EPCO-34. IMAGE-BASED PHENOTYPIC HIGH-CONTENT CRISPR-CAS9 SCREEN FOR CHARACTERIZING CANCER DRIVERS IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract

Abstract The genomic landscape of pediatric high-grade glioma is characterized by a high level of aneuploidy but few focal amplifications or homozygous deletions. Cancer aneuploidies are the principal source of tumor heterogeneity driving tumor growth and therapy resistance. Here, we deployed an image-based phenotypic high-content CRISPR-Cas9 screen to identify aneuploidy-associated pediatric glioma driver genes. Through integrated computational analyses combining whole-genome sequencing datasets from 106 pediatric gliomas and 81 canine gliomas, we identified a set of 460 genes present in regions of aneuploidy in both diseases. We designed a custom guideRNA library to systemically knockout the 460 gene set using an arrayed CRISPR-Cas9 knockout screen in two pediatric high-grade glioma cell lines. By deploying 72 of 384-well plates on automated confocal Opera Phenix High-Content Screening System and dyes staining cell membrane, nucleus, and apoptotic cells, we established 5 million live-cell images across three timepoints (day 7, 8, 9 post-transduction). Each gene was randomized to 32 randomized wells across four 384-well plates to gather robust morphological profiles. Through image-based nucleus and cell segmentation workflow, we characterized 50 million single cells across 27,648 wells over three time points. Using semi-supervised machine learning methods, we derived 1,050 cell morphological features to characterize knockout phenotypes, e.g., viability, growth, and morphology (size, shape, texture of nucleus and cells) across cells. The top ranked genes were then linked to oncogenes and tumor suppressors based on pathway and ontology analysis as well as functional in vitro and in silico validation using Pediatric Cancer Dependency Map. We find convergence of the most impactful molecular abnormalities - based on their knockout phenotypes - on candidate signaling pathways for the development of new drugs and repurposing of existing drugs for pediatric high-grade glioma.