Progression Elevated Gene-3 Research Articles

Conversion of a Non-Cancer-Selective Promoter into a Cancer-Selective Promoter.

Simple SummaryThe rat progression elevated gene-3 (PEG-3) promoter displays cancer-selective expression, whereas the rat growth arrest and DNA damage inducible gene-34 (GADD34) promoter lacks cancer specificity. PEG-3 and GADD34 minimal promoters display strong sequence homology except for two single point mutations. Since mutations are prevalent in many gene promoters resulting in significant alterations in promoter specificity and activity, we have explored the relevance of these two nucleotide alterations in determining cancer-selective gene expression. We demonstrate that these two point mutations are required to transform a non-cancer-specific promoter (pGADD) into a cancer-selective promoter (pGAPE). Additionally, we found GATA2 transcription factor binding sites in the GAPE-Prom, which regulates pGAPE activity selectively in cancer cells. This newly created pGAPE has all the necessary elements making it an appropriate genetic tool to noninvasively deliver imaging agents to follow tumor growth and progression to metastasis and for generating conditionally replicating adenoviruses that can express and deliver their payload exclusively in cancer.Progression-elevated gene-3 (PEG-3) and rat growth arrest and DNA damage-inducible gene-34 (GADD34) display significant sequence homology with regulation predominantly transcriptional. The rat full-length (FL) and minimal (min) PEG-3 promoter display cancer-selective expression in rodent and human tumors, allowing for cancer-directed regulation of transgenes, viral replication and in vivo imaging of tumors and metastases in animals, whereas the FL- and min-GADD34-Prom lack cancer specificity. Min-PEG-Prom and min-GADD34-Prom have identical sequences except for two single-point mutation differences (at −260 bp and +159 bp). Engineering double mutations in the min-GADD34-Prom produce the GAPE-Prom. Changing one base pair (+159) or both point mutations in the min-GADD34-Prom, but not the FL-GADD34-Prom, results in cancer-selective transgene expression in diverse cancer cells (including prostate, breast, pancreatic and neuroblastoma) vs. normal counterparts. Additionally, we identified a GATA2 transcription factor binding site, promoting cancer specificity when both min-PEG-Prom mutations are present in the GAPE-Prom. Taken together, introducing specific point mutations in a rat min-GADD34-Prom converts this non-cancer-specific promoter into a cancer-selective promoter, and the addition of GATA2 with existing AP1 and PEA3 transcription factors enhances further cancer-selective activity of the GAPE-Prom. The GAPE-Prom provides a genetic tool to specifically regulate transgene expression in cancer cells.

MRI detection of the malignant transformation of stem cells through reporter gene expression driven by a tumor-specific promoter

BackgroundExisting evidence has shown that mesenchymal stem cells (MSCs) can undergo malignant transformation, which is a serious limitation of MSC-based therapies. Therefore, it is necessary to monitor malignant transformation of MSCs via a noninvasive imaging method. Although reporter gene-based magnetic resonance imaging (MRI) has been successfully applied to longitudinally monitor MSCs, this technique cannot distinguish the cells before and after malignant transformation. Herein, we investigated the feasibility of using a tumor-specific promoter to drive reporter gene expression for MRI detection of the malignant transformation of MSCs.MethodsThe reporter gene ferritin heavy chain (FTH1) was modified by adding a promoter from the tumor-specific gene progression elevated gene-3 (PEG3) and transduced into MSCs to obtain MSCs-PEG3-FTH1. Cells were induced to undergo malignant transformation via indirect coculture with C6 glioma cells, and these transformed cells were named MTMSCs-PEG3-FTH1. Western blot analysis of FTH1 expression, Prussian blue staining and transmission electron microscopy (TEM) to detect intracellular iron, and MRI to detect signal changes were performed before and after malignant transformation. Then, the cells before and after malignant transformation were inoculated subcutaneously into nude mice, and MRI was performed to observe the signal changes in the xenografts.ResultsAfter induction of malignant transformation, MTMSCs demonstrated tumor-like features in morphology, proliferation, migration, and invasion. FTH1 expression was significantly increased in MTMSCs-PEG3-FTH1 compared with MSCs-PEG3-FTH1. Prussian blue staining and TEM showed a large amount of iron particles in MTMSCs-PEG3-FTH1 but a minimal amount in MSCs-PEG3-FTH1. MRI demonstrated that the T2 value was significantly decreased in MTMSCs-PEG3-FTH1 compared with MSCs-PEG3-FTH1. In vivo, mass formation was observed in the MTMSCs-PEG3-FTH1 group but not the MSCs-PEG3-FTH1 group. T2-weighted MRI showed a significant signal decrease, which was correlated with iron accumulation in the tissue mass.ConclusionsWe developed a novel MRI model based on FTH1 reporter gene expression driven by the tumor-specific PEG3 promoter. This approach could be applied to sensitively detect the occurrence of MSC malignant transformation.

