Abstract 13: Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro

Abstract

Abstract Despite the well-documented benefits of circulating tumor DNA (ctDNA) based liquid biopsy, including screening, prognostication, and treatment monitoring, there remain several concerns with its application. Chief among these concerns are imperfect sensitivity and specificity, particularly pre-diagnosis and after tumor resection, when ctDNA burden is low. Cell death, either through apoptosis or necrosis, has been the most widely accepted mechanism of DNA release. Recent studies have also suggested active release through extracellular vesicles as a paradigm for DNA release. However, this pathway remains highly debated in terms of its contribution, mechanism, and even credibility. To address the deficiencies in ctDNA detection in the clinic and the lack of basic mechanistic knowledge of cf/ctDNA release, we performed an in vitro CRISPR screen on MCF10A cells to identify regulators of DNA release. This normal breast epithelial cell line was chosen because MCF10A cells release DNA into cell culture media with a similar fragmentation pattern to that observed in human blood. After lentiviral infection with the genome-wide Brunello CRISPR-Cas9 knockout library, the population of knockouts identified in the genomic DNA of cells attached to the plates was compared to the population of knockouts found in the DNA released into the cell-depleted culture media. Genes with skewed representation were considered potential regulators of DNA release. Our analysis identified several genes in the TRAIL apoptotic pathway including FADD, as well as several genes encoding RNA-binding proteins including Sam68. Knockout of Sam68 and FADD in MCF10As, MDA-MB-468 breast cancer cells, and NCI-H841 lung cancer cells lead to decreases in DNA release. Previous studies have hypothesized that the fragment size of released DNA is dependent on release mechanism. However, the decreases observed in our knockout cell lines were not limited to a specific fragment size of DNA. Application of the small molecule apoptosis inhibitor Z-VAD-FMK also recapitulated the DNA release phenotype seen in our knockout cell lines. From these results, we conclude that apoptosis is the major source of DNA release from cancer cells, and that fragment size is not the indicator of release mechanism it has been presumed to be. Mechanistic studies of DNA release such as this could lead to the ability to modify the amount of ctDNA in circulation, which would significantly improve accurate detection in the clinic. Citation Format: Brad A. Davidson, Adam X. Miranda, Sarah Croessmann, Ben Ho Park. Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 13.