Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here,we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver)was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor.These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-α, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA-mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC. Clin Cancer Res; 16(13); 3378-89. ©2010 AACR.

Activating epidermal growth factor receptor (EGFR) mutations have been linked with sensitivity to gefitinib and erlotinib; however, there are no established predictive markers for response to the combination of EGFR inhibitors with standard chemotherapy in non -small cell lung cancer (NSCLC) patients. In this study, we characterized a panel of human EGFR wild-type and mutant NSCLC cells for their sensitivity to gefitinib alone and in combination with cisplatin or Taxol. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays. Cell cycle distribution was measured by flow cytometry. EGFR expression was measured by flow cytometry, real-time PCR, and Western blotting. EGFR/ Her2/Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation were measured by Western blotting. Two of nine EGFR wild type and one of two EGFR mutant NSCLC cells were sensitive to gefitinib, and this was associated with a decrease in phospho (p) -Akt and pErk1/2 following gefitinib exposure. There was no correlation between constitutive EGFR expression or activity and sensitivity to gefitinib nor was there a correlation between Her2/Akt and Erk1/2 activity and gefitinib sensitivity. However, in cells displaying a synergistic interaction between gefitinib and chemotherapy (cisplatin or Taxol), a dose-dependent increase in pEGFR was observed following chemotherapy exposure. In contrast, in cells where no change or a decrease in pEGFR following drug treatment was observed, we found an antagonistic or (at best) an additive interaction between the two compounds. Furthermore, the nature of this interaction was not dependent on the presence of a mutant EGFR. These novel findings suggest that modulation of EGFR activity following drug treatment determines response to gefitinib in combination with chemotherapy in NSCLC cells.

Background Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response. Methods To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R − ). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R − mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor ...

Oncolytic reovirus is currently under active investigation in a range of tumour types. Early phase studies have shown that this agent has modest monotherapy efficacy and its future development is likely to focus on combination regimens with cytotoxic chemotherapy. Indeed, phase I/II clinical trials have confirmed that reovirus can be safely combined with cytotoxic drugs, including a platin—taxane doublet regimen, which is currently being tested in a phase III clinical trial in patients with relapsed/metastatic head and neck cancer. Therefore, we have tested this triple (reovirus, cisplatin, paclitaxel) combination therapy in a panel of four head and neck cancer cell lines. Using the combination index (CI) method, the triple therapy demonstrated synergistic cytotoxicity in vitro in both malignant and non-malignant cell lines. In head and neck cancer cell lines, this was associated with enhanced caspase 3 and 7 cleavage, but no increase in viral replication. In vitro analyses confirmed colocalisation of markers of reovirus infection and caspase 3. Triple therapy was significantly more effective than reovirus or cisplatin—paclitaxel in athymic nude mice. These data suggest that the combination of reovirus plus platin—taxane doublet chemotherapy has significant activity in head and neck cancer and underpin the current phase III study in this indication.

BackgroundReovirus exploits aberrant signalling downstream of Ras to mediate tumor-specific oncolysis. Since ~90% squamous cell carcinomas of the head and neck (SCCHN) over-express EGFR and SCCHN cell lines are sensitive to oncolytic reovirus, we conducted a detailed analysis of the effects of reovirus in 15 head and neck cancer cell lines. Both pre- and post-entry events were studied in an attempt to define biomarkers predictive of sensitivity/resistance to reovirus. In particular, we analysed the role of EGFR/Ras signalling in determining virus-mediated cytotoxicity in SCCHN.MethodsTo test whether EGFR pathway activity was predictive of increased sensitivity to reovirus, correlative analyses between reoviral IC50 by MTT assay and EGFR levels by western blot and FACS were conducted. Inhibition or stimulation of EGFR signalling were analysed for their effect on reoviral oncolysis by MTT assay, and viral growth by TCID50 assay. We next analysed the effects of inhibiting signalling downstream of Ras, by specific inhibitors of p38MAPK, PI3-K or MEK, on reoviral killing examined by MTT assay. The role of PKR in reoviral killing was also determined by blockade of PKR using 2-aminopurine and assaying for cell survival by MTT assay. The apoptotic response of SCCHN to reovirus was examined by western blot analysis of caspase 3 cleavage.ResultsCorrelative analyses between reoviral sensitivity and EGFR levels revealed no association. Intermediate sub-viral and core particles showed the same infectivity/cytotoxicity as intact reovirus. Therefore, sensitivity was not determined by cell entry. In 4 cell lines, oncolysis and viral growth were both unaffected by inhibition or stimulation of EGFR signalling. Inhibition of signalling downstream of Ras did not abrogate reoviral oncolysis and, in addition, modulation of PKR using 2-aminopurine did not alter reovirus sensitivity in resistant cell lines. Caspase 3 cleavage was not detected in infected cells and oncolysis was observed in pan-caspase inhibited cells.ConclusionsIn summary, reovirus is potently oncolytic in a broad panel of SCCHN cell lines. Attempts to define sensitivity/resistance by analysis of the EGFR/Ras/MAPK pathway have failed to provide a clear predictive biomarker of response. Further analysis of material from in vitro and clinical studies is ongoing in an attempt to shed further light on this issue.

Oncogenic mutations in Kras occur in 40% to 45% of patients with advanced colorectal cancer (CRC). We have previously shown that chemotherapy acutely activates ADAM17, resulting in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. In this study, we examined the role of mutant Kras in regulating growth factor shedding and ADAM17 activity, using isogenic Kras mutant (MT) and wild-type (WT) HCT116 CRC cells. Significantly higher levels of TGF-a and VEGF were shed from KrasMT HCT116 cells, both basally and following chemotherapy treatment, and this correlated with increased pErk (phosphorylated extracellular signal regulated kinase)1/2 levels and ADAM17 activity. Inhibition of Kras, MEK (MAP/ERK kinase)1/2, or Erk1/2 inhibition abrogated chemotherapy-induced ADAM17 activity and TGF-a shedding. Moreover, we found that these effects were not drug or cell line specific. In addition, MEK1/2 inhibition in KrasMT xenografts resulted in significant decreases in ADAM17 activity and growth factor shedding in vivo, which correlated with dramatically attenuated tumor growth. Furthermore, we found that MEK1/2 inhibition significantly induced apoptosis both alone and when combined with chemotherapy in KrasMT cells. Importantly, we found that sensitivity to MEK1/2 inhibition was ADAM17 dependent in vitro and in vivo. Collectively, our findings indicate that oncogenic Kras regulates ADAM17 activity and thereby growth factor ligand shedding in a MEK1/2/Erk1/2-dependent manner and that KrasMT CRC tumors are vulnerable to MEK1/2 inhibitors, at least in part, due to their dependency on ADAM17 activity.

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