Nanoparticle-mediated tumor cell expression of mIL-12 via systemic gene delivery treats syngeneic models of murine lung cancers

Treatment of cancers in the lung remains a critical challenge in the clinic for which gene therapy could offer valuable options. We describe an effective approach through systemic injection of engineered polymer/DNA nanoparticles that mediate tumor-specific expression of a therapeutic gene, under the control of the cancer-selective progression elevated gene 3 (PEG-3) promoter, to treat tumors in the lungs of diseased mice. A clinically tested, untargeted, polyethylenimine carrier was selected to aid rapid transition to clinical studies, and a CpG-free plasmid backbone and coding sequences were used to reduce inflammation. Intravenous administration of nanoparticles expressing murine single-chain interleukin 12, under the control of PEG-3 promoter, significantly improved the survival of mice in both an orthotopic and a metastatic model of lung cancer with no marked symptoms of systemic toxicity. These outcomes achieved using clinically relevant nanoparticle components raises the promise of translation to human therapy.

D-103 Cancer terminator viruses: A strategy for targeting metastatic cancers

Our laboratory pioneered subtraction hybridization and used this elegant and effective gene identification strategy to clone novel genes of physiological relevance. This methodology defined differentially regulated genes in an unbiased manner that are modified when metastatic human melanoma cells are pharmacologically induced to terminally differentiate and lose irreversibly oncogenic potential following treatment with IFN-band the protein kinase C activator mezerein. This approach, called DISH (differentiation induction subtraction hybridization), identified melanoma differentiation associated (mda) genes relevant to growth control, cancer development, tumor progression, and cancer cell survival. One intriguing gene identified using the DISH scheme was mda-7, which based on chromosomal location, secretory properties and structure has been classified as interleukin-24 (IL-24). mda-7/IL-24 is a novel member of the IL-10 gene family that promotes apoptosis and toxic autophagy in a broad-spectrum of human tumors, exerts potent “bystander” antitumor activity, inhibits tumor angiogenesis, stimulates the immune system and synergizes with conventional therapies (including chemotherapy, radiation, monoclonal antibodies and immunomodulators) to promote selective tumor destruction. mda-7/IL-24 displays profound anti-cancer activity in preclinical animal models (including nude, syngeneic and transgenic mice) and following intratumoral administration using a type 5 adenovirus in a Phase I/II clinical trial in patients with advanced cancers. To enhance the therapeutic efficacy of mda-7/IL-24, we generated a novel class of adenoviruses, called Cancer Terminator Viruses (CTVs), that selectively replicate in cancer cells and simultaneously produce mda-7/IL-24, Ad.5-CTV. A further iteration of this virus employs a chimera between Ad.5 and Ad.3, Ad.5/3, that employs Coxsackie-Adenovirus Receptors (CAR) as well as desmoglien and CD46 to infect cells, thereby increasing its infectivity in cancer cells with reduced CAR. Ad.5/3-CTV displays profound anti-cancer activity in pre-clinical animal models, including GBM, melanoma, and prostate, colorectal and breast cancer. A clinical trial is in preparation to test the effect of the Ad.5/3-CTV in patients with recurrent glioma. To facilitate the delivery of adenoviruses systemically, avoiding immune clearance and liver trapping, we have refined the ultrasound-targeted microbubble-destruction (UTMD) approach. This strategy has allowed us to deliver therapeutic viruses and proteins intravenously to internal target organs, such as the prostate, to evoke pre-clinical antitumor activity. To enhance further the therapeutic efficacy of the UTMD approach, we are employing microbubbles that contain targeting ligands on their surface permitting accumulation in the tumor vasculature, that is, decorated microbubbles. Employing the same cancer-selective promoter used in the original CTV, the rat progression elevated gene-3 promoter (PEG-Prom), we also developed genetic-based imaging approaches for metastases. These approaches use the PEG-Prom (or another cancer-selective promoter) to drive an imaging gene (eg, luciferase or HSV-Tk) linked to a delivery vehicle PEI. These nanoparticle vectors when combined with bioluminescence imaging and SPECT-CT permit non-invasive imaging of tumors and metastases in pre-clinical animal models. As a further iteration of our CTV we have used 2 cancer-selective and a non-selective promoter to generate first in class tripartite Ads that selectively replicate in cancer cells, produce an imaging agent in cancer cells (Lucirerase or HSV-Tk) and simultaneously produce the therapeutic immunogenic cytokine mda-7/IL-24, Ad.Tri-CTV. These “theranostic viruses” permit tumor and metastasis imaging and treatment through application of a single vector. Overall, the novel reagents and methodologies we have developed are providing paradigm shifts in how cancers will be detected and treated in the future.

