Erratum: Improved oncolytic activity of a reovirus mutant that displays enhanced virus spread due to reduced cell attachment.

[This corrects the article DOI: 10.1016/j.omto.2023.100743.].

Oncolytic vaccinia virus immunotherapy antagonizes image-guided radiotherapy in mouse mammary tumor models.

Ionizing radiation (IR) and oncolytic viruses are both used to treat cancer, and the effectiveness of both agents depends upon stimulating an immune response against the tumor. In this study we tested whether combining image guided ionizing radiation (IG-IR) with an oncolytic vaccinia virus (VACV) could yield a better therapeutic response than either treatment alone. ΔF4LΔJ2R VACV grew well on irradiated human and mouse breast cancer cells, and the virus can be combined with 4 or 8 Gy of IR to kill cells in an additive or weakly synergistic manner. To test efficacy in vivo we used immune competent mice bearing orthotopic TUBO mammary tumors. IG-IR worked well with 10 Gy producing 80% complete responses, but this was halved when the tumors were treated with VACV starting 2 days after IG-IR. VACV monotherapy was ineffective in this model. The antagonism was time dependent as waiting for 21 days after IG-IR eliminated the inhibitory effect but without yielding any further benefits over IR alone. In irradiated tumors, VACV replication was also lower, suggesting that irradiation created an environment that did not support infection as well in vivo as in vitro. A study of how four different treatment regimens affected the immune composition of the tumor microenvironment showed that treating irradiated tumors with VACV altered the immunological profiles in tumors exposed to IR or VACV alone. We detected more PD-1 and PD-L1 expression in tumors exposed to IR+VACV but adding an αPD-1 antibody to the protocol did not change the way VACV interferes with IG-IR therapy. VACV encodes many immunosuppressive gene products that may interfere with the ability of radiotherapy to induce an effective anti-tumor immune response through the release of danger-associated molecular patterns. These data suggest that infecting irradiated tumors with VACV, too soon after exposure, may interfere in the innate and linked adaptive immune responses that are triggered by radiotherapy to achieve a beneficial impact.

Improved oncolytic activity of a reovirus mutant that displays enhanced virus spread due to reduced cell attachment.

Wild-type reovirus serotype 3 Dearing (T3wt), a non-pathogenic intestinal virus, has shown promise as a cancer therapy in clinical trials, but it would benefit from an increased potency. Given that T3wt is naturally adapted to the intestinal environment (rather than tumors), we genetically modified reovirus to improve its infectivity in cancer cells. Various reovirus mutants were created, and their oncolytic potency was evaluated in vitro using plaque size as a measure of virus fitness in cancer cells. Notably, Super Virus 5 (SV5), carrying five oncolytic mutations, displayed the largest plaques in breast cancer cells among the mutants tested, indicating the potential for enhancing oncolytic potency through the combination of mutations. Furthermore, in a HER2+ murine breast cancer model, mice treated with SV5 exhibited superior tumor reduction and increased survival compared with those treated with PBS or T3wt. Intriguingly, SV5 did not replicate faster than T3wt in cultured cells but demonstrated a farther spread relative to T3wt, attributed to its reduced attachment to cancer cells. These findings highlight the significance of increased virus spread as a crucial mechanism for improving oncolytic virus activity. Thus, genetic modifications of reovirus hold the potential for augmenting its efficacy in cancer therapy.

Radiation-Induced Cellular Senescence Reduces Susceptibility of Glioblastoma Cells to Oncolytic Vaccinia Virus.

Glioblastoma (GBM) is a malignant brain cancer refractory to the current standard of care, prompting an extensive search for novel strategies to improve outcomes. One approach under investigation is oncolytic virus (OV) therapy in combination with radiotherapy. In addition to the direct cytocidal effects of radiotherapy, radiation induces cellular senescence in GBM cells. Senescent cells cease proliferation but remain viable and are implicated in promoting tumor progression. The interaction of viruses with senescent cells is nuanced; some viruses exploit the senescent state to their benefit, while others are hampered, indicating senescence-associated antiviral activity. It is unknown how radiation-induced cellular senescence may impact the oncolytic properties of OVs based on the vaccinia virus (VACV) that are used in combination with radiotherapy. To better understand this, we induced cellular senescence by treating GBM cells with radiation, and then evaluated the growth kinetics, infectivity, and cytotoxicity of an oncolytic VACV, ∆F4LΔJ2R, as well as wild-type VACV in irradiated senescence-enriched and non-irradiated human GBM cell lines. Our results show that both viruses display attenuated oncolytic activities in irradiated senescence-enriched GBM cell populations compared to non-irradiated controls. These findings indicate that radiation-induced cellular senescence is associated with antiviral activity and highlight important considerations for the combination of VACV-based oncolytic therapies with senescence-inducing agents such as radiotherapy.

