Abstract 6103: Genomic instability delays cell death in PDAC after simultaneous double strand breaks

Abstract Apoptosis, the main cell death mechanism triggered by double strand breaks (DSBs), occurs as quickly as 2-3 hours, or 6-24 hours in vivo. However, when we transduced Cas9-expressing pancreatic cancer (PC) cell lines with multi-target sgRNAs, in which each sgRNA contains 2-16 target sites in the human genome, we found that most of the reduction in sgRNA tag counts did not occur in the first 7 days post transduction, but rather occurred between days 7 and 21. We demonstrated that CRISPR-Cas9 scission occurs over the course of days in our PC cells and peaked at days 3-5, consistent with another recent observation. Thus, we hypothesized that the mechanism of cell death was likely not due to DNA damage response pathways that were immediately and directly triggered by the multiple scission events, but rather was caused by a slower process. To test this, we treated the Cas9-expressing TS0111 PC cell line with a 14-target sgRNA and performed cytogenetic analysis on cells harvested from 0-21 days after transduction at 3-4 day intervals using a chromosome breakage assay. We observed various karyotypic abnormalities, such as formations of ring, dicentric, and tricentric chromosomes. These abnormalities accumulated over time and peaked at day 14, except for the chromatid and chromosome breaks in which the frequency was maintained through day 21, suggesting ongoing occurrence of breakage events. Analysis of breakpoints on dicentric and tricentric chromosomes showed that although breakpoints at sgRNA targeted regions predominated at early time points and decreased over time, non-targeted regions increased and peaked at day 14. A break-apart FISH assay was also implemented to confirm that these structural variants (SVs) were a direct result of CRISPR-Cas9 cuts, in which the number of cells with abnormal FISH patterns increased over time and also peaked at day 14. Additionally, we performed bioinformatics analyses on the whole genome sequencing data of surviving colonies post treatment of multi-target sgRNAs to identify novel SVs. We found that novel SVs increased as a function of the number of sgRNA target sites, and majority of the SVs were found at non-targeted sites, consistent with ongoing genomic instability. Interestingly, we found that cells responded to the 14-cutter by becoming polyploid, manifesting as extremely large nuclei or multinucleated giant cells. XY FISH showed that polyploidy peaked at day 10 and decreased by day 21. Finally, we assayed for apoptosis, which increased on days 7 and 14 but decreased by day 21. We concluded that cytotoxicity occurred following the induction of multiple DSBs that resulted in ongoing chromosomal rearrangements and polyploidization, ultimately leading to cell death via apoptosis and possibly other mechanisms. Citation Format: Selina Shiqing K. Teh, Eitan Halper-Stromberg, Laura Morsberger, Kirsten Bowland, Alexis Bennett, Robert B. Scharpf, Ying S. Zou, James R. Eshleman. Genomic instability delays cell death in PDAC after simultaneous double strand breaks. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6103.