Malignant brain tumors are among the most lethal of human tumors, with limited treatment options currently available. A complex array of recurrent genetic and epigenetic changes has been observed in gliomas that collectively result in derangements of common cell signaling pathways controlling cell survival, proliferation, and invasion. One important determinant of gene expression is DNA methylation status, and emerging studies have revealed the importance of a recently identified demethylation pathway involving 5-hydroxymethylcytosine (5hmC). Diminished levels of the modified base 5hmC is a uniform finding in glioma cell lines and patient samples, suggesting a common defect in epigenetic reprogramming. Within the tumor microenvironment, infiltrating immune cells increase oxidative DNA damage, likely promoting both genetic and epigenetic changes that occur during glioma evolution. In this environment, glioma cells are selected that utilize multiple metabolic changes, including changes in the metabolism of the amino acids glutamate, tryptophan, and arginine. Whereas altered metabolism can promote the destruction of normal tissues, glioma cells exploit these changes to promote tumor cell survival and to suppress adaptive immune responses. Further understanding of these metabolic changes could reveal new strategies that would selectively disadvantage tumor cells and redirect host antitumor responses toward eradication of these lethal tumors.

Replication-competent oncolytic adenoviruses hold considerable promise for treating malignant gliomas. The toxicity of the clinically tested E1B-55 kDa mutant virus is negligible; however, its full clinical potential is still being evaluated. The purpose of the present study is to compare the antiglioma activity in vitro and in vivo between Delta-24, an E1A mutant adenovirus, and RA55, an E1B-55 kDa mutant adenovirus. We selected human glioma cell lines that were tumorigenic in nude mice and express wild-type p53 (U-87 MG, D54 MG) or mutant p53 (U-251 MG, U-373 MG) protein. Our studies demonstrated that Delta-24 induced a more potent antiglioma effect in vitro than RA55. Moreover, Delta-24 replicated markedly more efficiently than RA55 in both wild-type and mutant p53 scenarios. Importantly, direct intratumoral injection of Delta-24, but not RA55, significantly suppresses tumor growth in intracranial (U-87 MG, U-251 MG) or subcutaneous (D54 MG) animal models. Staining for hexon protein detected replicating adenoviruses in xenografts infected with Delta-24, but not with RA55. Collectively, these data indicate that E1A mutant adenoviruses targeting the Rb pathway are more powerful putative agents for antiglioma therapy than E1B mutant adenoviruses, and suggest that E1A mutant adenoviruses should be tested in the clinical setting for patients with malignant gliomas.

Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of E1A protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CR1 (Delta-39), CR2 (Delta-24) regions, or both regions (Delta-24/39) of the E1A protein. Delta-39 and Delta-24 induced a cytopathic effect with similar efficiency in glioma cells and a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, E1A protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CR1 mutant E1A proteins, and inhibition of the proteasome activity resulted in the striking rescue of E1A levels. We conclude that the level of E1A protein is a critical determinant of oncolytic phenotype and we propose a completely novel strategy for the design and construction of conditionally replicative adenoviruses.

