Abstract LB357: CRISPR-Cas9 targeting of somatic mutations selectively kills cancer cells

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 3% of all cancers but is the 3rd leading cause of cancer death in the United States. This staggering discrepancy is partly because most pancreatic cancers evade early detection and patients most commonly present with metastatic disease. As cancer initiates from a single transformed cell, passenger mutations that accumulate over the course of the patient’s lifetime in that cell are then clonally propagated to the growing tumor and metastases, providing a targetable molecular signature throughout the cancer. Here we propose a CRISPR-Cas9 gene therapy aimed at these tumor-specific somatic mutations. Using whole genome sequencing of rapid autopsy tumor and normal samples from five patient cases, we identified single base substitutions present in tumor tissues and absent in patient-matched normal tissue. A subset (6.6% on average) of these mutations lead to novel protospacer adjacent motif (PAM) sites. These tumor specific PAM sites are necessary for Cas9 binding and DNA scission and are therefore selectively targetable by CRISPR-Cas9 systems. Using a hybrid capture/NGS approach, we demonstrate in patients with multiple metastases that the majority of these novel PAM sites, ~70%, are maintained among the different metastases. Using multitarget sgRNA which cut at a known number of sites in the human genome, we determined a relationship between number of CRISPR-Cas9 cuts and growth inhibition in PDAC cells. We found that 8-10 cut sites in non-coding regions are sufficient to induce >95% cancer cell death. Finally, using cocultures of fluorescently labeled PDAC cells and a multiplexed sgRNA array targeting multiple tumor specific PAMs, we demonstrate selective cell killing of the targeted cell line. 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 to 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, Selina Shiqing K. Teh, Alyza Skaist, Ralph H. Hruban, Nicholas J. Roberts, Chrisine Iacobuzio-Donahue, James R. Eshleman. CRISPR-Cas9 targeting of somatic mutations selectively kills cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB357.