DMG-08. Single-Cell Analysis of ONC201 and Paxalisib Resistance Mechanisms in Diffuse Intrinsic Pontine Glioma

Abstract Background Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), constitutes a uniformly fatal group of pediatric brain tumors harboring a median overall survival of 9–11 months post-diagnosis. Palliative radiotherapy remains the sole standard treatment for this disease. Brain-penetrant drugs, such as ONC201 and PI3K/mTOR inhibitor paxalisib, have shown promise in improving the clinical outcomes for patients with progressive DMG NCT05009992. Despite these advances, the highly adaptive nature of DMG portends robust capability for therapeutic resistance. Thus, there is an urgent need to understand adaptation mechanisms to inform the next effective therapies. Methods We conducted scRNA-seq and scATAC-seq on immunocompetent Pdgframut, p53mut, H3.3K27M (PPK) DMG mouse model treated with ONC201 (125 mg/kg) and paxalisib (5 mg/kg), alone or in combination. Additionally, brain tissues from RA-055 (H3.3K27M, TP53mut, PDGFRAamp, MYCNamp) tumor bearing xenografts treated with optimized paxalisib (5 mg/kg/b.i.d. with 150 mg/kg/day metformin) were collected at four weeks and at endpoint and subjected to Xenium spatial transcriptomics. Results We identified two distinct trajectories of tumor adaptation in oligodendrocyte progenitor cell (OPC)-like populations specific to ONC201 and paxalisib treatments, with increased activation of the mitogen-activated protein kinase (MAPK) signaling pathway alongside these trajectories. Statistical analyses confirmed significant over-activation of the MAPK in ONC201-treated vs untreated mice (GSEA: NES=1.50, padj=0.014; PROGENy: padj0.001). We show that ONC201 and paxalisib treatments enhance immune cell infiltration and antigen presentation with increased expression of MHC class II genes. However, this was accompanied by immunosuppressive features such as Cd274/PD-L1 and Cd74 expression, likely promoting immune escape. Conclusions Our study demonstrates the cellular and molecular adaptations of DMG cells to ONC201 and paxalisib within both tumor and immune compartments. Importantly, our data highlights the potential of immunotherapies and MAPK inhibitors as promising combination to improve clinical outcomes of DMG patients.