Abstract 7243: CRISPR-cas9 genetic targeting of metastatic pancreatic cancer
Authors: Bowland Kirsten , Lai Jiaying , Teh Selina Shiqing K. , Bennett Alexis , Skaist Alyza , Zhang Yan , Thompson Elizabeth , Hruban Ralph H. , Roberts Nicholas J. , Karchin Rachel , Iacobuzio-Donahue Christine A. , Eshleman James R. 
Published: March 2024

Abstract
Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here we test the hypothesis that metastatic cancer can be genetically targeted by exploiting somatic single base substitution mutations that occur as part of normal aging prior to transformation. These passenger mutations are targetable because ~10% of them form novel tumor-specific “NGG” protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9. We studied five patients with pancreatic cancer (pc) who underwent a rapid autopsy to determine areas of evolutionary conservation that were maintained throughout all metastases in each patient’s cancer. Using whole genome sequencing validated by high-depth capture sequencing, we found that 90% of somatic PAM targets were maintained between primary carcinomas and metastases. We identified regions of loss of heterozygosity (LOH) surrounding tumor suppressor genes and oncogenes in the primary tumor where PAMs were 100% conserved in metastases. These regions of truncal LOH represent genetic vulnerabilities in these carcinomas and a CRISPR-based gene therapy approach targeting PAMs in these regions may be a novel way to genetically target metastatic cancer. Using multitarget sgRNA which cut at a known number of sites in the human genome, we determined a relationship between the number of CRISPR-Cas9 cuts and growth inhibition in PC cells. We found that 8-10 target sites in non-coding regions were sufficient to induce &gt;95% cancer cell death. Furthermore, only 2-3 targets were required to achieve a comparable level of cell death in the presence of a DNA double strand break inhibitor. Finally, using cocultures of PC cells transduced with sgRNA targeting multiple tumor specific PAMs, we demonstrate selective cell killing of three different PC cell lines. Importantly, deep sequencing of patient-matched normal lymphocytes lacking the tumor-specific PAMs treated with the same sgRNA array exhibited no editing at the targeted loci. This work establishes a bioinformatic platform for the discovery of tumor-specific CRISPR targets in the context of metastatic disease and demonstrates selective killing of cancer cells while sparing the patient’s normal cells. These data establish the proof of principle for a gene therapy approach targeting metastatic disease.
Citation Format: Kirsten Bowland, Jiaying Lai, Selina Shiqing K. Teh, Alexis Bennett, Alyza Skaist, Yan Zhang, Elizabeth Thompson, Ralph H. Hruban, Nicholas J. Roberts, Rachel Karchin, Christine A. Iacobuzio-Donahue, James R. Eshleman. CRISPR-cas9 genetic targeting of metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7243.