Abstract Diffuse midline gliomas (DMGs) are malignant tumors characterized by pronounced tumor heterogeneity, rendering standard-of-care (SOC) radiotherapy ineffective for their treatment. In recent years, treatment with the imipridone ONC201 has shown prolonged survival (2 years) in some patients, while the rest remain resistant. To elucidate the mechanism of resistance towards ONC201 and identify adjuvant therapies, we profiled postmortem specimens from DMGs treated with ONC201 and SOC via single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase-chromatin by sequencing (scATAC-seq). We leveraged a cohort of n = 22 post-mortem specimens from pontine and thalamic tumors, including cases treated with SOC (N = 11), ONC201 as an adjuvant (N = 10), untreated cases (N = 1), and subjected them to snRNA-seq and snATAC-seq (sub-cohort). We investigated cellular composition, gene expression, and epigenetic regulation. Metabolism, stemness, and other signatures previously implicated in ONC201 responses were evaluated and correlated with intra-tumor paracrine signaling networks. SnRNA-seq and snATAC-seq results were integrated to identify cis-regulatory enhancers and the transcription factors that bind them. We also investigated the effects of ONC201 treatment on mitochondrial biogenesis of DMG cells. Our findings show that ONC201-treated tumors demonstrated reduced mesenchymal phenotypes and fewer myeloid-derived suppressive cells. Enhancer activity was globally diminished and the treatment increased PGC1a expression, promoting mitochondrial biogenesis, while reducing immunosuppressive signaling. Tumor cells upregulated TRAIL signaling and mitochondrial biogenesis genes, while heat-shock proteins and negative regulators of antigen presentation were broadly downregulated in both tumor and immune cells. ONC201-treated myeloid infiltrates exhibited downregulation of IL10, TGF-β, and other immune-suppressive genes, indicating a reduction in immunosuppressive paracrine signaling. Overall, the results show that ONC201 increases antigen presentation and may synergize with immunotherapy. However, mitochondrial biogenesis may act as a resistance mechanism to ONC201 treatment. As the mechanism by which ONC201 regulates myeloid cell activation remains unclear, future studies with preclinical models or longitudinal biopsies are warranted.
Okada et al. (Fri,) studied this question.