HGG-36. Elucidating the role of long non-coding RNAs in pediatric high grade gliomas

Abstract

Abstract BACKGROUND: Genomic and transcriptomic studies have elucidated new insights into the landscape of diffuse intrinsic pontine glioma (DIPG). However, the role of long non-coding RNAs (lncRNAs) has not been explored at depth in these tumors, and there have not been studies focused on how lncRNAs interact with the K27M histone mutation. In a recent analysis of nearly 200 DIPGs and pediatric high-grade gliomas (pHGG), we previously detected a novel, recurring structural variant in the lncRNA CCDC26. This rearrangement occurs in nearly 10% of all DIPGs, and we have furthermore identified alterations in more than 100 lncRNAs in DIPG. METHODS: To identify lncRNAs required for proliferation of patient-derived DIPG cancer cells, we designed two custom genome-scale lncRNA libraries. We generated a genome-scale lncRNA CRISPR-Cas9 knockout pooled library, consisting of 45,766 single guide RNAs (sgRNAs). Additionally, we generated a genome-wide CRISPR interference pooled library consisting of 45,608 sgRNAs, targeting lncRNA transcription start sites (TSS). RESULTS: We utilized in vitro histone-mutant pHGG models as well as edited clones of these models with the K27M mutant corrected in order to compare lncRNA dependencies in these two contexts. We have successfully performed genome-scale CRISPR-Cas9 knockout and CRISPR interference screens targeting lncRNAs in these cell lines, revealing lncRNA dependencies. Candidate dependencies in our CRISPR-Cas9 knockout screen include LOC100507412, LOC105379524, and LINC02193. CONCLUSION: Genome-wide lncRNA CRISPR knock-out and CRISPR interference screens are a novel approach for the unbiased identification of lncRNAs that are required for pediatric high-grade glioma proliferation. Further validation of specific lncRNAs is required, and these lncRNA dependencies represent potential novel therapeutic targets.