Abstract 5094: Precision therapeutic targeting of oncogenic FGFR3 fusions using CRISPR-Cas9 genome-editing in urothelial cancer

Introduction: Fibroblast-growth factor receptor 3 (FGFR3) fusions occur in up to 11 % of urothelial carcinomas. These fusions drive ligand-independent receptor dimerization and downstream oncogenic signaling and thus represent attractive targets for gene therapy. We hypothesized that CRISPR-Cas9 gene-editing can be used to disrupt these somatic oncogenic fusions in cancer cells. Methods: RT112 and SW780 urothelial cancer cell lines harboring genomic fusions of FGFR3 to TACC3, or BAIAP2L1, respectively were grown in cultures. The fusion sequences were confirmed by Sanger sequencing and the NGG protospacer adjacent motif necessary for guide RNA (gRNA) targeting were identified. Unique guide RNAs spanning the fusion sequences in each cell line were designed. An all-in-one lentiviral vector was designed to deliver both the gRNA and the Cas9 nuclease. A scrambled non-targeting gRNA vector was used as a control. Following transduction, genomic DNA was extracted and PCR was performed using primer sequences flanking the fusion sequences followed by targeted sequencing on the Illumina MiSeq platform. CrispRVariants and CRISPResso bioinformatic tools were used to determine editing efficiency. Results: In SW780 cells transduced with the FGFR3-BAIAP2L1 fusion-targeting gRNA vector, 64229/207425 (23.6%) reads harbored CRISPR-induced edits of the fusion sequence compared to only 2009/313553(0.6%) reads in the scrambled gRNA vector-transduced cells. Similarly, in RT112 cells transduced with the FGFR3-TACC3 fusion-targeting gRNA vector, 74292/448381 reads (14.2%) contained random indels consistent with CRISPR-induced edits compared to 17785/428035 reads (4.0%) in RT112s cells transduced with the scrambled gRNA vector. In the SW780 cell line, the most common indel in CRISPR-edited fusions sequences was a deletion of a cytosine occurring in 40% of edited reads. The second most common indel was an extra cytosine insertion and the third most common was a 13-base deletion spanning the fusion point. In RT112 cell line, the most common indel was an insertion of a cytosine in 13% of reads. Sanger sequencing of the fusion sequences from SW780 single cell clones confirmed Miseq results. Experiments determining the effects of these indels on the function of the fusion protein and on the cancer phenotype are ongoing. Conclusions: To our knowledge, this is the first report of CRISPR/Cas9 use as a therapeutic strategy for targeting oncogenic fusions. Because the fusion sequences are unique to cancer cells, this approach can be potentially used for in-vivo gene therapy for urothelial carcinomas harboring FGFR3 fusions with no off-target edits in normal cells. This approach represents a significant advance towards precision gene-therapy by specifically targeting fusions sequences unique to each patient’s cancer. We envision extending this approach to several solid and hematological malignancies driven by oncogenic fusions. Citation Format: Bishoy M. Faltas, Rebecca Meyer, Ethan Shelkey, Charles J. Murphy, Olivier Elemento, Mark A. Rubin. Precision therapeutic targeting of oncogenic FGFR3 fusions using CRISPR-Cas9 genome-editing in urothelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5094. doi:10.1158/1538-7445.AM2017-5094