Abstract 2924: In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes
Authors: Zhang Daniel D. , Westcott Peter M. , Smith Olivia , Sacks Nate , Hauck Haley , Beytagh Mary C. , Jin Abbey , Jacks Tyler 
Published: July 2021

Microsatellite instability is a hallmark of DNA mismatch repair (MMR)-deficient colorectal cancer (CRC), and is accompanied by genome-wide somatic hypermutation and enhanced immunogenicity. Clinical trials have demonstrated remarkable efficacy of immune checkpoint blockade (i.e. anti-PD-1) in treating MMR-deficient cancers, which is thought to be driven by an enhanced burden of mutation-derived tumor specific antigens (neoantigens). Despite these successes, tumor mutation burden is an imperfect clinical predictor of immunotherapy response, and major questions remain regarding the mechanistic role of neoantigens and other features of MMR-deficiency in the anti-tumor immune response. Notably, most genetically engineered mouse models (GEMMs) fail to recapitulate the mutation burden of their associated human cancer and lack prominent immune involvement, precluding careful study of these questions. To model microsatellite instability in colon cancer, we developed novel autochthonous GEMMs utilizing CRISPR/Cas9-based knockout of DNA MMR genes in the distal colon. Colonoscopy-guided submucosal injection was used to deliver lentivirus containing two single-guide RNAs (sgRNAs) against Apc (the initiating event in the majority of colon cancers) and MMR pathway genes (Msh2, Mlh1, Msh3, Msh6). Whole-exome sequencing of MMR-targeted colon tumors revealed a high mutation burden with signatures consistent with those found in MMR-deficient human cancer. In addition, sequencing revealed a number of recurrently mutated microsatellites, including those within putative tumor suppressor genes Asxl1, Acvr2a, and Acvr1b. To interrogate the functional relevance of these genes in MMR-deficient colon cancer, we developed a novel ex vivo organoid competition assay. Specifically, Apc null organoids expressing Cas9 were infected with lentivirus expressing the fluorophore mScarlet and sgRNAs targeting the putative tumor suppressor genes. Flow cytometry was used to longitudinally assess kinetics of the mScarlet positive fraction. Preliminary results suggest a selective advantage following deletion of Asxl1 and Acvr2a. Further assessment of tumorigenesis in vivo will be performed to confirm the role of these putative tumor suppressors. Altogether, these genetically manipulable systems can be used for rapid functional interrogation of tumor suppressor genes, facilitating downstream mechanistic studies and preclinical testing of therapies in these important subsets of CRC.

Citation Format: Daniel D. Zhang, Peter M. Westcott, Olivia Smith, Nate Sacks, Haley Hauck, Mary C. Beytagh, Abbey Jin, Tyler Jacks. In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes [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 2924.