Abstract B33: CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method

Abstract

Abstract Chromosomal rearrangements of ALK, RET, ROS1, and NTRK1 collectively represent approximately 10% of oncogenic drivers in lung adenocarcinoma. These rearrangements result in the aberrant expression and constitutive activation of a chimeric fusion kinase, which promotes growth, proliferation and survival of the cancer cell. CRISPR/Cas9 technology can now be employed to generate these chromosomal rearrangements both in vitro and in vivo. sgRNAs can be designed to target the Cas9 endonuclease to introns within the 5′ and 3′ gene partners of the rearrangement. A unique advantage of this approach is that it is compatible with immunocompetent mouse models. We have developed a novel in vitro screening strategy to identify sgRNAs that successfully generated the intended rearrangements. Ba/F3 cells are a murine B-cell cell line whose proliferation is normally dependent on the exogenous addition of the cytokine interleukin 3 (IL3), but can be rendered IL3 independent if an oncogenic alteration is introduced. We hypothesized that IL3-independence in Ba/F3 cells could be used as a method to select for cells that had successfully created chromosomal rearrangements leading to oncogenic fusions. This strategy eliminates the need for single cell cloning to confirm that successful CRISPR genome editing has occurred. We transfected Ba/F3 cells with pairs of sgRNAs within the PX330 Cas9 containing plasmid and then withdrew IL3. Cells that became IL3 independent were tested for rearrangements. Using this screening system, we successfully generated Kif5b-Ret, Trim24-Ret, and Tpm3-Ntrk1 rearrangements in Ba/F3 cells. These rearrangements were confirmed using PCR or RT-PCR and sequencing using fusion-specific primers. Additionally, the Ret rearrangements and Ntrk1 rearrangements were far more sensitive to RET and TRK inhibitors, respectively, than parental Ba/F3 cells, further confirming that the Ba/F3 cells' growth was now dependent on signaling from the fusion kinase. RET protein expression was also detected with Western blotting in the two Ret rearranged Ba/F3 cell lines. Our future plans are to introduce adenovirus containing validated sgRNAs into immune-competent mice, and confirm that they generate tumors with the intended rearrangements. Citation Format: Laura Schubert, Anh T. Le, Stephen P. Malkoski, Raphael Nemenoff, Robert C. Doebele. CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B33.