Abstract 2466: Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines

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Abstract The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using CRISPR/Cas9 can be associated with low yields of single cell clones harboring the desired homozygous or heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process and thus, better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers, especially for Diffuse Intrinsic Pontine Glioma (DIPG). To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including truncating mutations within exon 6 of the gene, protein phosphatase 1D (PPM1D). Using our HRM analysis protocol, clones containing activating mutations in PPM1D were detectable and grouped distinctly from wild-type and non-functional clones; as validated through conventional Sanger sequencing and Ion Torrent Next Generation Sequencing. Importantly, multiple PPM1D mutant clones were generated in an expedited manner (< 4 weeks), using our unique HRM process-flow. We subsequently validated these clones in a collection of secondary assays, and we confirmed that the mutant proteins were functionally active. Several unique findings were identified in these studies, including substantial cell cycle-associated defects and basal activation of key proteins in the DNA damage response network. To our knowledge, we have created the first set of astrocyte cell lines harboring engineered PPM1D mutations at the endogenous gene locus. We are now testing these cell lines in high-throughput synthetic lethal screens to identify novel inhibitors of PPM1D-mutant DIPG tumors. These findings highlight the utility and power of our unique approach to rapidly create pertinent mutant cell lines for use in mutation-targeted biological assays. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future. Citation Format: Nathan R. Fons, Yulia Surovtseva, Gregory A. Breuer, Ranjini K. Sundaram, Ranjit S. Bindra. Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2466.