Gene Switch Technology Research Articles

ET-06 Suppression of glioblastoma through novel drug based on “Gene Switch Technology”

Glioblastoma (GBM) is the most common and aggressive malignancy primarily affecting adults. Despite intensive multimodal therapies, the prognosis of GBM is dismal and a novel therapy is needed. Here, we focused on RUNX, a transcription factor involved in the malignant transformation of GBM, and developed a novel Chlorambucil-conjugated PI-polyamides (Chb-M’), which “switches off” RUNX family. Chb-M’ specifically recognizes the consensus RUNX-binding sequences (TGTGGT) and alkylates it to inhibit transcription of the downstream gene of RUNX family. Chb-M’ has been shown to induce apoptosis and suppress proliferation in a variety of cancers including leukemia, and in this study, similar results were found for glioblastoma cells in vitro. Specific inhibition of RUNX1 led to a marked inhibition of tumor growth through cell cycle arrest and apoptosis. By using apoptosis array, we isolated several candidate genes which regulated by RUNX1. And some types of glioblastoma cell lines treated with Chb-M’ showed elevated expression of p21 and decreased survivin. From in silico analysis using glioma patient cohorts, survivin expression was significantly higher in GBM and it was possibly involved in maintaining the malignancy of GBM. Mechanistically survivin was found to be directly transcriptionally regulated by RUNX1 through ChIP assay and reporter assay. In addition, survivin K/D cells upregulated p21 expression and accelerated apoptosis. Taken together, we hypothesized that the RUNX1-survivin-p21 pathway can potentially be exploited in the management of this malignancy. Chb-M’ mediated regulation of RUNX1 can be a novel therapeutic strategy against GBM.

Abstract 2937: The regulation of FGFR signaling by RTK adaptor protein down-regulation through CROX (cluster regulation of RUNX) theory in DNPC

Abstract Up-regulation of the FGFR signaling is frequently-encountered in DNPC: AR-, NE- prostatic cancer and contributes to the maintenance of these cancer cells. Even though, the importance of active FGFR signaling was recognized as a biomarker in DNPC, still core oncogenic driver of DNPC remained unknown. We formerly reported the importance of Pan RUNX (RUNX1,2,3) as an oncogenic driver. Here, we added a new evaluation factors to the examination of CRPC patient cohort to categorize potentially existing DNPC clearly. Pan RUNX was up-regulated in DNPC patients. The inhibition of Pan RUNX reduced the proliferation of DNPC cells significantly, suggesting that Pan RUNX is functionally and critically involved in the cell proliferation mechanism of DNPC. The inhibition of Pan RUNX inhibited FGFR signaling dramatically. On the other hand, the inhibition of FGFR decreased the transactivation of Pan RUNX. To resolve this contradiction, we identified candidate genes which are mediating the crosstalk between FGFR signaling and Pan RUNX. In addition to our formerly reported S100A16, we isolated and examined the adaptor protein family responsible for RTK activation, which are transcriptionally regulated by the RUNX family. In addition, from the analysis by using the CRPC patient cohorts, Pan RUNX expression and SOS1 etc. genes levels were significantly higher in DNPC rather than those of the other subtypes. RUNX family regulates the FGFR signaling pathway in DNPC cells through transactivating expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, ChIP-qPCR assay and luciferase reporter experiments further validated RUNX family interacts with the RUNX binding DNA sequence located in the candidate genes promoter and positively regulates it. Knockdown of Pan RUNX led to the decreased expression of the adaptor genes as well as dephosphorylation of FGFR, subsequently suppressed the proliferation of DNPC cells. These data suggest that RUNX depletion-mediated growth inhibitory effect on DNPC cells depends on FGFR signal inactivation via transcriptional regulation of adaptor protein. To achieve cluster regulations of RUNX(CROX), We screened our library and isolated our original Pan RUNX inhibitor based on gene switch technology; Chlorambucil-conjugated Pyrrole Imidazole Polyamide (Chb-M'), which specifically binds to the common RUNX binding consensus sequence(TGTGGT) and inhibits RUNX target genes including the adaptor protein, consistently led to the deactivation of the FGFR signaling pathway and was effective against DNPC cell lines and was exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of DNPC. Taken together, our work identified a novel interaction of RUNX family and the FGFR signaling pathway in DNPC, which can potentially be exploited in the management of this malignancy. Citation Format: Asami Sasaski, Youhei Yanagida, Hiroshi Sugiyama, Souichi Adachhi, Yasuhiko Kamikubo. The regulation of FGFR signaling by RTK adaptor protein down-regulation through CROX (cluster regulation of RUNX) theory in DNPC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2937.