Development of a Transcriptional Amplification System Based on the PEG3 Promoter to Target Androgen Receptor-Positive and -Negative Prostate Cancer Cells.

Localized prostate cancer (PCa) is often curable, whereas metastatic disease treated by castration inevitably progresses toward castration-resistant PCa (CRPC). Most CRPC treatments target androgen receptor (AR) signaling. However, not all CRPC cells rely on AR activity for survival and proliferation. With advances in immunotherapy and fluid biopsies for cancer management, expression systems specific for both AR-positive and -negative PCa are required for virus-based vaccines and cell imaging. To target both AR-responsive and non-responsive cells, we developed a three-step transcriptional amplification (3STA) system based on the progression elevated gene-3 (PEG3) promoter named PEG3AP1-3STA. Notably, we report on different genetic modifications that significantly improved PEG3 promoter’s strength in PCa cells. Adenoviruses incorporating PEG3 promoter with and without transcriptional amplification systems were generated. The potential of PEG3AP1-3STA to target PCa cells was then evaluated in vitro and in vivo in androgen-responsive and non-responsive PCa cell lines. PEG3AP1-3STA was shown to be active in all PCa cell lines and not regulated by androgens, and its activity was amplified 97-fold compared to that of a non-amplified promoter. The PEG3AP1-3STA system can thus be used to target advanced AR+ and AR− cells for imaging or immunovirotherapy in advanced PCa.

Abstract 1481: Selective protein expression in tumor cells controlled by progression elevated gene-3 promoter

Abstract The progression elevated gene-3 (PEG-3) promoter is a rodent gene that has shown strong activity in tumor progression. Demonstrating tumor specificity and activity in at least five different human cancers, PEG-3 is considered a novel promoter for tumor-specific gene expression. Understanding how PEG-3 controls expression of reporter proteins is of biomedical significance because of the role it could play in new therapeutic strategies against certain types of cancer. In this study, we aim to use the PEG-3 promoter to observe the expression of firefly luciferase 2 (luc 2), a bioluminescent reporter protein, in the transfection of mammalian cells. Ultimately, we plan to use PEG-3 to express a therapeutic gene, cytosine deaminase, in cancer specific areas of breast, lung, and ovary. Citation Format: Ruben Pando, Tej Shrestha, Marla Pyle, Deryl Troyer. Selective protein expression in tumor cells controlled by progression elevated gene-3 promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1481.

Therapeutic effects of adenovirus-mediated CD and NIS expression combined with Na131I/5-FC on human thyroid cancer.

