both the vascular and avascular compartments of tumors. The two classes of microtubule inhibitors were found to exert markedly different effects. Some agents that inhibited microtubule synthesis, such as HTI-286 and vinorelbine, caused rapid, massive hemorrhagic necrosis when used in combination with C. novyi-NT. In contrast, agents that stabilized microtubules, such as the taxanes docetaxel and MAC-321, resulted in slow tumor regressions that killed most neoplastic cells. Remaining cells in the poorly perfused regions of tumors could be eradicated by C. novyi-NT. Mechanistic studies showed that the microtubule destabilizers, but not the microtubule stabilizers, radically reduced blood flow to tumors, thereby enlarging the hypoxic niche in which C. novyi-NT spores could germinate. A single intravenous injection of C. novyi-NT plus selected anti-microtubule agents was able to cause regressions of several human tumor xenografts in nude mice in the absence of excessive toxicity.

Tumor Regression and Curability of Preclinical Neuroblastoma Models by PEGylated SN38 (EZN-2208), a Novel Topoisomerase I Inhibitor

Purpose: Treatment of neuroblastoma is successful in less than half of patients with high-risk disease. The antitumor activity of a water soluble pegylated SN38 drug conjugate, EZN-2208, was compared with CPT-11 (a prodrug for SN38) in preclinical models of human neuroblastoma. Experimental Design: The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye– and Annexin V–positive cells, whereas its therapeutic efficacy was evaluated, in terms of survival, and antitumor and antiangiogenic activities, in s.c. luciferase-transfected, pseudometastatic, and orthotopic neuroblastoma animal models. Results: EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis and p53 expression and by reducing hypoxia-inducible factor (HIF)-1α/HIF-2α expression. EZN-2208 gave superior antitumor effects compared with CPT-11 in neuroblastoma xenografts. EZN-2208 treatment always resulted in lack of tumor detection at the end of trials whereas only small therapeutic effects were observed with CPT-11, as assessed by luciferase assay or tumor size, or even by staining histologic sections of tumors with antibodies recognizing neuroblastoma cells and cell proliferation. In a neuroblastoma model resistant to doxorubicin, cisplatin, vincristine, fenretinide, and topotecan, EZN-2208 induced 100% curability. It also blocked tumor relapse after topotecan-vincristine-doxorubicin combined treatment. Mechanistic experiments showed statistically significantly enhanced terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and Histone H2ax staining as well as decreased vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, and MMP-9 expression in tumors removed from EZN-2208–treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic membranes. Conclusions: EZN-2208 should be considered a most promising novel antineuroblastoma agent. An ongoing phase I study in pediatric patients should identify the optimal dose for a phase II study. Clin Cancer Res; 16(19); 4809–21. ©2010 AACR.

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.

Cellular and in Vivo Activity of JNJ-28871063, A Nonquinazoline Pan-ErbB Kinase Inhibitor That Crosses the Blood-Brain Barrier and Displays Efficacy against Intracranial Tumors

JNJ-28871063 is a potent and highly selective pan-ErbB kinase inhibitor from a novel aminopyrimidine oxime structural class that blocks the proliferation of epidermal growth factor receptor (EGFR; ErbB1)- and ErbB2-overexpressing cells but does not affect the growth of non-ErbB-overexpressing cells. Treatment of human cancer cells with JNJ-28871063 inhibited phosphorylation of functionally important tyrosine residues in both EGFR and ErbB2 and blocked downstream signal transduction pathways responsible for proliferation and survival. A single dose of compound reduced phosphorylation of ErbB2 receptors in tumor-bearing mice, demonstrating target suppression in vivo. Tissue distribution studies show that JNJ-28871063 crosses the blood-brain barrier and penetrates into tumors, where it is able to accumulate to higher levels than those found in the plasma. JNJ-28871063 showed oral antitumor activity in human tumor xenograft models that overexpress EGFR and ErbB2. In an intracranial ErbB2-overexpressing tumor model, JNJ-28871063 extended survival relative to untreated animals. The brain is a primary site of metastasis for EGFR-overexpressing lung cancers and ErbB2-overexpressing breast cancers. Therefore, the ability to penetrate into the brain could be an advantage over existing therapies such as trastuzumab (Herceptin) and cetuximab (Erbitux), which are antibodies and do not cross the blood-brain barrier. These results show that JNJ-28871063 is orally bioavailable, has activity against EGFR and ErbB2-dependent tumor xenografts, and can penetrate into the brain and inhibit ErbB2-overexpressing tumor growth.

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.

Anticancer Agents from Unique Natural Products Sources

The National Cooperative Natural Products Drug Discovery Group (NCNPDDG) “Anticancer Agents from Unique Natural Products Sources, CA 67786” was first awarded in September 1995. The goal of the project is to discover and develop novel anticancer agents from a variety of natural products sources. The key accomplishments of this NCDDG which will be highlighted in this manuscript include: Development of tools to probe fungi for the production of novel natural products by DNA-based probes. Discovery that the majority of these fungi can produce natural products via nonribosomal peptide synthetases, polyketide synthases, or both – a much larger percentage than current culturing techniques reveal. Identification of the MDR-selective cytotoxic agent austocystin D, and use of a novel yeast deletion strain approach to help identify its molecular target(s). Identification of hemiasterlin and other naturally occurring analogs as potent antimitotic agents with excellent in vivo activity against human solid tumors in mouse models. Development of a total synthesis of hemiasterlin. The utilization of this methodology to provide the first SAR for the hemiasterlin family of antimitotic agents and to identify the synthetic analog HTI-286, which is being examined in clinical trials as an anticancer agent. To provided technology transfer, educational opportunities and compensation to countries of origin for collection and study of their natural product resources. This NCNPDDG program has provided funding to research programs at the University of the Philippines, The University of the South Pacific in the Fiji Islands, Colombo University in Sri Lanka, the Instituto de Quimica de Sao Carlos, Universidade de Sao Paulo, Brazil, and the University of Papua New Guinea.

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 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