Radiation combined with oncolytic vaccinia virus provides pronounced antitumor efficacy and induces immune protection in an aggressive glioblastoma model.

Glioblastoma (GB) is a malignant and immune-suppressed brain cancer that remains incurable despite the current standard of care. Radiotherapy is a mainstay of GB treatment, however invasive cancer cells outside the irradiated field and radioresistance preclude complete eradication of GB cells. Oncolytic virus therapy harnesses tumor-selective viruses to spread through and destroy tumors while stimulating antitumor immune responses, and thus has potential for use following radiotherapy. We demonstrate that oncolytic ΔF4LΔJ2R vaccinia virus (VACV) replicates in and induces cytotoxicity of irradiated brain tumor initiating cells in vitro. Importantly, a single 10 Gy dose of radiation combined with ΔF4LΔJ2R VACV produced considerably superior anticancer effects relative to either monotherapy when treating immune-competent orthotopic CT2A-luc mouse models-significantly extending survival and curing the majority of mice. Mice cured by the combination displayed significantly increased survival relative to naïve age-matched controls following intracranial tumor challenge, with some complete rejections. Further, the combination therapy was associated with an increased ratio of CD8+ effector T cells to regulatory T cells compared to either monotherapy. This study validates the use of radiation with an oncolytic ΔF4LΔJ2R VACV to improve treatment of this malignant brain cancer.

Genetic Modifications That Expand Oncolytic Virus Potency.

Oncolytic viruses (OVs) are a promising type of cancer therapy since they selectively replicate in tumor cells without damaging healthy cells. Many oncolytic viruses have progressed to human clinical trials, however, their performance as monotherapy has not been as successful as expected. Importantly, recent literature suggests that the oncolytic potential of these viruses can be further increased by genetically modifying the viruses. In this review, we describe genetic modifications to OVs that improve their ability to kill tumor cells directly, to dismantle the tumor microenvironment, or to alter tumor cell signaling and enhance anti-tumor immunity. These advances are particularly important to increase virus spread and reduce metastasis, as demonstrated in animal models. Since metastasis is the principal cause of mortality in cancer patients, having OVs designed to target metastases could transform cancer therapy. The genetic alterations reported to date are only the beginning of all possible improvements to OVs. Modifications described here could be combined together, targeting multiple processes, or with other non-viral therapies with potential to provide a strong and lasting anti-tumor response in cancer patients.

DREAM, a possible answer to the estrogen paradox of the Women's Health Initiative Trial.

Estrogen is thought to cause proliferation of all estrogen receptor positive (ER+) breast cancers. Paradoxically, in the Women's Health Initiative Trial, estrogen-only hormone replacement therapy reduced the incidence and mortality of low grade, ER+, HER2- breast cancer. We gave estradiol to 19 post-menopausal women with newly diagnosed low-grade, ER+, HER2- breast cancer in a prospective window of opportunity clinical trial and examined the changes in proliferation and gene expression before and after estradiol treatment. Ki67 decreased in 13/19 (68%) patients and 8/13 (62%) showed a decrease in Risk of Recurrence Score. We chose three prototypical estrogen responders (greatest decrease in ROR) and non-responders (no/minimal change in ROR) and applied a differential gene expression analysis to develop pre-treatment (PRESTO-30core) and post-treatment (PRESTO-45surg) gene expression profiles. The PRESTO-30core predicted adjuvant benefit in a published series of tamoxifen, the partial estrogen agonist. Of the 45 genes in the PRESTO-45surg, thirty contain the Cell cycle genes Homology Region (CHR) motif that binds the class B multi-vulva complex (MuvB) a member of the DREAM (Dimerization partner, retinoblastoma-like proteins, E2F, MuvB) complex responsible for reversible cell cycle arrest or quiescence. There was also near uniform suppression (89%) of the remaining DREAM genes consistent with estrogen induced activation of the DREAM complex to mediate cell cycle block after a short course of estrogens. To our knowledge, this is the first report to show estrogen modulation of DREAM genes and suggest involvement of DREAM pathway associated quiescence in endocrine responsive post-menopausal ER+ breast cancers.

Novel colchicine derivative CR42-24 demonstrates potent anti-tumor activity in urothelial carcinoma.

