Gene-21. The Ketogenic Diet Alters the Epigenetic Landscape of GBM to Potentiate the Effects of Chemo and Radiotherapy

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brainstem tumor with no efficacious treatment. The most prevalent mutation occurs on the amino terminal tail at Lysine 27 (K27M) resulting in global hypomethylation of H3K27. The H3.1-K27M mutation is highly co-expressed with an activating mutation (G328V) in Activin-A receptor Type 1 (ACVR1), a bone morphogenetic protein (BMP) receptor. We used the Sleeping Beauty Transposase (SB) system to deliver plasmids encoding NRASV12 and a short hair pin for TP53 (shp53) in combination with ACVR1-G328V or H3.1-K27M, or both, into the lateral ventricle of neonatal mice, to generate endogenous tumors with DIPG mutations. Tumor development was monitored by measuring luciferase activity in vivo. Moribund stage animals were perfused and processed for histology. Additionally, tumor neurospheres (NS) were generated from moribund stage tumors. H3.1 K27M (MS=99 dpi) did not have a statistically significant effect on survival compared to the control group NRAS/ shp53 (MS=80), however, tumors induced with NRAS/shp53/ACVR1 G328V (MS=119 dpi) had enhanced survival compared to control group NRAS/ shp53 (p=0.0029). Tumors harboring NRAS/shp53/ACVR1 G328V/H3.1 K27M (MS=154) exhibit a synergistic increase in survival compared to the NRAS/shp53/H3.1 group (p<0.0001) and the NRAS/shp53/ACVR1G328V group (p=0.0137). In vitro and in vivo, tumors or tumor NS harboring ACVR1-G328V exhibit elevated phopho-Smad1/5, transducer of the BMP pathway, while those harboring H3.1-K27M exhibit decreased H3K27me3 and increased H3K27 acetylation levels compared to controls. In conclusion, the SB transposase system can be used to develop a mouse model that accurately represents the molecular biology of DIPGs with ACVR1-G328V and H3.1-K27M mutations. ACVR1-G328V and H3.1-K27M may induce a protective effect that prolongs survival through upregulation of the BMP-Smad1/5 pathway and epigenetic deregulation. In the future we aim to elucidate the mechanisms by which ACVR1 and H3.1-K27M mutations contribute to tumor initiation and progression to develop targeted therapies for DIPG.