Irradiation enhances the therapeutic effect of the oncolytic adenovirus XVir-N-31 in brain tumor initiating cells.

Virotherapy using oncolytic viruses is an upcoming therapy strategy for cancer treatment. A variety of preclinical and clinical trials have indicated that adenoviruses may be used as potent agents in the treatment of a variety of cancers, and also for the treatment of brain tumors. In these studies, it has also been shown that oncovirotherapy is safe in terms of toxicity and side effects. In addition, previous studies have presented evidence for a significant role of oncovirotherapy in the activation of anti­tumor immune responses. With regard to oncolytic adenoviruses, we have demonstrated previously that the multifunctional protein Y­box binding protein­1 (YB­1) is a potent factor that was used to develop an YB­1­dependent oncolytic adenovirus (XVir­N­31). XVir­N­31 provides the opportunity for tumor­selective replication and exhibited marked oncolytic properties in a mouse glioma tumor model using therapy­resistant brain tumor initiating cells (BTICs). In a number of, but not all, patients with glioma, YB­1 is primarily located in the nucleus; this promotes XVir­N­31­replication and subsequently tumor cell lysis. However, in certain BTICs, only a small amount of YB­1 has been identified to be nuclear, and therefore virus replication is suboptimal. YB­1 in BTICs was demonstrated to be translocated into the nucleus following irradiation, which was accompanied by an enhancement in XVir­N­31 production. R28 glioma spheres implanted in living organotypic human brain slices exhibited a significantly delayed growth rate when pre­irradiated prior to XVir­N­31­infection as compared with single treatment methods. Consistent with the in vitro data, R28 glioma­bearing mice exhibited a prolonged mean and median survival following single tumor irradiation prior to intratumoral XVir­N­31 injection, compared with the single treatment methods. In conclusion, the present study demonstrated that in an experimental glioma model, tumor irradiation strengthened the effect of an XVir­N­31­based oncovirotherapy.

Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses.

Oncolytic viruses (OVs) are unique anticancer agents based on their pleotropic modes of action, which include, besides viral tumor cell lysis, activation of antitumor immunity. A panel of diverse viruses, often genetically engineered, has advanced to clinical investigation, including phase 3 studies. This diversity of virotherapeutics not only offers interesting opportunities for the implementation of different therapeutic regimens but also poses challenges for clinical translation. Thus, manufacturing processes and regulatory approval paths need to be established for each OV individually. This review provides an overview of clinical-grade manufacturing procedures for OVs using six virus families as examples, and key challenges are discussed individually. For example, different virus features with respect to particle size, presence/absence of an envelope, and host species imply specific requirements for measures to ensure sterility, for handling, and for determination of appropriate animal models for toxicity testing, respectively. On the other hand, optimization of serum-free culture conditions, increasing virus yields, development of scalable purification strategies, and formulations guaranteeing long-term stability are challenges common to several if not all OVs. In light of the recent marketing approval of the first OV in the Western world, strategies for further upscaling OV manufacturing and optimizing product characterization will receive increasing attention.

CDK4/6 Inhibition Controls Proliferation of Bladder Cancer and Transcription of RB1.

PURPOSE: The retinoblastoma signaling network is frequently altered in advanced bladder cancer. We investigated the potential of CDK4/6 as a therapeutic target and determined biomarkers for patient stratification. MATERIALS AND METHODS: Genetic alterations were analyzed using public databases, including TCGA (The Cancer Genome Atlas), COSMIC (Catalogue of Somatic Mutations in Cancer) and CCLE (Cancer Cell Line Encyclopedia). Effects of the CDK4/6-inhibitor PD-0332991 or LY2835219 were examined in 10 bladder cancer cell lines by immunoblot, cell viability, apoptosis and cell cycle progression. Efficacy of the PD-0332991 and cisplatin combination was analyzed using the combination index. Gene expression level was determined by quantitative polymerase chain reaction. Cytomegalovirus promoter regulated recombinant retinoblastoma was used for reconstitution. Three-dimensional xenografts were grown on chicken chorioallantoic membrane and analyzed by measuring tumor weight and immunohistochemical expression of total retinoblastoma and Ki-67. RESULTS: PD-0332991 treatment decreased the proliferation of retinoblastoma positive bladder cancer cell lines and was synergistic in combination with cisplatin. PD-0332991 or LY2835219 treatment decreased the phosphorylation, total protein and transcript level of retinoblastoma. Treatment resulted in a decrease in E2F target gene expression (CCNA2 and CCNE2) and cell cycle progression from G0/G1 to the S-phase but did not affect apoptosis. In retinoblastoma negative cells reconstituted with recombinant retinoblastoma PD-0332991 affected only phosphorylation and not the total retinoblastoma level. These cells remained resistant to treatment. In 3-dimensional retinoblastoma xenografts, treatment resulted in reduced tumor weight and decreased expression of total retinoblastoma and Ki-67. CONCLUSIONS: We provide preclinical evidence that CDK4/6 inhibition is a potential therapeutic strategy for retinoblastoma positive bladder cancer that probably acts by negatively regulating retinoblastoma transcription.

EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene.

We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by (123)I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. (124)I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of (131)I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e131; doi:10.1038/mtna.2013.58; published online 5 November 2013.

Systemic image-guided liver cancer radiovirotherapy using dendrimer-coated adenovirus encoding the sodium iodide symporter as theranostic gene.

UNLABELLED: Currently, major limitations for the clinical application of adenovirus-mediated gene therapy are high prevalence of neutralizing antibodies, widespread expression of the coxsackie-adenovirus receptor (CAR), and adenovirus sequestration by the liver. In the current study, we used the sodium iodide symporter (NIS) as a theranostic gene to investigate whether coating of adenovirus with synthetic dendrimers could be useful to overcome these hurdles in order to develop adenoviral vectors for combination of systemic oncolytic virotherapy and NIS-mediated radiotherapy. METHODS: We coated replication-deficient (Ad5-CMV/NIS) (CMV is cytomegalovirus) and replication-selective (Ad5-E1/AFP-E3/NIS) adenovirus serotype 5 carrying the hNIS gene with poly(amidoamine) dendrimers generation 5 (PAMAM-G5) in order to investigate transduction efficacy and altered tropism of these coated virus particles by (123)I scintigraphy and to evaluate their therapeutic potential for systemic radiovirotherapy in a liver cancer xenograft mouse model. RESULTS: After dendrimer coating, Ad5-CMV/NIS demonstrated partial protection from neutralizing antibodies and enhanced transduction efficacy in CAR-negative cells in vitro. In vivo (123)I scintigraphy of nude mice revealed significantly reduced levels of hepatic transgene expression after intravenous injection of dendrimer-coated Ad5-CMV/NIS (dcAd5-CMV/NIS). Evasion from liver accumulation resulted in significantly reduced liver toxicity and increased transduction efficiency of dcAd5-CMV/NIS in hepatoma xenografts. After PAMAM-G5 coating of the replication-selective Ad5-E1/AFP-E3/NIS, a significantly enhanced oncolytic effect was observed after intravenous application (virotherapy) that was further increased by additional treatment with a therapeutic dose of (131)I (radiovirotherapy) and was associated with markedly improved survival. CONCLUSION: These results demonstrate efficient liver detargeting and tumor retargeting of adenoviral vectors after coating with synthetic dendrimers, thereby representing a promising innovative strategy for systemic NIS gene therapy. Moreover, our study-based on the function of NIS as a theranostic gene allowing the noninvasive imaging of NIS expression by (123)I scintigraphy-provides detailed characterization of in vivo vector biodistribution and localization, level, and duration of transgene expression, essential prerequisites for exact planning and monitoring of clinical gene therapy trials that aim to individualize the NIS gene therapy concept.

Adenoviral vectors coated with PAMAM dendrimer conjugates allow CAR independent virus uptake and targeting to the EGF receptor.

Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)ß(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

Elevated YB-1 expression is a new unfavorable prognostic factor in non-Hodgkin's lymphomas.