CROX (Cluster Regulation of RUNX) as a Potential Novel Therapeutic Approach.

Comprehensive inhibition of RUNX1, RUNX2, and RUNX3 led to marked cell suppression compared with inhibition of RUNX1 alone, clarifying that the RUNX family members are important for proliferation and maintenance of diverse cancers, and “cluster regulation of RUNX (CROX)” is a very effective strategy to suppress cancer cells. Recent studies reported by us and other groups suggested that wild-type RUNX1 is needed for survival and proliferation of certain types of leukemia, lung cancer, gastric cancer, etc. and for their one of metastatic target sites such as born marrow endothelial niche, suggesting that RUNX1 often functions oncogenic manners in cancer cells. In this review, we describe the significance and paradoxical requirement of RUNX1 tumor suppressor in leukemia and even solid cancers based on recent our findings such as “genetic compensation of RUNX family transcription factors (the compensation mechanism for the total level of RUNX family protein expression)”, “RUNX1 inhibition-induced inhibitory effects on leukemia cells and on solid cancers through p53 activation”, and “autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells”. Taken together, these findings identify a crucial role for the RUNX cluster in the maintenance and progression of cancers and suggest that modulation of the RUNX cluster using the pyrrole-imidazole polyamide gene-switch technology is a potential novel therapeutic approach to control cancers.

Genetic regulation of the RUNX transcription factor family has antitumor effects.

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent-conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

Development of a Glycoprotein D-Expressing Dominant-Negative and Replication-Defective Herpes Simplex Virus 2 (HSV-2) Recombinant Viral Vaccine against HSV-2 Infection in Mice

Using the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4(+) and CD8(+) T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease.

Development of a Regulatable Oncolytic Herpes Simplex Virus Type 1 Recombinant Virus for Tumor Therapy

Oncolytic viruses are genetically modified viruses that preferentially replicate in host cancer cells, leading to the production of new viruses and, ultimately, cell death. Currently, no oncolytic viruses that are able to kill only tumor cells while leaving normal cells intact are available. Using T-REx (Invitrogen, Carlsbad, CA) gene switch technology and a self-cleaving ribozyme, we have constructed a novel oncolytic HSV-1 recombinant, KTR27, whose replication can be tightly controlled and regulated by tetracycline in a dose-dependent manner. Infection of normal replicating cells as well as multiple human cancer cell types with KTR27 in the presence of tetracycline led to 1,000- to 250,000-fold-higher progeny virus production than in the absence of tetracycline, while little viral replication and virus-associated cytotoxicity was observed in infected growth-arrested normal human cells. We show that intratumoral inoculation with KTR27 markedly inhibits tumor growth in a xenograft model of human non-small-cell lung cancer in nude mice. It is shown further that replication of KTR27 in the inoculated tumors can be efficiently controlled by local codelivery of tetracycline to the target tumors at the time of KTR27 inoculation. Collectively, KTR27 possesses a unique pharmacological feature that can limit its replication to the targeted tumor microenvironment with localized tetracycline delivery, thus minimizing unwanted viral replication in distant tissues following local virotherapy. This regulatory mechanism would also allow the replication of the virus to be quickly shut down should adverse effects be detected.

