scholarly journals Enhanced oncolytic adenoviral production by downregulation of death-domain associated protein and overexpression of precursor terminal protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jihyun Lee ◽  
Geun-Hyeok Oh ◽  
Jeong A. Hong ◽  
Soojin Choi ◽  
Hye Jin Choi ◽  
...  
Keyword(s):  
Viral Replication ◽  
Tumor Regression ◽  
Human Cancer ◽  
Virus Production ◽  
Death Domain ◽  
Viral Production ◽  
Tumor Cell Death ◽  
Terminal Protein ◽  
Enhanced Production

AbstractAdequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.

Alkene Metathesis: Synthesis of Kainic Acid, Pladienolide B and Amphidinolide Y

2011 ◽  
Author(s):  
Douglass Taber
Keyword(s):  
Kainic Acid ◽  
Second Generation ◽  
Tumor Regression ◽  
Human Cancer ◽  
Cross Coupling ◽  
Side Reaction ◽  
Chiral Auxiliary ◽  
Crotonyl Chloride ◽  
The University

As alkene metathesis has developed into one of the tools of organic synthesis, many practical questions have arisen. In the course of a synthesis (Organic Lett. 2007, 9, 1635) of the important neuropharmacological tool (-)-kainic acid 7, Tohru Fukuyama of the University of Tokyo prepared the key intermediate 1 by chiral auxiliary mediated coupling of crotonyl chloride with acetadehyde. Dibal reduction gave the hemiaminal, which underwent reductive amination with glycine methyl ester, leading to the alkene 2. Alkene metathesis of the derived ester 3 to form the unsaturated lactone 4 was then examined in detail. It was found that 0.5 mol % of the second generation Hoveyda catalyst was sufficient to cyclize 3 to 4 in 92% yield. With 0.8 mol %, the yield was 99%. The key to the efficacy of this cyclization was the use of 1,2-dichloroethane at reflux as the reaction solvent. The macrolide pladienolide D 14 induces in vivo tumor regression in several human cancer xenograft models. This activity was important enough that a team at Esai Co., Ltd in Tsukuba headed by Yoshihiko Kotake undertook the total synthesis (Angew. Chem. Int. Ed. 2007, 46, 4350). Their approach used two alkene metathesis steps. To prepare the substrate for the macrolide construction, the alcohol 8 and the acid 9, each prepared by chiral auxiliary control, were coupled to give the ester 10. An extensive investigation led to alkene metathesis conditions that were satisfactory, the use of the second generation Hoveyda catalyst in refluxing toluene. A significant competing side reaction was the migration of the monosubstituted alkene of 10 to make the alkenyl ether. The second alkene metathesis step was the coupling of 12 with 13. The most effective catalyst in this case was the second generation Grubbs. Note that the free alcohol 13 participated successfully in the cross-coupling. According to the authors, this is one of just a few examples of successful cross coupling of an alkene adjacent to a quaternary center. The stereocontrolled construction of trisubstituted alkenes by metathesis is a particular challenge.

Imaging and therapy for Epstein-Barr virus-associated gastric cancer

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 4644-4644
Author(s):  
D. Fu ◽  
J. Chong ◽  
C. Foss ◽  
J. Fox ◽  
S. Wang ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Tumor Regression ◽  
Human Cancer ◽  
Nucleoside Analogs ◽  
Scid Mice ◽  
Tumor Xenografts ◽  
Barr Virus ◽  
Bortezomib Treatment ◽  
Epstein Barr

