Abstract B06: Knock-down of H3.3 K27M mutant protein in DIPG cells delays, but does not abrogate, tumor growth in vivo

Abstract Genome-wide sequencing efforts of pediatric high grade gliomas (HGG) identified recurrent and hotspot mutations in the N-terminal regulatory tail of histone H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B/C). K27M histone mutations are mainly observed in midline tumors, including 78% of diffuse intrinsic pontine gliomas (DIPGs). By contrast, G34R/V mutations are predominantly restricted to 15% of the hemispheric tumours. This illustrates a highly specific selective pressure for each mutation in different cell types during brain development. The K27M mutation induces a dominant negative loss of trimethylated K27 (H3K27me3) marks in the bulk H3 pool. H3K27me3 interacts with Polycomb repressive complex 2 (PRC2) to repress transcription of target genes, a key process in the regulation and development of stem cells. Global gene expression analysis revealed that many significant differences between DIPGs (mostly K27M mutants) and non-brainstem pediatric HGG (mostly wild-type) are in genes that encode transcription factors and genes associated with developmental processes. Importantly, there was particular enrichment of genes exclusively expressed in distinct developing brain locations. Hence, it is challenging to define a clear gene expression signature associated with K27M mutations in DIPGs. In this study, in order to identify factors directly regulated by K27M, we aimed to knock-down the mutated protein in a DIPG-derived xenograft line. This H3.3 K27M line is maintained by successive implants of luciferase+/YFP+ tumor cells in the cortex of CD-1 nude immunocompromised mice. Resected tumor cells were transduced with lentiviral vectors expressing H3F3A specific shRNAs, selected with puromycin in neural stem cell culture conditions, and either kept under culture or re-implanted into mice. Cells in culture expressing any of three distinct shRNAs showed 92-96% H3F3A mRNA knock-down, 82-90% K27M protein knock-down and a 4-7 fold increase in H3K27me3 levels. Knock-down of H3F3A with any of three shRNAs in vivo significantly extended the time for tumor growth and the survival of xenografted mice. Notably, tumor growth was not completely abrogated. We observed an average of 79% H3F3A mRNA knock-down and increased levels of H3K27me3 by immunofluorescence in experimental tumors. Global gene expression profiling revealed modulation of a common subset of genes by all three shRNAs in vivo. We will present analysis of the gene expression changes associated with loss of the K27M mutation in DIPG cells. Overall, we show efficient knock-down of H3F3A in a H3.3 K27M mutant DIPG-derived xenograft line, leading to increased accumulation of H3K27me3, modulation of gene expression, and increased survival in vivo. Citation Format: André B. Silveira, Xiaoyan Zhu, Chunxu Qu, Suzanne Baker. Knock-down of H3.3 K27M mutant protein in DIPG cells delays, but does not abrogate, tumor growth in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B06.