Abstract A34: Gene editing with CRISPR-Cas9: Investigating novel therapeutic targets for the treatment of pediatric high-grade glioma

Abstract

Abstract Introduction: Pediatric high-grade glioma (pHGG) are a group of aggressive and difficult-to-treat childhood brain tumors (1), including grade III and IV anaplastic astrocytoma, and diffuse intrinsic pontine glioma (DIPG); the latter occurs almost exclusively in children. Aggressive, multidisciplinary treatment approaches incorporate surgery (where possible) followed by adjuvant chemo- and radiotherapy. Despite this treatment regimen, clinical prognosis remains poor, characterized by drug resistance and significant toxicity (2). There is an urgent need to identify novel therapeutic approaches that are more effective with reduced side effects. pHGG are increasingly characterized into distinct molecular subgroups, based on gene expression and epigenetic regulation (3). Critical to identifying novel therapeutic agents (and associated targets) for pHGG patients is understanding the complex biology of tumor cells further and elucidating the mechanisms pHGG utilize to proliferate and survive (4). The identification of these critical “survival” genes facilitates the design of pHGG target-specific compounds, potentially improving clinical prognosis. Methods and Results: Using a CRISPR-Cas9 Loss of Function (LOF) genome-wide gene engineering technology, we have silenced 19,050 genes across the human genome in a pHGG in vitro model. Silencing genes that are essential for survival results in cell death and the loss of this gene from the population. This LOF is identified by next-generation sequencing (NGS) of the CRISPR-Cas9 constructs in the Reference (immediately after initial CRISPR-Cas9 edit) and Experiment (following 6 cell doublings) population. We have analyzed these data using a custom-designed bioinformatics pipeline to identify genes absent from the experiment population, and further, to identify signaling pathways based on gene cluster analysis, that are essential for pHGG cell survival. With these results, we will select both novel and repurposed chemotherapeutics that interact with (by activating or repressing target genes, and their associated proteins) for further characterization, analysis, and study in patient-derived cell lines and primary biopsy tissue. Future Work: This work will identify novel pHGG survival genes and substantially contribute to our understanding of pHGG tumor biology, facilitating the identification of novel therapeutics or repurposed agents for progression into the clinic. We hypothesize that targeting multiple genes within key survival pathways with a combined treatment regimen will lead to more complete pathway abrogation, increasing treatment efficacy, circumventing resistance, and reducing adverse toxicity. Key Message: pHGG are highly aggressive and resistant to current therapeutics. There is an urgent need to identify novel therapeutics for progression to the clinic. Using CRISPR-Cas9 technology we have silenced 19,050 pHGG genes to determine which genes are specific to pHGG survival. Identification of these genes will aid the design of novel therapeutics for pHGG. We hypothesize that using a combined drug treatment approach for complete pathway disruption will increase efficacy and minimize adverse side effects.