Effective treatments are urgently needed for the paediatric brain tumour Diffuse Midline Glioma (DMG). This aggressive cancer most commonly occurs in children aged 5-9, with a median survival time of 8-11 months following diagnosis and a 5-year survival of <1%. DMGs have driver Histone H3 mutations (H3K27M), which produce extensive epigenetic dysregulation by inhibiting PRC2-mediated trimethylation of H3K27 (H3K27me3), resulting in a permissive chromatin state and aberrant transcription, central to DMG tumorigenesis. Thus, targeting the epigenome is a promising avenue of treatment. We found that targeting the histone chaperone complex Facilitates Chromatin Transcription (FACT) with the curaxin drug CBL0137 has potent pre-clinical efficacy against DMG, leading to a paediatric Phase I/II clinical trial for CBL0137 for DMG (NCT04870944). This project aims to elucidate the molecular mechanisms driving the activity of CBL0137 in DMG, to find effective combination therapies.
We mapped the genome-wide binding of the FACT subunit SPT16 using Cleavage Under Targets & Release Using Nuclease (CUT&RUN), and regions of open chromatin using Assay for Transposase-Accessible Chromatin (ATAC-seq) in patient-derived DMG cells. We found FACT to be enriched at accessible genes involved in development and transcription (e.g. SOX2, SMARCA2, MYC and POU2F1). Given the role of the BET protein BRD4 as a chromatin reader in driving oncogenic transcription in DMG and other cancers, we next examined the interaction between FACT and BRD4. Using co-immunoprecipitation and CUT&RUN, we found FACT to directly interact with BRD4 on chromatin, suggesting co-operation between these epigenetic regulators in DMG. Consistently, we found that dual targeting of FACT with CBL0137 and BRD4 with the BET inhibitor JQ1 to be cytotoxic against DMG in vitro and in vivo, with a significant increase in survival in orthotopic pre-clinical patient-derived xenograft (PDX) mouse models of DMG. To further investigate the mechanism of these epigenetic drugs, we performed ATAC-seq and RNA-seq in DMG cells treated with CBL0137 and JQ1 alone and in combination. We found that combination treatment reduced chromatin accessibility and transcription of genes involved in RNA-related processes such as RNA methylation, splicing, and transcription. This work has identified a promising novel epigenetic combination therapy – CBL0137 + BET inhibition – while uncovering intriguing new insights into DMG epigenetics and pathobiology.