4644 Background: Epstein-Barr virus (EBV) has been identified in a wide variety of malignancies, including gastric carcinomas. The virus encodes kinases that phosphorylate nucleoside analogs such as 2’-deoxy-2’-fluoro-5-iodo-1-beta-D- arabinofuranosyluracil (FIAU). We hypothesized that it might be possible to use the viral enzyme to specifically concentrate [125I]FIAU or [131I] FIAU in tumor cells harboring virus and thus deliver imaging and therapeutic radiation. Bortezomib is a potent stimulator of viral kinase expression in EBV tumor cell lines. Methods: We imaged lytic induction in vivo and evaluated the effect of [131I] FIAU on human cancer xenografts in SCID mice. These include a tumor line engineered to constitutively express the EBV thymide kinase (EBVTK), and a control engineered with a sham vector (SHAM), as well one EBV-associated human gastric tumor (KT tumor). Mice were treated with buffer, bortezomib (2μg/g), or radiolabeled FIAU or radiolabeled FIAU and bortezomib in combination. For imaging, mice, [125I]-FIAU and SPECT/CT were used. For therapy, 131I-FIAU was used and tumor dimensions were monitored with calipers. Results: SPECT/CT imaging with [125I]-FIAU of tumor-bearing SCID mice showed selective concentration of radiotracer in tumor tissue in EBVTK (3/3) and in EBV-associated KT tumors (3/3) when animals were pretreated with bortezomib. Treatment with buffer had no effect on 3 EBVTK tumors and 3 SHAM tumors all of which increased in volume. Treatment with 1.6 mCi of [131I]-FIAU alone led to tumor response in 3/3 mice with EBVTK tumors and 0/3 mice with SHAM tumors. Treatment with [131I]-FIAU alone had no effect on EBV KT tumor xenografts (0/3) and all tumors increased in volume. Treatment with bortezomib induced modest responses in all KT tumors. However, treatment with bortezomib and [131I]-FIAU led to marked tumor regression (>80%) in EBV-associated KT tumors (3/3). Conclusions: Treatment with bortezomib leads to selective concentration of radiolabeled FIAU in the EBV-associated tumor xenografts. In combination with [131I]-FIAU it leads to tumor regression. No significant financial relationships to disclose.

Cyclin-Dependent Kinase Pathways As Targets for Cancer Treatment

2006 ◽  
Vol 24 (11) ◽  
pp. 1770-1783 ◽  
Author(s):  
Geoffrey I. Shapiro
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Tumor Regression ◽  
Human Cancer ◽  
Cell Types ◽  
Therapeutic Benefit ◽  
Cell Cycle Regulators

Cyclin-dependent kinases (cdks) are critical regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor–dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B–responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, especially in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compounds potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors.

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

2010 ◽  
Vol 84 (16) ◽  
pp. 8163-8171 ◽  
Author(s):  
Feng Yao ◽  
Nao Murakami ◽  
Oliver Bleiziffer ◽  
Pengwei Zhang ◽  
Natali V. Akhrameyeva ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cancer ◽  
Tumor Therapy ◽  
Xenograft Model ◽  
Virus Production ◽  
Cell Types ◽  
Oncolytic Viruses ◽  
Progeny Virus ◽  
Dependent Manner ◽  
Simplex Virus

ABSTRACT 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.

scholarly journals Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

2019 ◽  
Author(s):  
Tadashi Watanabe ◽  
Mayu Nishimura ◽  
Taisuke Izumi ◽  
Kazushi Kuriyama ◽  
Yuki Iwaisako ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Kaposi's Sarcoma ◽  
Virus Production ◽  
Kaposi’S Sarcoma ◽  
Early Gene ◽  
Immediate Early ◽  
Late Gene ◽  
Viral Production ◽  
Late Gene Expression

ABSTRACTKaposi’s sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the virus specific pre-initiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a BAC system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased viral production, viral DNA replication was unaffected. CHIP analysis showed that ORF66 bound to the promoters of late gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine-repeat in the C-terminal region of ORF66 were essential for interaction with ORF34 and viral production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model, in which ORF66 serves as a critical vPIC component to promote late viral gene expression and viral production.IMPORTANCEKSHV ORF66, a late gene product, and vPIC are thought to contribute significantly to late gene expression during the lytic replication. However, the physiological importance of ORF66 in terms of viral replication and vPIC formation remains poorly understood. Therfore, we generated a ORF66-deficient BAC clone and evaluated its viral replication. Results showed that ORF66 played a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing ORF66 function in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacted with the transcription start site ofK8.1gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C-terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among β- and γ-herpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

Inhibiting primary effusion lymphoma by lentiviral vectors encoding short hairpin RNA

