Anti-invasive therapies may limit damage to healthy brain tissue caused by invading high-grade glioma (HGG) cells. However, the actin machinery that drives invasion is shared by cancer and normal cells, and therefore, there is a risk of on-target, off-tumor side effects. We compared omega 3 polyunsaturated fatty acid epoxide analog p-tolyl-ureidopalmitic acid (PTU) effects between HGG multicellular tumor spheroids (MCTSs) embedded in Matrigel and assembloids consisting of HGG MCTS with embryonic stem cell-derived cortical brain organoids. PTU inhibited cofilin and induced cell rounding consistent with a loss of filamentous (F-) actin and efficiently inhibited invasion in 3D Matrigel invasion assays, but effects were cell line specific in assembloids. Immunofluorescence and GeoMx Digital Spatial Profiling of assembloids revealed that PTU disrupts signaling in cancer cells and the "healthy" cortical organoid. This study demonstrates a methodological approach for in vitro testing of efficacy and safety for anti-invasive therapies for HGG.

BACKGROUND: Imipridone ONC201 is the first FDA-approved therapy for H3K27-altered diffuse midline glioma; however, clinical responses remain limited. Defining tumor-intrinsic determinants and microenvironmental, extrinsic factors that shape sensitivity or resistance to imipridones will identify actionable therapeutic opportunities and inform improved clinical strategies. METHODS: To identify mechanisms of imipridone resistance, we obtained postmortem brain tissue from DMG patients who had received imipridones and/or standard care. Single-nucleus RNA and open-chromatin sequencing were performed on N = 22 cases. Immunofluorescence-based myeloid phenotyping was performed on N = 46 cases. Mitochondrial copy-number analysis was performed on N = 19 cases. Validation of imipridone sensitivity, its effect on mitochondrial density, and its synergy with inhibition of mitochondrial biogenesis were assessed in DMG primary cells. RESULTS: We established a single-cell RNA/open-chromatin atlas from postmortem DMG cases and found imipridone treatment resulting in regressed mesenchymal transition, reduced myeloid-derived suppressive cells, and reversed aberrant H3K27-altered enhancer activity. Resistant tumors showed increased mitochondrial density, turnover, and membrane potential. Mitochondrial biogenesis and PPARGC1A emerged as resistance biomarkers and actionable targets. CONCLUSIONS: These studies implicate mitochondrial biogenesis as a biomarker of imipridone resistance and a focus for the development of combinatorial strategies to provide effective therapeutic options for a challenging pediatric brain tumor. Diffuse midline gliomas are lethal brain tumors, mostly affecting children. The drug Dordaviprone was recently approved by the Food and Drug Administration as the first systemic therapy for this disease. However, it is not a cure. In this study, we asked why some patients stop responding to Dordaviprone and how we might overcome this resistance. By analyzing tumor samples from patients, we found that resistant tumors increase their number of mitochondria, structures that produce energy, helping them to survive treatment. Blocking this energy-producing process enhanced Dordaviprone’s effectiveness. Moreover, Dordaviprone enhanced anti-tumor immune responses and may thus synergize with emerging immunotherapies.

Heterozygous variants in the KAT6A gene encoding the histone lysine acetyltransferase KAT6A (MOZ, MYST3) cause Arboleda-Tham syndrome, a cognitive impairment syndrome. Histone acetylation is generally associated with active gene transcription. Genetic deletion of both alleles of the Kat6a gene in mice causes developmental defects including anterior homeotic transformation, cleft palate, interrupted aortic arch and cardiac septal defects. Loss of KAT6A impairs expression of HOX, DLX and TBX genes, which are essential for body segment identity specification, palate, heart and aortic arch development. However, the effects of loss of KAT6A on chromatin modifications and gene expression in neural cells, which are relevant to normal brain development and function, is still poorly understood. In this study, we used an automated high-throughput chromatin profiling method and RNA sequencing in mouse neural system and progenitor cells to assess the effects of loss of one or two alleles of Kat6a on gene expression, histone acetylation and methylation. We also assessed occupancy by a trithorax group protein and RNA polymerase II. Our data suggests two modes of action for KAT6A: (1) acetylation of histone H3 on lysine 23 at promoters and enhancers and (2) recruitment of the trithorax group protein MLL1 (KMT2A) to promote the expression of developmental genes, including SOX and homeodomain genes. Together, these two functions appear to be required for normal gene expression in neural progenitors and essential for proliferation and neuronal differentiation. Author summaryDuring embryonic development, specific families of transcription factors pattern the early embryo to lay down and define the body and organ structure. However, the mechanisms governing the onset of the expression of these developmental transcription factors in less well understood. KAT6A is thought to promote gene expression by acetylation histone proteins. Here we determine the effects of KAT6A on histone acetylation and gene expression in mouse neural stem and progenitor cells. Our data are relevant for the understanding of pathogenic genetic variants in one allele of the human KAT6A gene, which cause the Arboleda-Tham cognitive impairment syndrome.