MODL-12. A NOVEL GENETICALLY ENGINEERED H3.3G34R MODEL REVEALS COOPERATION WITH ATRX LOSS IN UPREGULATION OF HOXA CLUSTER GENES AND PROMOTION OF NEURONAL LINEAGE

Abstract

Abstract BACKGROUND Pediatric high-grade gliomas (pHGGs) are an aggressive CNS tumor which are often characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). A substitution of the Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described in a large cohort of pHGG samples and has been characterized as occurring in anywhere between 5-20% of pHGGs. Attempts to study the mechanisms of H3.3G34R have proven difficult due to the developmental nature of the disease and the requirement of co-occurring mutations for model development. METHODS We utilized the RCAS/tv-a system to develop a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the context of ATRX loss and ATRX presence in nestin expressing progenitor cells. RESULTS Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates the Hoxa cluster genes Hoxa2, Hoxa3, Hoxa5, and Hoxa7 (p < 0.05, unpaired t-test). H&E staining and EMA immune-staining indicate that ATRX loss is correlated with a higher incidence of ependymal differentiation in H3.3G34R expressing samples (p < 0.05, unpaired t-test). GSEA and RT-qPCR analysis demonstrate that H3.3G34R expression in the context of ATRX loss promotes increased expression in genes associated with neuronal lineage (FWER pval < 1.0) and exhibits upregulation of the neurofilament polypeptides Nefl, Nefm (p < 0.05, unpaired t-test) as well as the neuronal differentiation marker Stmn2 (p < 0.001, unpaired t-test). CONCLUSION Our study proposes a model in which cooperation between ATRX loss and H3.3G34R expression mediate major transcriptomic and histopathological changes in H3.3G34R pHGGs. Broadly, our work highlights the importance to study mechanisms of co-occurring genetic events separately and in combination.