Thyroid cancer is the most common type of malignant endocrine tumor diagnosed. Previous studies have indicated that gene therapy is the most promising and effective therapeutic method for thyroid cancer. Therefore, in the present study, Na131I/5-fluorocytosine (5-FC) treatment was combined with cytosine deaminase (CD, encoded by the CDA gene) and sodium iodide symporter (NIS, encoded by the SLC5A5 gene) to act together as a therapeutic tool for thyroid cancer. The present study explored the combined cytotoxic effects of adenovirus-mediated CD and NIS under the control of the progression elevated gene-3 (PEG-3) promoter (Ad-PEG-3-CD-NIS) with Na131I/5-FC against the human thyroid cancer TT cell line in vitro. The PEG-3 fragment was obtained by polymerase chain reaction (PCR) using rat genomic DNA as the template, and then Ad-PEG-3-CDA-SLC5A5 was constructed using XbaI. TT cells were transfected by recombinant adenovirus. The method of reverse transcription-quantitative PCR was performed to test the expression of CD and NIS at the level of transcription. The morphological change was assessed by fluorescence microscopy and investigated by western blot analysis. An MTT assay was used to determine the number of living cells inhibited by single or combination therapies on TT cells. The results indicated that the PEG-3 was successfully cloned, and was also positively regulated in 293 cells. CDA and SLC5A5 genes were highly expressed in TT cells. Na131I combined with 5-FC significantly decreased the human thyroid cancer cells. In conclusion, combination therapy of Ad-PEG3-CDA-SLC5A5 and Na131I/5-FC induces significantly more apoptotic characteristics than either single treatment with Ad-PEG-3-CDA-SLC5A5 or Na131I/5-FC, and low doses of Ad-PEG-3-CDA-SLC5A5 enhanced the cytotoxic effects.

677. Evaluation of Tumor Specific Promoters for Use in Conditionally Replicating Adenovirus Mediated Virotherapy of Canine Lymphoma

Conditionally Replicating Adenoviruses (CRAds) are genetically modified therapeutic viruses that incorporates transcriptional targeting of replication to promote selective killing of tumor cells. Transcriptional targeting utilizes tissue/tumor-specific promoters driving the expression of genes in a tissue- or tumor-specific manner to allow replication of the virus in tumor cells while sparing normal cells. Selection of appropriate intermediate animal models is a basic-requirement for successful cancer virotherapy. The dog is an outstanding animal model of cancer and other complex human diseases. The outbred nature of the dog, the heterogeneity of their tumors, their genomic similarity to humans and similar disease causation, progression and pathology support the use of this animal model. Previous studies have shown high levels of expression of several promoters, including human telomerase reverse transcriptase (hTERT), survivin, chemokine receptor 4 (CXCR4) and progression elevated gene 3 (PEG3) in a variety of human cancers and murine models. The exogenous promoter PEG3 from rats has not only shown tumor-specificity in the human model, but has also shown pan-tumor properties with active transcription occurring in almost all tumor cells. None of these promoters have been tested for their potential as a transcriptional targeting tool for canine cancers. Non-Hodgkin lymphoma accounts for 83% of all hematopoietic cancer and 6 % of all malignancies in the dog. Resistance to current treatments has emerged as a critical challenge for lymphoma treatment due to the presence of genetic diversity among tumor cells. These studies explore tumor-specific activity of these promoters with the goal of identifying a suitable canine lymphoma specific promoter to generate transcriptionally targeted CRAds facilitating viral replication in canine lymphoma, but not in normal cells. In this regard, a GFP reporter gene driven by the rat PEG3 promoter was evaluated for activity after transfection into canine lymphoma cells as well as normal canine cells. The activity of the endogenous canine promoters CXCR4, cTERT, and cSurvivin were examined using quantitative reverse transcriptase PCR. Results showed negligible expression differences between normal and lymphoma cells for cTERT and PEG3 whereas cSurvivin and cCXCR4 showed markedly higher expression in tumor cells when compared with most normal cells and tissues. However, cCXCR4 also showed a high level of expression in normal peripheral blood mononuclear cells (PBMC) cells. In contrast, cSurvivin showed increased expression in canine lymphoma cells, along with other canine tumors, with reduced expression in normal canine cells/tissues and canine PBMCs. These findings will be used to generate a canine lymphoma specific CRAd.

Tumor-specific expression and detection of a CEST reporter gene.

To develop an imaging tool that enables the detection of malignant tissue with enhanced specificity using the exquisite spatial resolution of MRI. Two mammalian gene expression vectors were created for the expression of the lysine-rich protein (LRP) under the control of the cytomegalovirus (CMV) promoter and the progression elevated gene-3 promoter (PEG-3 promoter) for constitutive and tumor-specific expression of LRP, respectively. Using those vectors, stable cell lines of rat 9L glioma, 9L(CMV-LRP) and 9L(PEG-LRP) , were established and tested for CEST contrast in vitro and in vivo. 9L(PEG-LRP) cells showed increased CEST contrast compared with 9L cells in vitro. Both 9L(CMV-LRP) and 9L(PEG-LRP) cells were capable of generating tumors in the brains of mice, with a similar growth rate to tumors derived from wild-type 9L cells. An increase in CEST contrast was clearly visible in tumors derived from both 9L(CMV-LRP) and 9L(PEG-LRP) cells at 3.4 ppm. The PEG-3 promoter:LRP system can be used as a cancer-specific, molecular-genetic imaging reporter system in vivo. Because of the ubiquity of MR imaging in clinical practice, sensors of this class can be used to translate molecular-genetic imaging rapidly.

