Abstract
Abstract INTRODUCTION Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children and is currently incurable. Clinical trials using immune checkpoint blockade (ICB) therapy have failed to show efficacy due to multifactorial mechanisms including limited lymphocyte infiltration, low immune checkpoint (IC) expression, and a low mutational burden. Adenosine deaminase acting on RNA (ADAR) is an RNA editor, which limits anti-tumor immunity by editing/preventing the sensing of endogenous double-stranded RNA (dsRNA). We have found that ADAR protein levels are significantly elevated in DMG patients, suggesting that ADAR’s immune-silencing function is relevant to DMG immune suppression. The onco-histone driver H3K27M promotes epigenetic dysregulation in DMGs, de-suppressing retroelement transcription, a major source of endogenous dsRNA. By inhibiting ADAR, we expect that the increased levels of dsRNA will stimulate intracellular type-1 interferon (IFN) responses, induce chemokine secretion, and promote antitumor CD8+ cytotoxicity. METHODS Human DMG cells and patient-derived glioma spheres were treated with silencing RNA to transiently knockdown (KD) ADAR levels for assays involving in vitro viability and proliferation. Affected pathways were identified using immunoblotting and quantitative PCR. RESULTS Our data shows that ADAR protein levels are elevated in DMG patient proteomic datasets and cell lines. ADAR-depleted patient-derived DMG cell lines exhibit significantly less proliferation compared to control siRNA and ADAR-depleted hTERT-astrocytes (Mann-Whitney test, P-value <0.0001, ***). CONCLUSION Our results demonstrate that DMG tumor cells have an ADAR selective dependency in vitro. In ongoing studies, we aim to determine if ADAR-depletion improves in-vivo survival in both immunodeficient and immunocompetent models in combination with immunotherapies against DMG.