ID #602 Investigating the neurodevelopmental origins of diffuse hemispheric glioma in hPSC-derived 3D brain organoids

Abstract

Paediatric high-grade gliomas (pHGGs) are uniformly fatal childhood central nervous system tumours. More than 30% of pHGGs are classified as diffuse hemispheric glioma (DHG) H3.3 G34R/V mutant and arise in the cerebral hemispheres of adolescents and young adults. The median survival rate ranges from 15-22 months post treatment. DHG is molecularly defined by a heterozygous driver mutation in the H3F3A gene which encodes the histone variant H3.3, resulting in the substitution of a glycine for an arginine or valine at residue 34. H3.3 G34R/V driver mutations can co-occur with other mutations including TP53, ATRX and amplification of PDGFRA and MYCN. DHGs have recently been defined by transcriptional profiles and tumour cells recapitulate GABAergic interneuron precursor signatures. During human forebrain development, newborn GABAergic interneurons migrate tangentially from the ventral forebrain to populate the dorsal neocortex, thereby contributing to the balance of inhibitory and excitatory neurons.

We have employed a model to recapitulate the DHG neurodevelopmental milieu with human pluripotent stem cell derived ventral forebrain organoids (vFOs). Through CRISPR/Cas9 gene editing techniques, we have generated a vFO model that expresses the H3.3G34R epigenetic driver mutation of DHG. We aim to investigate the impact of the H3.3G34R mutation on brain development by analysing transcription profiles and chromatin accessibility to uncover the mechanisms involved in DHG formation. In parallel, we have employed NKX2.1-GFP reporter cell lines to track differentiation of vFOs and show expression beginning at Day 14. We have also established a whole mount imaging protocol to validate expression of stem cell neural progenitor markers including NESTIN, FOXG1, GABA and DLX2. Further, we have characterised whole transcriptome gene expression across three timepoints in vFOs with RNA sequencing. By employing a human specific model of ventral forebrain development, this research aims to uncover the mechanisms involved in the formation of DHG.