Therapy of prostate cancer using a novel cancer terminator virus and a small molecule BH-3 mimetic.

Despite recent advances, treatment options for advanced prostate cancer (CaP) remain limited. We are pioneering approaches to treat advanced CaP that employ conditionally replication-competent oncolytic adenoviruses that simultaneously produce a systemically active cancer-specific therapeutic cytokine, mda-7/IL-24, Cancer Terminator Viruses (CTV). A truncated version of the CCN1/CYR61 gene promoter, tCCN1-Prom, was more active than progression elevated gene-3 promoter (PEG-Prom) in regulating transformation-selective transgene expression in CaP and oncogene-transformed rat embryo cells. Accordingly, we developed a new CTV, Ad.tCCN1-CTV-m7, which displayed dose-dependent killing of CaP without harming normal prostate epithelial cells in vitro with significant anti-cancer activity in vivo in both nude mouse CaP xenograft and transgenic Hi-Myc mice (using ultrasound-targeted microbubble (MB)-destruction, UTMD, with decorated MBs). Resistance to mda-7/IL-24-induced cell death correlated with overexpression of Bcl-2 family proteins. Inhibiting Mcl-1 using an enhanced BH3 mimetic, BI-97D6, sensitized CaP cell lines to mda-7/IL-24-induced apoptosis. Combining BI-97D6 with Ads expressing mda-7/IL-24 promoted ER stress, decreased anti-apoptotic Mcl-1 expression and enhanced mda-7/IL-24 expression through mRNA stabilization selectively in CaP cells. In Hi-myc mice, the combination induced enhanced apoptosis and tumor growth suppression. These studies highlight therapeutic efficacy of combining a BH3 mimetic with a novel CTV, supporting potential clinical applications for treating advanced CaP.

Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.

Chemoprevention Gene Therapy (CGT): Novel Combinatorial Approach for Preventing and Treating Pancreatic Cancer

Pancreatic cancer remains one of the deadliest of all cancers despite aggressive surgical treatment combined with adjuvant radiotherapy and chemotherapy. Chemoresistance and radioresistance are the principal causes of failure of pancreatic cancer patients to respond to therapy. Conditionally replication competent adenovirus (CRCA)-based cancer gene therapy is an innovative strategy for treating cancers displaying inherent resistance to treatment. Limitations of current adenovirus (Ad)-based gene therapies for malignant tumors include lack of cancer-specificity, and effective and targeted delivery. To remedy this situation, CRCAs have been designed that express E1A, necessary for Ad replication, under the control of a cancer-specific progression elevated gene-3 promoter (PEG-Prom) with concomitant expression of an immunomodulatory cytokine, such as mda-7/IL-24 or interferon-γ (IFN-γ), under the control of a ubiquitous and strong cytomegalovirus promoter (CMV-Prom) from the E3 region. These bipartite CRCAs, when armed with a transgene, are called cancer terminator viruses (CTVs), i.e., Ad.PEG-E1A-CMV-mda-7 (CTV-M7) and Ad.PEG-E1A-CMV-IFN-γ (CTV-γ), because of their universal effectiveness in cancer treatment irrespective of p53/pRb/p16 or other genetic alterations in tumor cells. In addition to their selective oncolytic effects in tumor cells, the potent 'bystander antitumor' properties of MDA-7/IL-24 and IFN-γ embody the CTVs with expanded treatment properties for both primary and distant cancers. Pancreatic cancer cells display a "translational block" of mda-7/IL-24 mRNA, limiting production of MDA-7/IL-24 protein and cancer-specific apoptosis. Specific chemopreventive agents abrogate this "translational block" resulting in pancreatic cancer-specific killing. This novel chemoprevention gene therapy (CGT) strategy holds promise for both prevention and treatment of pancreatic cancers where all other strategies have proven ineffective.

