Immuno-modulating Diffuse Midline Glioma by Epi-transcriptomic Reprogramming - Project Summary Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children with a 5-year survival rate of <1%. DMG characteristically has an immunologically cold tumor microenvironment (TME) leading to poor clinical responses and resistance to immunotherapy. To overcome scarce lymphocyte infiltration and limited immune checkpoint (IC) expression in cold TMEs, our team and others have established novel viral mimicry activating therapies that leverage the accumulation of immunogenic dsRNAs and anti-viral innate immune response to change the TME and sensitize tumors to immunotherapies. Traditionally, drugs that modulate the epigenome have been used to activate the viral mimicry response in tumors. Unfortunately, these drugs have lacked efficacy in DMG clinical trials amid dosing limitations secondary to systemic toxicities and poor neuropenetrance. Thus, novel targets to activate viral mimicry and potentiate the immune response against DMG are critically needed. We propose a paradigm shift to focus on targeting epi-transcriptomic (RNA modifications) regulators, which promise to be more effective with less off-target effects in DMG. Via an unbiased screen, we have found that the RNA editor ADAR is significantly upregulated in DMG. Interestingly, ADAR-mediated RNA editing has been shown to help some cancer cells escape antitumor immune responses. In patient-derived DMG cell lines, our preliminary results show that ADAR knockdown (KD) increases immunogenic RNA species, activates an IFN response, reduces cell proliferation, and increases MHC-I and IC ligand levels on the tumor cell surface. The objective of this proposal will be to determine the mechanism for the observed ADAR-dependency in DMG cell lines, and to assess if this promising epitranscriptomic target activates viral mimicry to synergize with immunotherapies against DMG in vivo. To determine modulators of ADAR-dependency, I will perform a genome-wide CRISPR screen in DMG cells with a doxycycline-inducibleADAR KD background. This will inform us what genes cause sensitization or resistance to ADAR-modulation in DMG. Next, I will assess if ADAR knockdown synergistically enhances immune checkpoint inhibition (ICI, aPD-1) in a novel syngeneic orthotopic immunocompetent murine DMG model. Utilizing a previously validated panel to characterize the DMG TME by flow cytometry, we will assess the viral mimicry response by intracellular markers such as dsRNA levels and cell surface markers such as CD45 and CD3 to determine immune cell infiltration, namely tumor-infiltrating lymphocytes. This project seeks to understand the role of upregulated epitranscriptomic regulators in DMG from an immunologic perspective and assess their efficacy as targets for viral mimicry therapy. The results of these studies will open the doors to novel multi-modal regimens involving epitranscriptomic reprogramming and inform potential clinical trials for immune modulation in DMG.
