DIPG-32. COMBINATION OF ChIP-SEQ AND RNA-SEQ ANALYSIS FOR TARGET DISCOVERY REVEAL PROMISING CANDIDATES FOR VALIDATION

摘要

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) has one of the lowest overall survival amongst pediatric brain cancers. DIPG can be stratified into molecular subtypes dictated by histone mutational status: H3.3K27M, H3.1K27M, and H3 wildtype. Mutant histone proteins drive cellular proliferation by reprogramming epigenetic landscape. To interrogate the DIPG epigenome, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in 5 DIPG tumors (H3.3K27M and H3 wildtype) and 3 healthy pons. This is the first report of ChIP-seq in DIPG tissue with H3 wildtype tumor. METHODS: ChIP-Seq was performed on DIPG tumors and control healthy pons for histone modifications indicative of active promoter (H3K4me3), repressive promoter (H3K27me3), and enhancer chromatin (H3K4me1 and H3K27ac). We focused on targets active in both H3K27M and H3 wildtype. Already existing RNA-seq data from DIPG upfront biopsy specimens (n=25) were used to narrow targets with increased transcripts. Immunohistochemistry (IHC) was performed to show protein expression. Human DIPG primary cell lines were assessed using Western blot analysis. RESULTS: Multiple targets were identified from ChIP-seq with activated histone including Notch1, CSPG4, and B7-H3. Focusing on B7-H3, RNA-seq confirmed high transcript levels at 43 Transcripts Per Million (TPM) in DIPG tumor and was significantly elevated compared to adjacent healthy brain at 5.5 TPM (p < 6x10-6). IHC analysis of 3 DIPGs showed strong B7-H3 staining compared to adjacent normal brain. To show the fidelity of preclinical models to human donor, primary DIPG cell lines (n=6) were interrogated by western blot assay. A 4.9 fold increase in B7-H3 (p=0.011) in DIPG cell lines was found when compared to protein lysates from healthy brain tissue (n=3). CONCLUSION: Using primary DIPG tumor tissue for ChIP-Seq analysis is novel and our study shows that epigenetic analysis by ChIP-seq coupled with transcriptional analysis by RNA-seq can be utilized to find new targets for DIPG therapy.