Histone Deacetylase Inhibitors Interact with Melanoma Differentiation Associated-7/Interleukin-24 to Kill Primary Human Glioblastoma Cells

We presently demonstrate that histone deacetylase inhibitors (HDACIs) enhance toxicity of melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) in invasive primary human glioblastoma multiforme (GBM) cells. Additionally, a method is described to augment the efficacy of adenoviral delivery of mda-7/IL-24 in these cells. HDACIs synergized with melanoma differentiation-associated (MDA)-7/IL-24 killing GBM cells. Enhanced lethality correlated with increased autophagy that was dependent on the expression of ceramide synthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation of reactive oxygen species and Ca(2+). Quenching of reactive oxygen species and Ca(2+) blocked HDACI and MDA-7/IL-24 killing. In vivo MDA-7/IL-24 prolonged the survival of animals carrying orthotopic tumors, and HDACIs enhanced survival further. A serotype 5/3 adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than a serotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus that conditionally replicates in tumor cells expressing MDA-7/IL-24, in which the adenoviral early region 1A (E1A) gene was driven by the cancer-specific promoter progression elevated gene-3 [Ad.5/3 (INGN 241)-PEG-E1A-mda-7; also called Ad.5/3-CTV (cancer terminator virus)]. Ad.5/3-CTV increased the survival of mice carrying GBM tumors to a significantly greater extent than did a nonreplicative virus Ad.5/3-mda-7. Ad.5/3-CTV exhibited no toxicity in the brains of Syrian hamsters. Collectively our data demonstrate that HDACIs enhance MDA-7/IL-24 lethality, and adenoviral delivery of mda-7/IL-24 combined with tumor-specific viral replication is an effective preclinical GBM therapeutic.

Abstract 4535: Molecular-genetic imaging of prostate cancer.

Abstract Cancer-specific promoters, such as the progression elevated gene-3 or the astrocyte elevated gene promoters, PEG-Prom and AEG-Prom, respectively, can be used as molecular genetic imaging instruments for tumor-selective imaging of cancer. We have previously shown this in experimental models of metastatic breast cancer and melanoma using bioluminescence imaging (BLI) and single photon emission computed tomography with computed tomography (SPECT/CT). Here we show that we can express PEG-Prom and AEG-Prom selectively in PC3-ML (human bone metastatic derived prostate cancer) cells and use these constructs for imaging prostate cancer in vivo. PC3-ML cells were transfected with firefly luciferase (FLuc) or the herpes simplex type I thymidine kinase (HSV1-tk), and validated for suitable transfection through bioluminescence and [125I]FIAU cell uptake assays, respectively. PC3-ML cells were administered through intra-cardiac injection to allow widespread metastases. A linear polyethyleneimine (l-PEI) nanoparticle was used for intravenous (IV) delivery of the promoter constructs to animals bearing metastases. PEG-Prom- and AEG-Prom-driven imaging reporter gene expression was monitored by small animal BLI and by SPECT/CT after IV administration of D-luciferin or [125I]FIAU, respectively. Both BLI and SPECT/CT imaging demonstrated tumor-specific PEG-Prom and AEG-Prom activity in the model of metastatic prostate cancer but not in control. Using the thoracic cavity and tibia as the regions of interest (ROI), quantification of the BLI data (n=6) at the 48 h time point after reporter gene delivery showed a range of 16- to 19-fold higher accumulation of FLuc in the metastasis group compared to control for PEG-Prom and AEG-Prom, respectively. BLI results were confirmed by histopathology, immunohistochemistry and western blot. Quantification of the SPECT/CT data (n=5) at the 56 h time point after reporter gene and 8 h after [125I]FIAU delivery showed a 3- to 11-fold higher accumulation of [125I]FIAU, depending on the tumor burden, in the group bearing metastases compared to control for AEG-Prom. Metastatic nodules detected by the SPECT/CT imaging were confirmed by pathology. The results demonstrate the potential of systemically delivered, PEG-Prom- and AEG-Prom-driven constructs for enabling the imaging of metastatic prostate cancer in vivo. Citation Format: Akrita Bhatnager, Kathleen Gabrielson, Paul B. Fisher, Martin G. Pomper. Molecular-genetic imaging of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4535. doi:10.1158/1538-7445.AM2013-4535

Selected Approaches for Rational Drug Design and High Throughput Screening to Identify Anti-Cancer Molecules

