Molecular Determinants of Diffuse Midline Glioma of the Pons Vulnerability to the Histone Deacetylase Inhibitor, Quisinostat

Abstract

Diffuse Midline Glioma of the Pons (DMG/Pons) represents a highly aggressive pediatric high-grade glioma, standing as a predominant cause of brain tumor-related fatalities in children. Despite persistent efforts over the last four decades, no clinically approved therapy has emerged, leaving fractionated focal radiation as the sole standard of care. Genomic and molecular scrutiny has unraveled pivotal mutations in histone-encoding genes, genetic drivers, and alterations in DNA methylation patterns within DMGs, offering critical insights into tumorigenic mechanisms. The concurrent development of in vitro and in vivo DMG models has opened avenues for investigating novel therapeutic interventions. Notably, treatment-naive patient-derived xenograft (PDX) models, characterized by their untreated status, provide a unique opportunity to explore the natural state of DMGs before any intervention. The hallmark epigenetic anomaly in DMGs involves a histone H3 mutation, specifically a substitution of lysine with methionine at residue 27 (H3K27M). This mutation instigates profound reprogramming and alterations in the tumor microenvironment. Histone deacetylase inhibitors (HDACi), exemplified by Quisinostat, have exhibited clinical efficacy across various cancer types, inducing apoptosis, growth arrest, and regression of oncogenic phenotypes. Our study sets out to probe the effects of Quisinostat on DMGs, with a keen focus on its influence on the genome, transcriptome, and patterns of cell-free DNA fragmentation. In our investigation, two DMG models, PBT-22 and PBT-29, both carrying H3K27M and TP53 mutations, displayed a remarkable 30-fold difference in response to Quisinostat treatment. Western blot analysis showcased heightened protein expression of H3K27ac and H3K27me3, while H3K27M expression remained largely unchanged in both cell lines. RNA sequencing further delineated distinct gene expression profiles for each cell line, featuring commonalities and differences. Upregulated genes orchestrated cell signaling cascades, cell growth, collagen production, stemness, chromatin remodeling, and transcription. Conversely, downregulated genes were associated with chromatin remodeling, lysine and DNA methyltransferases, cell cycle, apoptosis, ATP-binding cassette, and immune response. Validation experiments with additional cell lines affirmed these findings. Crucially, examination of cell-free DNA fragmentation patterns pre- and post-Quisinostat treatment disclosed discernible differences in fragment lengths. Treated groups exhibited shorter fragments than controls, with the most resistant cell line, PBT-29, displaying the shortest fragment lengths. In summary, this comprehensive study delves into the multifaceted impact of Quisinostat on DMGs, unraveling alterations at the genomic, transcriptomic, and cell-free DNA fragmentation levels. These revelations furnish crucial insights into potential therapeutic avenues for addressing the complexities of this formidable and aggressive brain tumor.