H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo

Abstract

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27Mdependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. https://www.springer.com/aamterms-v1 *

Invited Review: Emerging functions of histone H3 mutations in paediatric diffuse high‐grade gliomas

“…While the tumour composition varied between individuals, the OPC‐like subset was consistently the largest in DIPGs, and when compared to IDH mutant HGGs, DIPGs were found to contain more cycling and undifferentiated cells. H3K27M knockdown in xenografts from three different DIPGs consistently decreased expression of the AC‐like and increased expression of the OC‐like programs described by Filbin et al , and decreased expression of neural tube progenitor signatures from normal midline brain development . Interestingly, H3K27M mutations also occur at much lower frequency in acute myeloid leukaemia, and were shown to increase the frequency of functional haematopoietic stem cells and alter differentiation .…”

“…Two recent studies have used orthotopic xenografts into mouse to look at the effect of H3K27M loss on tumour growth against an isogenic DIPG background. One study used lentiviral delivery of H3F3A or nonsilencing shRNAs to three different H3.3K27M DIPG patient‐derived xenografts (PDX) and assessed the ability of the cells to form tumours in vivo . While loss of the H3K27M mutation increased survival for all three DIPG PDXs, the H3F3A knockdown tumours that grew out still had strong reduction in H3K27M and high H3K27me3, suggesting DIPG tumours can still grow with significantly depleted H3K27M.…”

“…In addition to effects on tumour growth and self‐renewal, H3K27M alters differentiation programs. Gene ontology analysis of genes differentially expressed in response to H3K27M frequently show enrichment for lists associated with neural lineage differentiation . In cultured cells, expression of H3K27M blocked astrocytic and impaired oligodendrocytic differentiation of hNPCs expressing constitutively active PDGFRA with p53 loss .…”

“…PRC2‐mediated repression has been shown to be critical to maintaining stemness and preventing differentiation in embryonic stem (ES) cells and H3K27M‐dependent loss of H3K27me3 may act on the cell of origin to repress differentiation and/or reset it to an earlier stem‐like state. Evidence from most experimental studies shows an H3K27M‐dependent increase in expression of stemness genes in neural precursor (NPC) or gliomas, while the bulk of DIPG tumour cells show expression signatures similar to oligodendrocyte precursor (OPC) cells .…”

XenoCP: Cloud-based BAM cleansing tool for RNA and DNA from Xenograft

“…Next, we used XenoCP to remove mouse reads from RNA-Seq. We knocked down the H3F3A (K27M) gene in a diffuse intrinsic pontine glioma (DIPG) 9 xenograft sample (referred to as sh K27M ) (Supplementary Methods). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) confirmed the robust knockdown of H3F3A relative to a control (K27M).…”