Bladder cancers, and specifically urothelial carcinoma, have few effective treatment options, and tumors typically develop resistance against standard of care chemotherapies leading to significant mortality. The development of alternative therapies with increased selectivity and improved tolerability would significantly impact this patient population. Here, we investigate a novel colchicine derivative, CR42-24, with increased selectivity for the ßIII tubulin subtype as a treatment for urothelial carcinoma. ßIII tubulin is a promising target due to its low expression in healthy tissues and its clinical association with poor prognosis. This study demonstrated that CR42-24 is selectively cytotoxic to several cancer cell lines at low nanomolar IC50, with high activity in bladder cancer cell lines both in vitro and in vivo. CR42-24 monotherapy in an aggressive urothelial carcinoma xenograft model results in effective control when treated early. We observed significant ablation of large tumors and patient-derived xenografts at low doses with excellent tolerability. CR42-24 was highly synergistic in combination with the standard of care chemotherapies gemcitabine and cisplatin, further increasing its therapeutic potential as a novel treatment for urothelial carcinoma.

Inhibition of triple negative breast cancer metastasis and invasiveness by novel drugs that target epithelial to mesenchymal transition.

Invasive breast cancer (BrCa) is predicted to affect 1 in 9 women in a lifetime;1 in 32 will die from this disease. The most aggressive forms of BrCa, basal-like/triple-negative phenotype (TNBC), are challenging to treat and result in higher mortality due high number of metastatic cases. There is a paucity of options for TNBC treatment, which highlights the need for additional innovative treatment approaches. NIH-III mice were injected in the abdominal mammary fat pad with luciferase-expressing derivative of the human TNBC cell line, MDA-MB-231 cells. Animals were gavage-fed with nitrofen at the doses of 1, 3 or 6 mg/kg/alternate days. However, several structural properties/components of nitrofen raise concerns, including its high lipophilicity (cLogP of nearly 5) and a potential toxophore in the form of a nitroarene group. Therefore, we developed analogues of nitrofen which lack the nitro group and/or have replaced the diaryl ether linker with a diarylamine that could allow modulation of polarity. In vitro anti-invasiveness activity of nitrofen analogues were evaluated by quantitative determination of invasion of MDA-MB-231-Luciferase cells through Matrigel using a Boyden chamber. Our in vivo data show that nitrofen efficiently blocks TNBC tumor metastasis. In vitro data suggest that this is not due to cytotoxicity, but rather is due to impairment of invasive capacity of the cells. Further, using an in vitro model of EMT, we show that nitrofen interferes with the process of EMT and promotes mesenchymal to epithelial transformation. In addition, we show that three of the nitrofen analogues significantly reduced invasive potential of TNBC cells, which may, at least partially, be attributed to the analogues' ability to promote mesenchymal to epithelial-like transformation of TNBC cells. Our study shows that nitrofen, and more importantly its analogues, are significantly effective in limiting the invasive potential of TNBC cell lines with minimal cytotoxic effect. Further, we demonstrate that nitrofen its analogues, are very effective in reversing mesenchymal phenotype to a more epithelial-like phenotype. This may be significant for the treatment of patients with mesenchymal-TNBC tumor subtype who are well known to exhibit high resistance to chemotherapy.

Procaspase-Activating Compound-1 Synergizes with TRAIL to Induce Apoptosis in Established Granulosa Cell Tumor Cell Line (KGN) and Explanted Patient Granulosa Cell Tumor Cells In Vitro.

Granulosa cell tumors (GCT) constitute only ~5% of ovarian neoplasms yet have significant consequences, as up to 80% of women with recurrent GCT will die of the disease. This study investigated the effectiveness of procaspase-activating compound 1 (PAC-1), an activator of procaspase-3, in treating adult GCT (AGCT) in combination with selected apoptosis-inducing agents. Sensitivity of the AGCT cell line KGN to these drugs, alone or in combination with PAC-1, was tested using a viability assay. Our results show a wide range in cytotoxic activity among the agents tested. Synergy with PAC-1 was most pronounced, both empirically and by mathematical modelling, when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This combination showed rapid kinetics of apoptosis induction as determined by caspase-3 activity, and strongly synergistic killing of both KGN as well as patient samples of primary and recurrent AGCT. We have demonstrated that the novel combination of two pro-apoptotic agents, TRAIL and PAC-1, significantly amplified the induction of apoptosis in AGCT cells, warranting further investigation of this combination as a potential therapy for AGCT.

Incomplete resection of necrotic bowel may increase mortality in infants with necrotizing enterocolitis.

