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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.

PIK3CA is the most commonly mutated gene in breast cancer, and in breast cancer there are two hotspot mutations that represent an outsized proportion of PIK3CA mutations. Previous research suggests that there are distinct prognostic and cell signaling differences induced by these mutational isoforms, however there is currently only one targeted treatment approved for PIK3CA mutant breast cancer. The goal of our research is to leverage a comprehensive multi-lineage isogenic breast cell line model to further interrogate the unique effects of these PIK3CA mutations on breast cells using assays to assess gene expression and chromatin state. Insights gained from these assays shed light on disrupted pathways that may prove to be new targets for PIK3CA mutation-specific treatment of breast cancer. Citation Format: Adam X. Miranda, Emily Hodges, Ben Ho Park. Functional genomic dissection of PIK3CA hotspot mutations using a multi-lineage isogenic breast cell model [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 3726.

Background: Three-dimensional organization of chromatin within the nucleus is an important and dynamic regulator of cellular transcription. Changes in chromatin topology are implicated in processes like cell differentiation and cell fate decisions, but have been largely unstudied with regards to mutations and cancer cell transformation. Our goal is to assess the changes in chromatin topology that occur in response to a single oncogenic mutation and how these changes may influence cancer cell transformation. Methods: Using AAV-mediated homologous recombination, a E545K “hotspot” PIK3CA mutation was incorporated into MCF10A breast epithelial cells and conversely, corrected back to wild-type in MCF7 breast cancer cells that natively harbor a E545K PIK3CA mutation. To interrogate the chromatin topology of these cell lines and the unmodified versions we will use a technique called tethered chromosome conformation capture (TCC). TCC maps chromatin topology by combining the proximity ligation of interacting DNA segments with next generation sequencing to create a matrix of interacting DNA elements. We can analyze and overlay these matrices to look for significant differences in topologically associating domains (TADs). Results: Preliminary results comparing MCF7 parental cells and MCF7 cells with the PIK3CA exon 9 mutation corrected suggest rearrangement of TADs at many key cancer-associated genes. One of these rearrangements is the formation of a de novo TAD on chromosome 10 near PTEN, a tumor suppressor gene that acts antagonistically to PIK3CA. Rearrangement of chromatin topology at this locus could lead to downregulation of a negative actor on this pathway further stimulating downstream elements of the PIK3CA pathway. Conclusions: Oncogenic mutations can cause changes that can have a drastic impact on transcription. Rearrangement of chromatin topology is a key mechanism by which the transcriptional program of a cell can be modified. Our experiments reveal key sites of chromatin rearrangement caused by a single oncogenic mutation that may promote a cancer phenotype in our cells. We plan to investigate the degree to which the expression of PTEN is altered by chromosomal rearrangements as well as perform CRISPR-KO of the interacting loci to determine the transcriptional effect of removing the loop. These assays will also be conducted for other sites of chromatin rearrangement. Results from these experiments will reveal the influence oncogenic mutations may have on chromatin topology and expression. Citation Format: Adam X. Miranda, Ben H. Park, Emily Hodges. Assessing the impact of PIK3CA mutation on the three-dimensional organization of chromatin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-171.