H3.3G34W promotes growth and impedes differentiation of osteoblast-like mesenchymal progenitors in Giant Cell Tumour of Bone [ChIP-Seq]

Glycine 34 to tryptophan (G34W) substitutions in H3.3 arise in ~90% of giant cell tumour of bone (GCT). Here, we show H3.3G34W is necessary for tumour formation. Profiling the epigenome, transcriptome and secreted proteome of patient samples and tumour-derived cells CRISPR/Cas9-edited for H3.3G34W shows that H3.3K36me3 loss on mutant H3.3 induces a shift of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes the redistribution of antagonistic chromatin marks and aberrant downregulation of contractile myofibroblast-associated genes altering cell fate in mesenchymal progenitors. Single-cell transcriptomics reveals that H3.3G34W stromal cells recapitulate a neoplastic trajectory from an SPP1+ osteoblast progenitor-like population towards an ACTA2+ myofibroblast population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors which promotes neoplastic growth, pathological recruitment of giant osteoclasts, and bone destruction.