BACKGROUND: Infants with advanced necrotizing enterocolitis (NEC) often need surgical resection of necrotic bowel. We hypothesized that incomplete resection of NEC lesions, signified by the detection of necrotic patches in margins of resected bowel loops, results in inferior clinical outcomes. METHODS: We reviewed the medical records of infants with surgical NEC in the past 15 years for demographic, clinical, and histopathological data. We also developed statistical models to predict mortality and hospital stay. RESULTS: Ninety infants with surgical NEC had a mean (±standard error) gestational age of 27.3 ± 0.4 weeks, birth weight 1008 ± 48 g, NEC onset at 25.2 ± 2.4 days, and resected bowel length of 29.2 ± 3.2 cm. Seventeen (18.9%) infants who had complete resection of the necrosed bowel had fewer (4; 23.5%) deaths and shorter lengths of hospital stay. In contrast, a group of 73 infants with some necrosis within the margins of resected bowel had significantly more (34; 46.6%) deaths and longer hospital stay. The combination of clinical and histopathological data gave better regression models for mortality and hospital stay. CONCLUSION: In surgical NEC, incomplete resection of necrotic bowel increased mortality and the duration of hospitalization. Regression models combining clinical and histopathological data were more accurate for mortality and the length of hospital stay. IMPACT: In infants with surgical NEC, complete resection of necrotic bowel reduced mortality and hospital stay. Regression models combining clinical and histopathological information were superior at predicting mortality and hospital stay than simpler models focusing on either of these two sets of data alone. Prediction of mortality improved with the combination of antenatal steroids, chorioamnionitis, and duration of post-operative ileus, with severity of inflammation and hemorrhages in resected intestine. Length of hospital stay was shorter in infants with higher gestational ages, but longer in those with greater depth of necrosis or needing prolonged parenteral nutrition or supervised feedings.

The Impact of Radiation-Induced DNA Damage on cGAS-STING-Mediated Immune Responses to Cancer.

Radiotherapy is a major modality used to combat a wide range of cancers. Classical radiobiology principles categorize ionizing radiation (IR) as a direct cytocidal therapeutic agent against cancer; however, there is an emerging appreciation for additional antitumor immune responses generated by this modality. A more nuanced understanding of the immunological pathways induced by radiation could inform optimal therapeutic combinations to harness radiation-induced antitumor immunity and improve treatment outcomes of cancers refractory to current radiotherapy regimens. Here, we summarize how radiation-induced DNA damage leads to the activation of a cytosolic DNA sensing pathway mediated by cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING). The activation of cGAS-STING initiates innate immune signaling that facilitates adaptive immune responses to destroy cancer. In this way, cGAS-STING signaling bridges the DNA damaging capacity of IR with the activation of CD8+ cytotoxic T cell-mediated destruction of cancer-highlighting a molecular pathway radiotherapy can exploit to induce antitumor immune responses. In the context of radiotherapy, we further report on factors that enhance or inhibit cGAS-STING signaling, deleterious effects associated with cGAS-STING activation, and promising therapeutic candidates being investigated in combination with IR to bolster immune activation through engaging STING-signaling. A clearer understanding of how IR activates cGAS-STING signaling will inform immune-based treatment strategies to maximize the antitumor efficacy of radiotherapy, improving therapeutic outcomes.

Clinical determinants of postoperative outcomes in surgical necrotizing enterocolitis.

OBJECTIVE: Investigate predictors of postoperative morbidity and mortality in surgical NEC. STUDY DESIGN: We analyzed the clinical outcomes of infants with surgical NEC from the years 2000-2015. RESULTS: Ninety infants born at gestation (mean ± standard deviation, SD; standard error of mean, SEM) 27.3 ± 6.6 weeks (SEM ± 0.07 weeks) and weighing 1008 ± 456 g (SEM ± 48 g) developed NEC on 25.2 ± 22.4 days (SEM ± 2.4 days). Average bowel resection was 29.2 ± 30.5 cm (SEM ± 3.2 cm). Postoperative Ileus lasted 16.5 ± 12.2 days (SEM ± 1.3 days), and was significantly longer in infants with higher gestation and birth weight, age at onset of NEC, length of intestinal resection, maternal chorioamnionitis, and need for pressors. Thirty-eight (42.2%) infants died. Mortality was higher at gestation <31 weeks. CONCLUSION: Gestational age, birth weight, age at NEC onset, and length of resected bowel determined postoperative morbidity and mortality in NEC. Length of hospital stay was affected by above factors, and also the duration of postoperative ileus and parenteral nutrition.

