Amanda R. Oliveira

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F.

Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1–S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

Oncolytic adenovirus expressing interferon alpha in a syngeneic Syrian hamster model for the treatment of pancreatic cancer

BACKGROUND The addition of interferon (IFN) alpha to adjuvant chemoradiotherapy regimens resulted in remarkable improvements in survival for pancreatic cancer patients. However, systemic toxicities and insufficient levels of IFN at the tumor sites have limited its widespread adoption in treatment schemes. We have previously developed an IFN-expressing conditionally replicative oncolytic adenovirus and demonstrated its therapeutic effects both in vitro and in vivo. Here, the same vectors were tested in a syngeneic and immunocompetent Syrian hamster model to better understand the roles of adenoviral replication and of the pleiotropic effects of IFN on pancreatic tumor growth suppression. METHODS Oncolytic adenoviruses expressing human or hamster IFN were designed and generated. Viral vectors were tested in vitro to determine qualitative and quantitative cell viability, cyclooxygenase 2 (Cox2) promoter activity, and IFN production. For the in vivo studies, subcutaneous hamster pancreatic cancer tumors were treated with 1 intratumoral dose of virus. Similarly, 1 intraperitoneal dose of virus was used to prolong survival in a carcinomatosis model. RESULTS All cell lines tested demonstrated Cox2 promoter activity. The oncolytic potential of a replication competent adenovirus expressing the IFN cytokine was clearly demonstrated. These viruses resulted in significant tumor growth suppression and survival increases compared with controls in a hamster model. CONCLUSION The profound therapeutic potential of an IFN-expressing oncolytic adenovirus for the treatment of pancreatic cancer was demonstrated in a syngeneic Syrian hamster model. These results strongly suggest the potential application of our viruses as part of combination regimens with other therapeutics.

321. Oncolytic Adenovirus Expressing IFN as a Tool To Eliminate Pancreatic Cancer Stem Cells

Pancreatic cancer is the 4th leading cause of cancer related death in the US, and curative resection of tumors is the most effective treatment against the disease. Unfortunately, the majority of patients are diagnosed in the advanced stage of the disease, and palliative treatment with Gemcitabine is the therapy of choice. In both cases, curative resection of tumors or chemotherapy, effectiveness can be limited by the presence of pancreatic cancer stem cells. Cancer stem cells (CSC) are correlated with drug resistance, tumor recurrence, and metastasis. CSC are usually located in the hypoxic center of the tumors in a quiescent stage and are not affected by conventional chemotherapeutics that target highly replicating cells, such as Gemcitabine. CSC are also called tumor initiating cells (TIC), and as low as 100 cells are reported to form tumors in pre-clinical models.Inclusion of IFN alpha (IFN) in combination therapy protocols against pancreatic cancer can be highly beneficial to tackle this problem. IFN is reported to be cytotoxic to cancer cells, have antiangiogenic properties, stimulate anti-tumor immunity, and sensitize cancer cells to chemoradiation. In addition, IFN is reported to induce activation of quiescent CSC making them susceptible to chemotherapy drugs that target highly replicating cells. Phase II and III clinical trials combining IFN with 5-FU and radiation in an adjuvant therapy setting reported a 35% increase in the five year overall survival of pancreatic cancer patients. Stimulated by the promising results reported in IFN clinical trials we developed an oncolytic adenovirus expressing IFN (OAd-IFN). Our aim is to use the virus to improve IFN therapeutic effects in combination therapy by restricting high levels of IFN expression to the tumor. In vitro data testing triple combination of OAd-IFN + radiation+ 5-FU using MIA PACA-2 and S2103 pancreatic cancer cells in colony formation assay (CFA) showed that OAd-IFN combination was more efficacious in inhibiting colony formation than triple combination with OAd-LUC, a counterpart of OAd-IFN expressing luciferase. Triple combination with OAd-IFN was also more efficient than double therapies with 5-FU+radiation, OAd-LUC + radiation, and OAd-LUC+5-FU. As CFA is an assay frequently used to access the proliferating capacity of single cells in vitro, our data suggests that IFN expressed virus decreased the number of TIC/CSC. This is supported by our in vivo studies showing that inclusion of OAd-IFN in combination with radiation or with 5-FU+radiation results in superior tumor shrinkage than all therapies not involving IFN or including OAd-LUC. In addition, tumors treated with OAd-IFN combination have a longer recurrence interval compared with all other treatments suggesting reduced levels of TIC.Although further studies are necessary to understand the impact of our OAd-IFN virus in pancreatic cancer stem cells, we believe IFN expressed by our virus made quiescent CSC more susceptible to treatments. Because our virus can express high levels of IFN restricted to the tumor site, we believe that a higher percentage of these cells will be affected by IFN delaying clinical recurrence of tumors, and improving therapy effectiveness.