Background: Our drug discovery program has led to the development of a series of diverse, unique small molecule drugs targeting the key oncogenic transcription factor STAT3 which drives diverse human cancers. WP1066 was selected as a lead drug because of its drug-like properties, therapeutic window and validated in vivo activity in a broad range of tumor models. During the process of WP1066 development as a clinical phase new agent, we developed and further characterized an orally bioavailable formulation of WP1066 using a spray dried oral formulation. Methods: The spray dried formulation of WP1066 (SDD1) was prepared using Hypermellose acetate succinate (HPMCAS) as an enteric coating film. HPMCAS is a mixture of acetic and monosuccinic acid esters of hydroxypropylmethyl cellulose. While insoluble in gastric fluids, the SDD1 formulation swells and leads to rapid dissolution in the higher pH found in upper small intestine, and subsequent release of the drug. For theses studies SDD1 was characterized for size, stability and purity. Single and multiple-dose toxicity studies were done in female CD-1 mice, using 5% dextrose as a vehicle. Single dose PK and organ distribution analysis was performed after oral gavage administration of 200 mg/kg after 12 hour fasting. Animals were sacrifice at different time-points (2 min to 24 hours). Plasma, brain and pancreas were isolated and the apparent concentration of WP1066 was determined using HPLC/MS/MS analysis. For multi-dose PK analysis, animals were fasted for six hours, dosed with SDD1 at 200 mg/kg. Animals were euthanized at different time intervals after the second dose (given six hours after first dose). Results: Mice dosed with SDD1 did not show any apparent toxicity when administered up to 200 mg/kg. For multiple doses, mice received 50, 100 or 200 mg/kg of WP1066 in SDD1 every day for 28 days. No apparent toxicity or significant weight lost was observed. In a similar way, animals dosed twice a day with 200 mg/kg of WP1066 (SDD1) for 15 doses did not show any symptoms of toxicity. Pilot PK analysis confirmed dose-dependent absorption of WP1066. Single-dose PK analysis revealed rapid GI absorption with a TMAX = 45 min and CMAX in plasma 1.5 μg/ml (which corresponds to ∼4 μM). The estimated half-life was approximately 3 hrs. Similar parameters were obtained from PK analysis performed in canines. SDD1 dosing led to significant accumulation of WP1066 in intact brain and pancreatic tissues in mice. These results support further preclinical development of SDD1 formulated WP1066 aimed the initiation of clinical studies in pancreatic, brain cancer and melanoma metastasis to brain patients. Acknowledgment: These studies were supported in part by the grants: Brain SPORE (2 P50 CA127001), Melanoma SPORE (2 P50 CA093459); Viragh Foundation, Houston Pharmaceuticals. Citation Format: Rafal Zielinski, Aleksandra Rusin, Timothy Madden, Charles Conrad, Mary Johansen, Izabela Fokt, Stanislaw Skora, Arumugam Jayakumar, Amy Heimberger, Waldemar Priebe. Development of orally bioavailable formulation of WP1066 and its evaluation in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2015-4540

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ and CD8+ T cells, has adjuvant properties for establishing effective T-cell immunity against cancer. The hypothesis of this study is that oncolytic viruses expressing OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement. We will also test whether Delta-24-RGD induces DAMPs, and systemic immunity against the tumor. To test the hypothesis we modified Delta-24-RGD to express the OX40 ligand during replication, the new virus was called Delta-24-RGDOX. Flow-cytometry analyses of human glioma U-87MG and mouse glioma GL261 cells infected with Delta-24-RGDOX detected the expression of OX40L by 48h after infection. Importantly, infection of glioma cells with Delta-24-RGDOX resulted in the production of DAMPs (The damage-associated molecular pattern high-mobility group box 1, calreticulin, HSP70 and HSP90 proteins) as assessed by western blot and ELISA analyses, indicating immunogenic cell death. In addition, using a syngeneic animal model, we showed that intratumoral injection of Delta-24-RGDOX caused infiltration of innate and adaptive immune cells, instigating a Th1 immunity at the tumor site which resulted in specific anti-glioma immunity, shrunken tumor and prolonged animal survival. Importantly, Delta-24-RGDOX treatment showed superior efficacy that the treatment with Delta-24-RGD (28.5 vs. 17 days; P, 0.0001). Moreover, we showed that splenocytes from mice treated with both therapies were stimulated against infected and non-infected GL261 cells in a significantly greater degree than splenocytes of mice treated with the combination of Delta-24-RGD and OX40 antibodies (P,0.001). Consistent with the antitumor immune hypothesis, treatment with Delta-24-RGDOX did not prolong the life of immunosuppressed mice bearing intracranial GL261 tumors (P.0.01). These results support the concept that the immunemediated anti-gliomas effects of Delta-24-RGD can be greatly enhanced by Delta-24-RGD-OX40L and warrant the clinical testing of the new immunetargeted adenovirus in patients with malignant gliomas.