Abstract AP16: MODELING ENDOMETRIOID AND HIGH GRADE SEROUS CARCINOMAS IN THE MOUSE USING CRISPR/CAS9-MEDIATED SOMATIC GENE EDITING IN FALLOPIAN TUBE EPITHELIUM

Abstract

Abstract Genetically engineered mouse models (GEMMs) have contributed significantly to our understanding of the role of specific gene defects in human cancers. GEMMs that recapitulate many of the molecular and biological characteristics of various histologic subtypes of ovarian carcinomas have already been developed. However, conventional methods of GEMM development based on Cre-lox technology require the time-consuming and labor-intensive processes of transgenic line production and cross-breeding, a problem that is exacerbated when developing cancer GEMMs based on multiple genetic defects. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system potentially offers a more rapid and versatile alternative platform for tumor modeling in the mouse, as it can be used to target multiple genomic loci simultaneously by specifying a 20-nt targeting sequence for each gene of interest. The development of gynecological cancer GEMMs using CRISPR/Cas9-mediated conditional gene editing has not yet been reported. We have tested this approach to generate endometrioid carcinoma (EC) and high-grade serous carcinoma (HGSC) in the mouse oviductal epithelium. We first selected potentially optimal 20-nt sequences (guide RNAs) targeting the murine orthologs of the Apc and Pten tumor suppressor genes for the EC model, and the Brca1, Trp53, Rb1, and Nf1 genes for the HGSC model. Efficient cutting mediated by each guide RNA was validated in vitro by the Surveyor nuclease assay in NIH3T3 mouse fibroblasts. Next, we generated two transgenic mouse lines: one carrying a transgene with guide RNAs targeting Apc and Pten in tandem as a single guide RNA (sgAP) and a second carrying a transgene with a single guide RNA targeting Brca1, Trp53, Rb1, and Nf1 (sgBPRN). These transgenic lines were then crossed with Ovgp1-iCre-ERT2 and Rosa26LSL-Cas9-EGFP (Jackson laboratory) mice to allow for conditional (Tamoxifen-regulated) inactivation of Apc-Pten or Brca1-Trp53-Rb1-Nf1 specifically in the FTE. Endometrioid carcinomas were identified in Ovgp1-iCreERT2;Rosa26LSL-Cas9-EGFP;sgAP mice by 20 weeks post tamoxifen and early HGSC was present in an Ovgp1-iCreERT2; Rosa26LSL-Cas9-EGFP;sgBPRN mouse 34 weeks post tamoxifen treatment. The tumors derived from Cre-CRISPR/Cas9-sgRNA technology showed similar morphology and immunophenotypic characteristics to tumors arising in the models using Cre-lox technology based on the same genetic defects. Insertions/deletions (Indels) in tumor DNA were found in all of the targeted genes near the expected cut-sites, and confirmed by Sanger sequencing of cloned PCR products. All of the Indels resulted in predicted premature protein truncation and loss of function. Our results show that CRISPR/Cas9-sgRNA system genome editing can be used successfully to model gynecological cancers in mice. Citation Format: Rong Wu, Carmine Stolfi, Yali Zhai, Eric R. Fearon, and Kathleen R. Cho. MODELING ENDOMETRIOID AND HIGH GRADE SEROUS CARCINOMAS IN THE MOUSE USING CRISPR/CAS9-MEDIATED SOMATIC GENE EDITING IN FALLOPIAN TUBE EPITHELIUM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP16.