Background
Oncolytic virotherapy is a special case of cancer immunotherapy that mounts a significant inflammatory immune response along with oncolysis. We recently demonstrated that Delta-24-RGD, an oncolytic adenovirus, can be safely combined with radiotherapy to treat pediatric Diffuse Intrinsic Pontine Glioma in a phase I clinical trial (NCT03178032). In this trial, combining Delta-24-RGD with radiotherapy increased the median survival of the patients to 17.8 months. Histone hyperacetylation is an epigenetic signature of Diffuse Midline Glioma (DMG). Since the early adenoviral protein, E1A binds to the histone acetyltransferase protein, P300 to redirect acetylation, we hypothesized that combing Delta-24-RGD with a P300 acetyltransferase inhibitor would induce a stark shift in the acetylome of DMG, enhancing the oncolytic and immune effect of the virus.
Methods
Experiments were performed on human and murine DMG cell lines with H3.1 and H3.3 K27M mutations that are predominantly observed in the clinic. Western blot analyses were performed to investigate changes in H3K27ac levels following virus infection. HPLC and mass spectrometry were performed to evaluate the post-translational modifications induced by virus infection. Additionally, Cell Titer Blue cell viability assay along with SynergyFinder analyses were performed to assess the optimal concentration of C646, a P300 HAT inhibitor, and Delta-24-RGD that induces potent oncolysis. Orthotopic supratentorial and brainstem injections were performed with murine DMG cells in immunocompetent backgrounds to study the combinatorial effect on survival and the change in the post-translational landscape of the tumor in vivo.
Results
We observed that Delta-24-RGD induced a time-dependent reduction in H3K27ac with over 89% reduction in H3K27ac at 48h post-infection in TP54 human DMG cell line. We also observed that virus-induced H3K27 hypoacetylation was E1A-dependent and was not observed when DMG cells were infected with E1A-deficient adenovirus. Additionally, infecting DMG cells with adenovirus expressing E1A with P300-deficient binding does not reduce H3K27ac indicating E1A-P300 binding is essential for virus-induced H3K27 hypoacetylation. Interestingly, HPLC analyses also showed potentially altered levels of H3 in virus-infected DMG cells compared to the non-infected cohort. Mass spectrometry analyses revealed reduction in acetylation of H3 subunits in virus-infected cells, reiterating our western blot observations. Combining Delta-24-RGD with C646 was associated with a synergistic effect on cell viability in vitro and reduced tumor burden in mice observed via luminescence imaging.
Conclusions
Overall, our results show the potent effect of combing Delta-24-RGD with P300 inhibitors, providing a strong rationale to test this combination in a clinical trial for pediatric DMG.
Acknowledgements
MD Anderson Cancer Center Brain Tumor Center Animal Core.