Deciphering the Immunomodulatory Capacity of Oncolytic Vaccinia Virus to Enhance the Immune Response to Breast Cancer.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8+ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

Breast Tumor-Associated Metalloproteases Restrict Reovirus Oncolysis by Cleaving the σ1 Cell Attachment Protein and Can Be Overcome by Mutation of σ1.

Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyomavirus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein σ1, which diminished virus binding to sialic acid (SA)-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated σ1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231, and HS578T breast cancer cell lines recapitulated σ1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers and that breast cancer cells by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of SA levels on a panel of cancer cells showed that truncated σ1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in σ1 that prevents σ1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, toxicity equivalent to that of wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data show that tumor microenvironments have the potential to reduce infectivity of reovirus.IMPORTANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have a negative impact on proteinaceous cancer therapies, such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

Targeting Nucleotide Biosynthesis: A Strategy for Improving the Oncolytic Potential of DNA Viruses.

The rapid growth of tumors depends upon elevated levels of dNTPs, and while dNTP concentrations are tightly regulated in normal cells, this control is often lost in transformed cells. This feature of cancer cells has been used to advantage to develop oncolytic DNA viruses. DNA viruses employ many different mechanisms to increase dNTP levels in infected cells, because the low concentration of dNTPs found in non-cycling cells can inhibit virus replication. By disrupting the virus-encoded gene(s) that normally promote dNTP biosynthesis, one can assemble oncolytic versions of these agents that replicate selectively in cancer cells. This review covers the pathways involved in dNTP production, how they are dysregulated in cancer cells, and the various approaches that have been used to exploit this biology to improve the tumor specificity of oncolytic viruses. In particular, we compare and contrast the ways that the different types of oncolytic virus candidates can directly modulate these processes. We limit our review to the large DNA viruses that naturally encode homologs of the cellular enzymes that catalyze dNTP biogenesis. Lastly, we consider how this knowledge might guide future development of oncolytic viruses.

Assessment of Specificity of an Adenovirus Targeted to HER3/4.

Gene therapy with viral vectors, such as adenovirus (Ad), targeted to the human epidermal growth factor receptors 3 and 4 (HER3/4) are potentially useful for cancer therapy. Testing the expression of a reporter gene from these viruses in target cells is essential to determine functionality of the targeted virus. A competition assay with a relevant ligand (heregulin, HRG) can provide convincing evidence that blocking binding to the HER3/4 receptor results in decreased reporter gene expression. Labeling individual viruses with a fluorescent molecule allows examination of the targeted virus in specific steps in the infection. Virus internalization into cell lines can be determined using antibody-labeled receptors, and the virus colocalization with receptors can also be visualized. Characterization of a targeted virus in this fashion is important to demonstrate that the targeting of the virus functions in an expected manner, and provides support for larger-scale testing of the virus. Information acquired in these experiments may also be useful to inform and improve on the design of future targeted viruses.

Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti-tumor immunity with superior safety in bladder cancer models.

Bladder cancer has a recurrence rate of up to 80% and many patients require multiple treatments that often fail, eventually leading to disease progression. In particular, standard of care for high-grade disease, Bacillus Calmette-Guérin (BCG), fails in 30% of patients. We have generated a novel oncolytic vaccinia virus (VACV) by mutating the F4L gene that encodes the virus homolog of the cell-cycle-regulated small subunit of ribonucleotide reductase (RRM2). The F4L-deleted VACVs are highly attenuated in normal tissues, and since cancer cells commonly express elevated RRM2 levels, have tumor-selective replication and cell killing. These F4L-deleted VACVs replicated selectively in immune-competent rat AY-27 and xenografted human RT112-luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. It was also observed that rats cured of AY-27 tumors by VACV treatment developed anti-tumor immunity as evidenced by tumor rejection upon challenge and by ex vivo cytotoxic T-lymphocyte assays. Finally, F4L-deleted VACVs replicated in primary human bladder cancer explants. Our findings demonstrate the enhanced safety and selectivity of F4L-deleted VACVs, with application as a promising therapy for patients with BCG-refractory cancers and immune dysregulation.

2-aminopurine enhances the oncolytic activity of an E1b-deleted adenovirus in hepatocellular carcinoma cells.

Adenoviruses with deletions of viral genes have been extensively studied as potential cancer therapeutics. Although a high degree of cancer selectivity has been demonstrated with these conditionally replicating adenoviruses, low levels of virus replication can be detected in normal cells. Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells. Recent studies have shown that co-administration of chemotherapeutic drugs may increase the activity of these viruses without affecting their specificity. We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines. Furthermore, we show that 2-aminopurine (2'AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2'AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2'AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.