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2510-2518 ◽  
Author(s):  
Andrew Godfrey ◽  
John Anderson ◽  
Antigoni Papanastasiou ◽  
Yasu Takeuchi ◽  
Chris Boshoff
Keyword(s):  
Rna Interference ◽  
Human Cancer ◽  
Primary Effusion Lymphoma ◽  
Kaposi Sarcoma ◽  
Lentiviral Vectors ◽  
Death Domain ◽  
Inhibitory Protein ◽  
Endogenous Gene

AbstractWe use lentiviral-delivered RNA interference (RNAi) to inhibit the growth of a model of primary effusion lymphoma (PEL) in vitro and in vivo. RNAi is a phenomenon allowing the sequence-specific targeting and silencing of exogenous and endogenous gene expression and is being applied to inhibit viral replication both in vitro and in vivo. We show that silencing of genes believed to be essential for the Kaposi sarcoma-associated herpesvirus (KSHV) latent life cycle (the oncogenic cluster) has a varied effect in PEL cell lines cultured in vitro, however, concomitant silencing of the viral cyclin (vcyclin) and viral FLICE (Fas-associating protein with death domain-like interleukin-1β-converting enzyme) inhibitory protein (vFLIP) caused efficient apoptosis in all PEL lines tested. We demonstrate that in a murine model of PEL, lentiviral-mediated RNA interference both inhibits development of ascites and can act as a treatment for established ascites. We also show that the administered lentiviral vectors are essentially limited to the peritoneal cavity, which has advantages for safety and dosage in a therapeutic setting. This shows the use of lentiviral-mediated RNA interference in vivo as a potential therapeutic against a virally driven human cancer.

CTNI-49. PHASE 1 STUDY OF ST101, A FIRST-IN-CLASS PEPTIDE ANTAGONIST OF CCAAT/ENHANCER-BINDING PROTEIN ß, IN PATIENTS WITH ADVANCED SOLID TUMORS, WITH A PHASE 2 EXPANSION IN RECURRENT GLIOBLASTOMA MULTIFORME

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi71-vi71
Author(s):  
Stefan Symeonides ◽  
Jeff Evans ◽  
Hendrik-Tobias Arkenau ◽  
Meredith McKean ◽  
Nehal Lakhani ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Solid Tumors ◽  
Human Cancer ◽  
Phase 1 ◽  
Single Agent ◽  
Phase 2 ◽  
Tumor Cell Death ◽  
Recurrent Glioblastoma Multiforme ◽  
Peptide Antagonist

Abstract BACKGROUND C/EBPβ is a transcription factor that is active during embryofetal development but held in an inactive state in most mature cells (Zahnow 2009). C/EBPβ is upregulated or overactivated in multiple cancers, where it inversely correlates with disease prognosis and survival due to activation of a gene signature that promotes tumor cell proliferation and survival. ST101 is a cell-penetrating peptide antagonist of C/EBPβ. ST101 exposure leads to selective tumor cell death in multiple human cancer cell lines, including GBM, without impacting normal cell viability. In vivo, ST101 displays rapid uptake into multiple organs, the ability to cross the blood-brain barrier, and a long plasma half-life due to its resistance to degradations. It has potent anti-tumor activity in multiple GBM models, as a single agent or in combination, which supported moving into clinical development. TRIAL DESIGN This phase 1-2 study is enrolling patients ≥ 18 years of age with advanced, unresectable metastatic solid tumors refractory to or intolerant of other therapeutic options. We began recruitment in August 2020. The primary objective of phase 1 is to evaluate safety and tolerability of ST101. Secondary objectives include the recommendation of a dose and regimen of ST101 for further evaluation, pharmacokinetics, several pharmacodynamic measures, and preliminary efficacy. Patients receive intravenous ST101 once weekly in a standard 3 + 3 design. Enrollment is ongoing, and by 21 May 2021, 15 patients have been recruited in four dose-escalation cohorts up to 4 mg/kg; a 5th cohort (6 mg/kg) is ongoing. The recommended phase 2 dose will be used in a 15-30 patient GBM expansion cohort, with a Simon 2-stage design, which requires one response or two patients with PFS6 in the first cohort to continue the study. Up to 120 patients are planned in a total of four expansion cohorts, which should be enrolling by Q3 2021.