BACKGROUND: Y-Box-binding protein-1 (YB-1) acts as a transcription factor for multiple genes and is linked to DNA replication and repair, cell proliferation and resistance to cytostatic drugs. PATIENTS AND METHODS: The prognostic value of YB-1 expression in primarily untreated malignant non-Hodgkin's lymphomas (NHLs) was examined using immunohistochemistry. RESULTS: Expression of YB-1 was detected in 48 out of 56 NHLs, and the immunohistochemical reaction was localized exclusively in the cytoplasm. Expression of YB-1 did not correlate with clinicopathological variables. Patients with higher YB-1 expression had shorter progression-free survival during the entire period of observation (p=0.0434), as well as in the course of 30 months' observation (p=0.0253). Additionally, in the course of 50 months' observation, patients with higher expression of YB-1 demonstrated a shorter overall survival time (p=0.0383) and a shorter progression-free survival (p=0.0309). CONCLUSION: Elevated YB-1 expression may represent a new unfavorable prognostic factor.

YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma.

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting.

Inhibition of the multidrug-resistant phenotype by targeting YB-1 with a conditionally oncolytic adenovirus: implications for combinatorial treatment regimen with chemotherapeutic agents.

Bearing in mind the limited success of available treatment modalities for the therapy of multidrug-resistant tumor cells, alternative and complementary strategies need to be developed. It is known that the transcriptional activation of genes, such as MDR1 and MRP1, which play a major role in the development of a multidrug-resistant phenotype in tumor cells, involves the Y-box protein YB-1. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In addition, it has been reported previously that YB-1 is an important factor in adenoviral replication because it activates transcription from the adenoviral E2-late promoter. Here, we report that an oncolytic adenovirus, named Xvir03, expressing the viral proteins E1B55k and E4orf6, leads to nuclear translocation of YB-1 and in consequence to viral replication and cell lysis in vitro and in vivo. Moreover, we show that Xvir03 down-regulates the expression of MDR1 and MRP1, indicating that recruiting YB-1 to the adenoviral E2-late promoter for viral replication is responsible for this effect. Thus, nuclear translocation of YB-1 by Xvir03 leads to resensitization of tumor cells to cytotoxic drugs. These data reveal a link between chemotherapy and virotherapy based on the cellular transcription factor YB-1 and provide the basis for formulating a model for a novel combined therapy regimen named Mutually Synergistic Therapy.

Novel three-pronged strategy to enhance cancer cell killing in glioblastoma cell lines: histone deacetylase inhibitor, chemotherapy, and oncolytic adenovirus dl520.

Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency. The objective of this study was to determine whether the chemotherapeutic agent irinotecan, by enhancing nuclear localization of YB-1, and the histone deacetylase inhibitor trichostatin A, by upregulating coxsackievirus-adenovirus receptor (CAR) expression, could augment replication of and cell lysis by adenovirus dl520 in glioblastomas in vitro. We found that trichostatin A upregulated CAR expression and that irinotecan caused increased nuclear localization of YB-1 in both glioblastoma cell lines. Irinotecan alone, and trichostatin A alone, enhanced replication of and cell lysis by dl520. Importantly, when combining both agents, the replication efficiency (maximum, 27-fold) and induction of cytopathic effect (maximum, 3.8-fold) of dl520 were further augmented significantly. These results support the hypothesis that the enhanced oncolytic effect of dl520, after incubation with chemotherapeutic agents, is mediated by an increased accumulation of YB-1 in the nucleus (due to irinotecan) and by upregulation of CAR (due to trichostatin A). Thus, therapy combining virotherapy, chemotherapy, and histone deacetylase inhibitor treatment is a novel approach to enhance the oncolytic efficacy of dl520.

Multidrug-resistant cancer cells facilitate E1-independent adenoviral replication: impact for cancer gene therapy.

Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In drug-resistant cancer cells and in adenovirus-infected cells YB-1 is found in the nucleus. Nuclear accumulation of YB-1 in adenovirus-infected cells is a function of the E1 region, and we have shown that YB-1 facilitates adenovirus replication. Here we report that E1A-deleted or mutant adenovirus vectors, such as Ad312 and Ad520, replicate efficiently in multidrug-resistant (MDR) cancer cells and induce an adenovirus cytopathic effect resulting in host cell lysis. Thus, replication-defective adenoviruses are a previously unrecognized vector system for a selective elimination of MDR cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors for the treatment of MDR malignant diseases in the clinical setting.

YB-1 relocates to the nucleus in adenovirus-infected cells and facilitates viral replication by inducing E2 gene expression through the E2 late promoter.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.