No abstract

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.

Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage–tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis–induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.

Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in Phase I clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may only be efficacious in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. As such, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe “cytokine storm”. Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFNβ), tumor necrosis factor alpha (TNFα) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.

Understanding the mechanism of cisplatin (CDDP) action may improve therapeutic strategy for ovarian cancer. Although p53 and FLICE-like inhibitory protein (FLIP) are determinants of CDDP sensitivity in ovarian cancer, the interaction between p53 and FLIP remains poorly understood. Here, using two chemosensitive ovarian cancer cell lines and various molecular and cellular approaches, we show that CDDP induces p53-dependent FLIP ubiquitination and degradation, and apoptosis in vitro. Moreover, we showed that Itch (an E3 ligase) forms a complex with FLIP and p53 upon CDDP treatment. These results suggest that p53 facilitates FLIP down-regulation by CDDP-induced FLIP ubiquitination and proteasomal degradation. [Cancer Res 2008;68(12):4511-7]

To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.

d Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. Brain tumors are particularly life-threatening due to their sensitive anatomical location. Recently, temozolomide plus radiotherapy has provided a measurable survival benefit to a subset of patients (1), but more effective therapies are still needed. In this regard, oncolytic viruses (OVs) seem particularly promising, as they display higher tumor specificity and possibly fewer side effects than standard therapies (2). One of our OV candidates, attenuated Semliki Forest virus (SFV), was able to fully eradicate orthotopic U87 xenografts in 100% of treated nude mice following a single systemic injection (3). However, in other models, we and others have identified limitations to oncolytic virotherapy; in particular, innate antiviral defenses limit virus replication in tumor cells (4,5).In order to probe further and overcome the mechanisms of glioma resistance to oncolytic SFV, we combined it with oncolytic vaccinia virus (VV), which itself has shown promise in glioma targeting and also has the capacity to facilitate replication of type I interferon (IFN)-sensitive OVs by antagonizing innate antiviral defenses (6)(7)(8). First, we demonstrate efficient killing of DBT mouse glioma cells in vitro but not in vivo by SFV alone, mirroring the results we observed in our previous rat model (4) (Fig. 1A and B). Lack of efficacy could not be explained simply by the immune competence of the animals, as SFV successfully eradicated another type of syngeneic tumor (CT26LacZ) at a similar dose (not shown). Next, we observed that SFV limits its own spread in DBT cells under spatially restrictive conditions (under agarose) and that this limitation could be lifted by coinfecting cells with VV or neutralizing type I IFN using polyclonal antibody (anti-beta interferon [IFN-␤]) or recombinant vaccinia virus-encoded B18R protein (Fig. 1C). Facilitation of SFV spread in DBT cells by VV is dependent on B18R, as we did not see enhancement when B18R-deleted VV was used. While the spread of SFV under agarose was enhanced when SFV was combined with VV, replication of VV itself was strongly inhibited (Fig. 1C), which was also confirmed by quantifying virus output from coinfected DBT cells in free culture (Fig. 1D). DBT cell killing by the virus combinations was synergistic, as calculated by the Chou-Talalay method (Fig. 1E). However, combination of SFV with VV in an orthotopic DBT model did not provide statistically significant improvement in survival compared to the next best therapy, VV alone, by either systemic injection or d...

Normal values for healthy young men are now provided for the normal and the sensitized state. The percentage of subjects sensitized after acid stimulation are thoroughly documented, and depends on stimulation type and the cut-off value chosen.