632. Oncolytic Adenoviruses Targeted to the HPV E6 and E7 Oncoproteins as a Novel Treatment for Head & Neck Squamous Cell Carcinomas

Introduction:Recent reports have shown that the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has been steadily increasing. HNSCCs are known to have a high recurrence rate and compared to their HPV-negative counterparts, these tumors have a unique biology which necessitates the development of novel treatment modalities. The HPV E6 and E7 oncoproteins are attractive therapeutic targets as they interact with key cell cycle regulatory components, namely p53 and pRb. Our research group has applied conditionally replicating oncolytic adenoviruses (CRAd) with modifications in the E1a region of the genome allowing for selective replication in E6 and E7 expressing HNSCCs.Methods:The in vitro transduction efficiency of E1-deleted luciferase expressing vectors with fiber modifications was assessed in multiple HPV-positive and negative cell lines. The CRAds were designed with a distinct deletion in the E1a region of the adenoviral genome (D24 and CB016) intended to allow for selective replication in HPV-positive HNSCC cells. Additionally, the CRAds have a luciferase transgene in the E3 region that is expressed in a replication dependent manner. By using a luciferase assay, the degree of viral replication was analyzed in numerous HNSCC cell lines. In vitro cell viability following viral infection was analyzed with crystal violet and MTS assays. A HPV-positive cell line (UPCI SCC 090) was used to establish subcutaneous tumors in female nude mice. They were subsequently treated with either one intratumoral viral injection or four injections (given every fourth day).Results:The 5/3 fiber modification significantly increased viral infectivity in all tested HNSCC cell lines. The 5/3 CB016 vector replicated selectively in HPV-positive cell lines, while the 5/3 D24 virus replicated in all cell lines regardless of HPV status. Both of the vectors demonstrated profound cytocidal effects in the crystal violet and MTS assays. For the in vivo experiments, a single intratumoral injection of virus demonstrated an anti-tumor effect for only one week following injection. Given this limited effect, an additional in vivo experiment was performed to analyze the efficacy of multiple intratumoral injections (3.5 x1011 vp/injection). This setup resulted in statistically significant tumor growth suppression at day 26 when compared to the saline control group (p<0.05 for 5/3 CB016, p<0.01 for 5/3 D24).Conclusion:CRAds designed to target HPV-positive HNSCCs demonstrated tremendous, yet selective in vitro cytocidal effects. The profound tumor growth suppression in a mouse model (particularly by multiple injections) shows the excellent potential of our viruses to be clinically translated as a new therapeutic entity for this emerging disease process.

436. Oncolytic Adenoviruses Targeted to Esophageal Adenocarcinoma

In the United States and other Western countries, the incidence of esophageal adenocarcinoma (EAC) has increased in recent years. Given the poor overall survival of these patients, the need for novel therapeutics has never been greater. Our research group has designed conditionally replicative oncolytic adenoviruses (CRAd) which use the cyclooxygenase-2 (Cox2) promoter to control the tissue specificity of viral replication. EAC, along with many other human gastrointestinal malignancies, has a high expression of Cox2. We hypothesize that our vectors will selectively replicate within EAC cells, thereby minimizing potential deleterious effects on normal tissues.A chimeric 5/3 fiber was employed to maximize infectivity. Viral replication was under control of the Cox2 promoter. In addition, the E3 region was modified to allow for expression of either a luciferase or interferon alpha (IFN) transgene. The vectors were thoroughly tested in vitro using both crystal violet and MTS assays to assess cell viability following viral infection in multiple EAC cell lines. Furthermore, the in vitro cytocidal effect was also characterized when the Cox2-IFN vector was combined with cisplatin and radiation. Additionally, a nude mouse model was used to test the in vivo antitumor effect. Finally, the vectors were tested ex vivo using tissue samples derived from resected human EAC specimens. A Krumdiek tissue slicer was used to prepare the samples, which were then infected with virus and subsequently analyzed.Systemic IFN has demonstrated some success in the treatment of EAC, but its use is limited by systemic side effects. Our vectors provide a way of delivering IFN locally to the tumor. Here, the Cox2-IFN virus demonstrated a strong cytocidal effect at early time points in all tested cell lines. In fact, it caused significantly more cell death than an otherwise identical vector expressing luciferase (p<0.05), thereby demonstrating the profound effect of IFN expression. Additionally, there was a dose-dependent increase in cytocidal effect when the Cox2-IFN virus was combined with cisplatin and radiation. In vivo, that same tumor-specific IFN-expressing vector demonstrated a profound anti-tumor effect when compared to the groups treated with a wild type virus and a saline control. Replication dependent reporter expression (luciferase) was measured to assess the viral replication in the patient-derived tissue slices. Both the Cox2-controlled vector and the vector lacking a promoter showed strong replication in the EAC tissue slices as expected. Importantly, in the normal esophagus samples, there was significantly less replication in the Cox2-controlled group (p<0.05). Viral copy number (using the E4 primer) was also used to quantify viral replication. Here, a similar trend was observed as there was virtually no replication of the Cox2 virus within the normal tissue samples.We have demonstrated that a CRAd driven by the Cox2 promoter has a strong oncolytic effect both in vivo and in vitro. Moreover, our Cox2-controlled vector has demonstrated cancer-specific replication. We believe that our novel vector possesses both therapeutic efficacy and specificity, which makes it an ideal candidate for clinical translation.

Tu2021 Interferon Expressing Oncolytic Adenovirus for Esophageal Cancer

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFNs properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinomas low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.