Abstract 2037: Targeting chromosome rearrangements in cancer with CRISPR

Abstract Background: We have developed a novel precision and personalized cancer therapeutic approach to specifically target cancer cells using CRISPR technology. This approach is universal to all cancers and not dependent on the unique biology of the tumor. The innovation of this approach is that chromosome rearrangement junctions (CRJs), which typically exist in the hundreds in each tumor cell, are targeted by guide RNAs that bring together two parts of a dCas9-fused endonuclease, Fok1, leading to the induction of DNA double strand breaks (DSBs) at sites of the targeted CRJs in cancer cells specifically. Materials and Methods: HCT116 colon cancer cells and UMUC-3 bladder cancer cells were engineered to express Fok1-dCas9 and two pairs of CRJ-targeting gRNA under doxycycline regulation. Induction of DSBs at CRJs were assessed by γH2AX foci and γH2AX ChIP-PCR and cell death was assessed by apoptosis (PARP1 & caspase cleavage and flow cytometry for sub-G1 DNA content and Annexin V staining) and clonogenic survival. Results: Induction of Fok1-dCas9 lead to the induction of 1-2 DSBs per cell and promoted cell death when accompanied with the expression of pairs of CRJ-targeting gRNAs. However, no DSBs or cell death was observed with expression of Fok1-dCas9 alone or in combination with non-targeting gRNAs. Conclusions: We have shown that we can direct two parts of a dCas9-fused Fok1 endonuclease to sites of CRJs in cancer cells using CRISPR gRNAs leading to the induction of DSBs and loss of cell survival specifically in cancer cells. Recent breakthroughs in CRISPR delivery using lipid nanoparticles will allow us to next explore the utility of our precision CRISPR approach in animal models. If successfully implemented, the impact of our universal approach targeting CRJs in cancer with CRISPR could be transformative for cancer therapy. Citation Format: Huibin Yang, Radhika Suhas-Hulbatte, Mats Ljungman. Targeting chromosome rearrangements in cancer with CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2037.