High-Level Expression of Glycoprotein D by a Dominant-Negative HSV-1 Virus Augments its Efficacy as a Vaccine against HSV-1 Infection

Using the T-REx (Invitrogen, Carlsbad, CA) gene switch technology, we previously generated a dominant-negative herpes simplex virus (HSV)-1 recombinant, CJ83193, capable of inhibiting its own replication as well as that of wild-type HSV-1 and HSV-2. It has been further demonstrated that CJ83193 is an effective vaccine against HSV-1 infection in a mouse ocular model. To ensure its safety and augment its efficacy, we generated an improved CJ83193-like HSV-1 recombinant, CJ9-gD, which contains a deletion in an HSV-1 essential gene and encodes an extra copy of gene-encoding glycoprotein D (gD) driven by the tetO-bearing human cytomegalovirus major immediate-early promoter. Unlike CJ83193, which exhibits limited plaque-forming capability in Vero cells and expresses little gD in infected cells, CJ9-gD is completely replication defective, yields high-level expression of gD following infection, and cannot establish detectable infection in mouse trigeminal ganglia following intranasal and ocular inoculation. Mice immunized with CJ9-gD produced 3.5-fold higher HSV-1 neutralizing antibody titer than CJ83193-immunized mice, and were completely protected from herpetic ocular disease following corneal challenge with wild-type HSV-1. Moreover, immunization of mice with CJ9-gD elicited a strong HSV-1-specific T-cell response and led to an 80% reduction in latent infection by challenge wild-type HSV-1 compared with the mock-immunized control.

Preface: Papers from the 16th International Ecdysone Workshop

AbstractWe are pleased to introduce a two‐issue set of papers by contributors to the 16th International Ecdysone Workshop held last July in Ghent, Belgium with 128 participants from 18 countries. It would be very difficult to understate the biological significance of ecdysteroids in insect science, and the papers in this Special Issue help us appreciate the continued and rapid progress in this important area. This is also reflected in the fact that nearly 2000 papers were published on “ecdysteroids” in ISI indexed peer‐reviewed international journals over the last 6 years since 2000.Ecdysteroids do not act on a one‐dimensional hormone‐effect axis. To the contrary, papers in this Special Issue report on ecdysteroid interactions with other signal moieties. Gruntenko et al. and Rauschenach et al. reveal important interactions between biogenic amines and ecdysteroid titers. Eizaguirre et al. bring in new information on interactions of JH and ecdysteroids, while Kamimura et al. write about the influence of ecdysteroids on JH esterase gene expression. Ecdysteroids influence cell death, however, this effect is modulated by JH, as seen in Lobbia et al. As this area develops, we are sure to see new entities—perhaps even eicosanoids—interacting with ecdysteroids.The mechanisms of ecdysteroids and ecdysteroid biosynthesis continue to fascinate many insect scientists. Dedos et al. report on calcium signal cascades in prothoracic glands. Localization and movement of the ecdysteroid receptor complex draws the attention of Cronauer et al. and Betanska et al. This receptor also has potential biotech application with gene switch technology, as discussed by Tavva et al.The significance of ecdysteroids in insect biology is highlighted by the number of parasitoids and plants that influence insect ecdysteroids. Rharrabe et al. outline the diversity of detoxification pathways for ingested ecdysteroids, and Bodin et al. report on host‐induced ecdysteroids in a parasitoid. Krishnan et al. report on the influence of ecdysteroids in oxidative stress damage.Together, the papers in this Special Issue advance our appreciation of the biological significance of ecdysteroids in insect biology and they celebrate the insect scientists who are working on the frontiers of our knowledge in this substantial area. We hope this body of work helps scientists who want to participate in this exciting research.

