Abstract 6371: KLIPP: Precision targeting of fusions and amplified oncogenes using CRISPR

Abstract Oncogenic fusion genes and amplified oncogenes are common drivers of cancer but there are currently no therapeutic approaches available that directly target these cancer-specific genomic features. Amplified oncogenes in cancer can be found on chromosomes as homologous staining regions (HSRs) or on extrachromosomal DNA (ecDNA). Presence of oncogenes on ecDNA poses a particular challenge because they are under strong positive selection in response to anti-cancer therapies leading to treatment resistance as their copy number can rapidly change due to uncontrolled segregation during cell division. To specifically target cells with fusions or amplified oncogenes, we have developed a CRISPR-based therapeutic approach, “KLIPP, ” which is designed to target structural variants junctions (SVJs) specific to cancer genomes, with few, or no off-target effects expected in normal cell. KLIPP uses a “split enzyme” approach consisting of a dead Cas9 endonuclease (dCas9) fused to the endonuclease Fok1 where two Fok1 endonucleases need to homodimerize to become active. To “nucleate” and activate these complexes, sgRNAs are designed to bind sequences flanking cancer-specific SVJs, bringing two Fok1-dCas9 complexes together to induce double-strand breaks (DSBs). Using the KLIPP approach, we have obtained strong proof-of-concept that it can specifically target cancer-unique SVJs and induce DSBs leading to cell death in cancer cells both in culture and in vivo. We have shown this for SVJs throughout cancer genomes as well as in oncogenic fusion genes such as EWS-FLI1 in Ewing sarcoma. Furthermore, we show that we can use KLIPP to target unique SVJs on amplified oncogenes resulting in strong induction of γH2AX and loss of cell survival. We are currently investigating the efficacy of KLIPP in targeting ecDNA in cancer cells. We believe that KLIPP could be a safe and cancer-specific precision therapy for targeting cancer cells driven by fusions and amplified oncogenes. Citation Format: Huibin Yang, Radhika Suhas Hulbatte, Natalie Gratsch, Ashley Sutter, Anne Urzynicok, Mayer Cusnir, Mario Ashaka, Aadi Shiv Malhotra, Ishwarya Venkata Narayanan, Michelle Paulsen, Anna Schwendeman, Tom Wilson, Mats Ljungman. KLIPP: Precision targeting of fusions and amplified oncogenes using CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6371.