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

Abstract 61: A new serotype chimera Cancer Terminator Virus (Ad.5/3-CTV) expands the efficiency and specificity of prostate cancer gene transfer and therapy

Abstract Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating prostate cancer (PC). We previously constructed a unique CRCA, Cancer Terminator Virus (CTV), in which the E1A gene, necessary for adenoviral replication, is controlled by the cancer-selective Progression Elevated Gene-3 (PEG-3) promoter and which simultaneously expresses melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) from the E3 region of the adenovirus. mda-7/IL-24, a novel member of the IL-10-related cytokine gene family, displays almost ubiquitous antitumor properties in vitro and in vivo, leading to its rapid entry into the clinic, where its safety and clinical efficacy, when administered by adenovirus (Ad.mda-7; INGN 241), was observed in a phase I clinical trial in humans with advanced carcinomas and melanomas. mda-7/IL-24 preferentially induces apoptosis in cancer cells while exerting no discernible toxic effects toward normal cells and it also elicits potent “antitumor bystander activity” in distant cancer cells as a consequence of autocrine and paracrine secretion of MDA-7/IL-24. The CTV was generated on a serotype 5 background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC in Hi-myc transgenic mice. Ad.5/3.CTV exerted a marked anti-tumor ‘bystander’ effect in vivo confirming therapeutic utility. In these contexts, and considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7, the use of Ad.5/3-CTV to administer therapeutic and cytotoxic mda-7/IL24 to selectively induce cytolysis and apoptosis in prostate tumor cells represents a viable treatment options. (Supported by R01 CA097318 and P01 CA104177) 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 61. doi:1538-7445.AM2012-61

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression

Molecular-genetic imaging is advancing from a valuable preclinical tool to guiding patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression, protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 promoter (PEG-Prom), derived from a rodent gene mediating the malignant phenotype, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in murine models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter, tumor specificity and capacity for clinical translation, PEG-Prom-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy.

Eradication of Therapy-resistant Human Prostate Tumors Using an Ultrasound-guided Site-specific Cancer Terminator Virus Delivery Approach

Intratumoral injections of a replication-incompetent adenovirus (Ad) expressing melanoma differentiation-associated gene-7/interleukin-24 (Ad.mda-7), a secreted cytokine displaying cancer-selective, apoptosis-inducing properties, profoundly inhibits prostate cancer (PC) growth in immune-incompetent animals. In contrast, Ad.mda-7 is ineffective in PCs overexpressing antiapoptotic proteins such as Bcl-2 or Bcl-x(L). However, intratumoral injections of a conditionally replication-competent Ad (CRCA) in which expression of the adenoviral E1A gene is driven by the cancer-specific promoter of progression-elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/interleukin (IL)-24 in the E3 region of the Ad (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV), is highly active in these cells. A major challenge for gene therapy is systemic delivery of nucleic acids directly into an affected tissue. Ultrasound (US) contrast agents (microbubbles-MBs) are viable candidates for gene delivery/therapy. Here, we show that MB/Ad.mda-7 complexes targeted to DU-145 cells using US dramatically reduced tumor burden in xenografted nude mice. Additionally, US-guided MB/CTV delivery completely eradicated not only targeted DU-145/Bcl-x(L)-therapy-resistant tumors, but also nontargeted distant tumors (established in the opposite flank), thereby implementing a cure. These findings highlight potential therapeutic applications of this novel image-guided gene therapy technology for advanced PC patients with metastatic disease.

Suppression of teratocarcinoma growth by soluble TRAIL gene driven by the progression-elevated gene-3 promoter

A plasmid expressing the soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligand sTRAIL amino acids 114-281 of TRAIL), driven by rat progression-elevated gene-3 (rPEG) promoter was constructed and evaluated. Transfection of embryonal carcinoma (EC) cells with the plasmid resulted in significant cellular apoptosis and elevated expression of death receptor 4 (DR4) and death receptor 5 (DR5). Direct intratumoral injection of DNA:liposome complexes suppressed tumor growth significantly and prolonged the survival of teratocarcinoma-bearing mice. Histological examination and serum analyses showed the absence of detectable toxicity in all examined tissues including liver. Our results demonstrate that sTRAIL gene expression driven by the rPEG promoter may enable effective gene therapy against teratocarcinoma.