Applications of EcR gene switch technology in functional genomics

Genetic engineering of plants using transgenic technology is targeted to enhance agronomic performance or improved quality traits in a wide variety of plant species, and has become a fundamental tool for basic research in plant biotechnology. Constitutive promoters are presently the primary means used to express transgenes in plants. However, inducible gene regulation systems based on specific chemicals have many potential applications in agriculture and for enhancing the basic understanding of gene function. As a result, several gene switches have been developed. The ecdysone receptor gene switch is one of the best inducible gene regulation systems available, because the chemical, methoxyfenozide, required for its regulation is registered for field use. An EcR gene switch with a potential for use in large-scale field applications has been developed by adopting a two-hybrid format. In a two-hybrid switch format, the GAL4 DNA binding domain (GAL4 DBD) was fused to the ligand binding domain (LBD) of the Choristoneura fumiferana ecdysone receptor (CfEcR); and, the VP16 activation domain (VP16 AD) was fused to the LBD of Locust migratoria retinoid X receptor (LmRXR). The sensitivity of the CfEcR gene switch was improved from micromolar to nanomolar concentrations of ligand by using the CfEcR:LmRXR two-hybrid switch. In this report, we demonstrate the utility of CfEcR:LmRXR two-hybrid gene switch in functional genomics applications for regulating the expression of a Superman-like single zinc finger protein 11 (ZFP11) gene in both Arabidopsis and tobacco transgenic plants.

Preface: Papers from the 16th International Ecdysone Workshop

AbstractWe are pleased to introduce a two‐issue set of papers by contributors to the 16th International Ecdysone Workshop held last July in Ghent, Belgium with 128 participants from 18 countries. It would be very difficult to understate the biological significance of ecdysteroids in insect science, and the papers in this Special Issue help us appreciate the continued and rapid progress in this important area. This is also reflected in the fact that nearly 2000 papers were published on “ecdysteroids” in ISI indexed peer‐reviewed international journals over the last 6 years since 2000.Ecdysteroids do not act on a one‐dimensional hormone‐effect axis. To the contrary, papers in this Special Issue report on ecdysteroid interactions with other signal moieties. Gruntenko et al. and Rauschenach et al. reveal important interactions between biogenic amines and ecdysteroid titers. Eizaguirre et al. bring in new information on interactions of JH and ecdysteroids, while Kamimura et al. write about the influence of ecdysteroids on JH esterase gene expression. Ecdysteroids influence cell death, however, this effect is modulated by JH, as seen in Lobbia et al. As this area develops, we are sure to see new entities—perhaps even eicosanoids—interacting with ecdysteroids.The mechanisms of ecdysteroids and ecdysteroid biosynthesis continue to fascinate many insect scientists. Dedos et al. report on calcium signal cascades in prothoracic glands. Localization and movement of the ecdysteroid receptor complex draws the attention of Cronauer et al. and Betanska et al. This receptor also has potential biotech application with gene switch technology, as discussed by Tavva et al.The significance of ecdysteroids in insect biology is highlighted by the number of parasitoids and plants that influence insect ecdysteroids. Rharrabe et al. outline the diversity of detoxification pathways for ingested ecdysteroids, and Bodin et al. report on host‐induced ecdysteroids in a parasitoid. Krishnan et al. report on the influence of ecdysteroids in oxidative stress damage.Together, the papers in this Special Issue advance our appreciation of the biological significance of ecdysteroids in insect biology and they celebrate the insect scientists who are working on the frontiers of our knowledge in this substantial area. We hope this body of work helps scientists who want to participate in this exciting research.

Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals

Zooecdysteroids (arthropod steroid hormones) regulate the development of arthropods and probably many other invertebrates. Phytoecdysteroids are analogues occurring in a wide range of plant species, where they contribute to the deterrence of phytophagous invertebrates. The purpose of this short review is to summarise findings on the occurrence, metabolism and pharmacological effects of ecdysteroids in mammalian systems and to draw attention to their potential applications, particularly in gene-switch technology, where ecdysteroid analogues (steroidal and non-steroidal) can be used as effective and potent elicitors.

Conditional Induction of Ovulation in Mice1

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.

Industry update

Industry update