Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and infiltrative paediatric brainstem tumour with universal lethality. Previous DIPG studies have revealed the presence of a recurrent heterozygous mutation (p.K27M) in the Histone H3 variant genes: H3.1 (HIST1H3B, HIST1H3C) or H3.3 (H3F3A) in >80% of cases. While H3K27M function is still under heavy investigation, accumulating molecular evidence suggests its tumorigenic role is driven through epigenetic dysregulation underpinned by loss of H3K27me3. Co-occurring mutations in known oncogenes and amplification in cancer-prone proliferative pathways further contribute to the DIPG’s diverse molecular landscape and may also represent therapeutic vulnerabilities.
This study aims to utilise a functional genomics pipeline to investigate the intrinsic molecular mechanisms underlying H3K27M DIPG. Identification of genetic dependencies and tumourigenic pathways contributing to disease progression serve as prospective therapeutic targets for clinical utility.
Sixteen established and validated patient-derived DIPG cell lines (10 H3.3K27M, 4 H3.1K27M and 2 Wt), were obtained from external collaborators and used in this study. High throughput drug screening with a panel of previously approved and investigatory molecular compounds (2048) was used to determine DIPG therapeutic sensitivity. Cell viability data measured 72 hours post-treatment were z-score transformed and “hits” identified based on a z score threshold (≤-1.5) and minimal effect in a neural stem cell (NSC) control line (>-1.5). Highly prevalent targets include HDAC, proteasome, topoisomerase and microtubule associated inhibitors. In parallel, a 300 gene pooled CRISPR/Cas9 loss of function screen was used to determine key DIPG survival dependencies (3 H3.3K27M, 3 H3.1K27M). Next Generation Sequencing (NGS) measured gRNA abundance, and significant gRNA depletion was set to a z-score threshold of ≤-1.5. Comparison between CRISPR and drug screens revealed 24 molecular targets with therapeutic sensitivity and DIPG cell survival dependency and secondary validation studies of these molecular targets are currently in progress. Collectively, we show a functional genomics approach can identify therapeutic dependencies in DIPG, potentially paving the way for molecularly informed personalised therapies for patients.