scholarly journals Mechanisms Conferring Resistance to Pro-Apoptotic Cancer Gene Therapy

2011 ◽  
Vol 4 ◽  
pp. JCD.S4686
Author(s):  
Shona T. Dougherty ◽  
Graeme J. Dougherty
Keyword(s):  
Tumor Cell ◽  
Plasmid Vector ◽  
Death Domain ◽  
Tumor Cell Death ◽  
Surface Receptors ◽  
Novel Approach ◽  
Cell Clones ◽  
Ligand Expression

Recently, we have described a novel approach to the treatment of cancer that employs a series of vectors that encode surface expressed chimeric proteins in which the cytoplasmic death domain of Fas is fused in-frame to the extracellular domain of one of a number of cell surface receptors that recognize and bind various ligands that are differentially expressed within the tumor microenvironment. Although the majority of tumor cells transduced with such vectors are killed in the presence of the corresponding cognate ligand, a small percentage survive and in vivo may go on to repopulate a treated tumor. In order to understand the mechanisms employed by tumors to escape the cytotoxic effects of pro-apoptotic signals triggered via Fas, we isolated a large number of 293 tumor cell clones that survive following transfection with a plasmid vector encoding Flk-1/Fas, a chimeric receptor that induces tumor cell death in the presence of the pro-angiogenic cytokine VEGF. Characterization of Flk-1/Fas-positive clones revealed that while survival can most often be attributed simply to the down-regulation of VEGF ligand expression, in cells that express both receptor and ligand, other proteins involved in the regulation of apoptosis may be targeted. Specifically, a Flk-1/Fas-positive, VEGF-positive clone was identified in which expression of APAF-1 was almost completely abrogated.

scholarly journals Sequences Just Upstream of the Simian Immunodeficiency Virus Core Enhancer Allow Efficient Replication in the Absence of NF-κB and Sp1 Binding Elements

1998 ◽  
Vol 72 (7) ◽  
pp. 5589-5598 ◽  
Author(s):  
Stefan Pöhlmann ◽  
Stefan Flöss ◽  
Petr O. Ilyinskii ◽  
Thomas Stamminger ◽  
Frank Kirchhoff
Keyword(s):  
Viral Replication ◽  
Binding Site ◽  
Simian Immunodeficiency Virus ◽  
Binding Sites ◽  
Regulatory Element ◽  
Virus Production ◽  
Enhancer Element ◽  
Immunodeficiency Virus ◽  
Efficient Replication

ABSTRACT Large deletions of the upstream U3 sequences in the long terminal repeats (LTRs) of human immunodeficiency virus and simian immunodeficiency virus (SIV) accumulate in vivo in the absence of an intact nef gene. In the SIV U3 region, about 65 bp just upstream of the single NF-κB binding site always remained intact, and some evidence for a novel enhancer element in this region exists. We analyzed the transcriptional and replicative capacities of SIVmac239 mutants containing deletions or mutations in these upstream U3 sequences and/or the NF-κB and Sp1 binding sites. Even in the absence of 400 bp of upstream U3 sequences, the NF-κB site and all four Sp1 binding sites, the SIV promoter maintained about 15% of the wild-type LTR activity and was fully responsive to Tat activation in transient reporter assays. The effects of these deletions on virus production after transfection of COS-1 cells with full-length proviral constructs were much greater. Deletion of the upstream U3 sequences had no significant influence on viral replication when either the single NF-κB site or the Sp1 binding sites were intact. In contrast, the 26 bp of sequence located immediately upstream of the NF-κB site was essential for efficient replication when all core enhancer elements were deleted. A purine-rich site in this region binds specifically to the transcription factor Elf-1, a member of the etsproto-oncogene-encoded family. Our results indicate a high degree of functional redundancy in the SIVmac U3 region. Furthermore, we defined a novel regulatory element located immediately upstream of the NF-κB binding site that allows efficient viral replication in the absence of the entire core enhancer region.

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