LAUSR

Human colorectal cancer (CRC) is characterized by a high burden of somatic mutations, particularly in tumors harboring defects in the DNA mismatch repair (MMR) pathway. However, genetically-engineered mouse models (GEMMs) of cancer to date harbor a very low burden of mutations. Importantly, recent clinical trials have shown that MMR deficient tumors respond favorably to anti-PD-1 immune checkpoint blockade, and it is thought that PD-1 suppression reinvigorates existing T cell clones against neo-antigens associated with hypermutation in these tumors. In order to develop a more clinically relevant GEMM of CRC in which to study the immune response, MMR genes will be knocked out via CRISPR-Cas9 to induce hypermutation. To identify optimal Cas9 single guide RNAs (sgRNAs) against the MMR genes Msh2, Msh3, Msh6, and Mlh1, sgRNAs were designed using the Broad Institute algorithm and knockout efficiency assessed in a reporter cell line by Western blot. In order to induce colon tumors in vivo, colonoscopy-guided submucosal injection was employed to deliver lentivirus encoding sgRNAs against a MMR gene and Apc into mice carrying a constitutive Cas9 allele. Apc is inactivated in over 80% of human CRC, and is an initiating event in CRC in humans and mice. Using a sgMsh2-sgApc dual targeting lentivirus, we were able to efficiently generate colon tumors harboring complete knockout of Apc and near complete knockout of Msh2. Deep sequencing of the targeted loci revealed predominantly loss-of-function frameshift mutations at Apc and Msh2, but also a high degree of polyclonality. Lower titer injections of virus will be explored to induce more clonal tumors. Mlh1, Msh3, and Msh6 will also be targeted in vivo, and the mutational effects of MMR gene loss will be assessed by whole-exome sequencing of tumors. This rapid and flexible GEMM of hypermutant CRC may facilitate a more clinically relevant assessment of the role of the immune system in CRC development, as well as provide improved platforms for testing immunotherapies. Citation Format: Abbey Y. Jin, Peter Westcott, Olivia Smith, Amanda Cruz, Mary Clare Beytagh, Tyler Jacks. Knockout of genes in the DNA mismatch repair pathway to model colorectal cancer hypermutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4091.