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

组蛋白H3.3中甘氨酸34向色氨酸的替换（Glycine 34 to tryptophan (G34W)）在约90%的骨巨细胞瘤（giant cell tumour of bone, GCT）中发生。本研究证实，H3.3G34W是肿瘤形成的必要条件。通过对患者样本以及经CRISPR/Cas9编辑引入H3.3G34W的肿瘤来源细胞的表观组、转录组与分泌蛋白质组进行表征分析，研究发现：突变型H3.3上的H3.3K36me3修饰缺失，会导致抑制性染色质修饰H3K27me3从基因间区向基因区发生偏移，且偏移范围超出H3.3的沉积区域。该现象会促进拮抗型染色质修饰的重分布，并异常下调收缩性肌成纤维细胞相关基因的表达，进而改变间充质祖细胞的细胞命运。单细胞转录组学分析显示，H3.3G34W阳性基质细胞重现了一条肿瘤发生轨迹：从分泌型磷蛋白1（secreted phosphoprotein 1, SPP1）阳性的成骨细胞祖细胞样群体，向平滑肌肌动蛋白α2（actin alpha 2, ACTA2）阳性的肌成纤维细胞群体分化；该肌成纤维细胞群体可分泌细胞外基质配体，据预测这类配体能够招募并激活破骨细胞。本研究结果表明，H3.3G34W通过维持成骨细胞样祖细胞的转化状态，促进肿瘤性生长、病理性招募巨型破骨细胞并引发骨破坏，最终导致骨巨细胞瘤的发生。