Abstract 4078: Sleeping Beauty transposon mutagenesis identifies genes involved in colorectal cancer progression

Abstract

Abstract Cancer genome sequencing studies have identified numerous genes mutated in colorectal cancers (CRC). Majority of genes, however, were infrequently mutated between tumors. Sorting out infrequent driver genes from passenger genes is still challenging, but important since these infrequent driver genes sometimes seed for metastasis or confer drug resistance. In the previous study, we performed Sleeping Beauty (SB) transposon mutagenesis screens in mice and identified 1,333 candidate driver genes in CRC. These gene included known CRC genes such as Apc, Smad4 and Trp53 as well as novel CRC genes. Using publicly available datasets for genes mutated in human CRC, we compared the genes commonly mutated between human and mice, thus we could enrich a set of candidate driver genes. Among them, we especially focused on candidate tumor suppressor genes since tumor suppressor genes play a fundamental role in CRC development. To validate these candidate tumor suppressor genes, we set up the experimental system using the CRISPR-Cas9 system to knockout the genes in the intestinal organoid culture system. We obtained the mouse intestinal tumor organoid carrying Apc mutations and a KrasG12D activating point mutation (AK organoid), and also obtained organoids derived from human CRC. First, we introduced a Cas9 to obtain Cas9 expressing AK organoids and then introduced a set of 10 gRNAs targeting candidate tumor suppressor genes by lentivirus. These organoids were subsequently transplanted to NSG mice subcutaneously. AK organoid with gRNAs induced tumors, in contrast, AK organoids without gRNA did not induced tumors within 1 month after transplantation showing gene knockout by CRISPR-Cas9 induced tumor development. Next we purified genome from tumors and analyzed the ratio for each gRNA. The gRNA with an expanded ratio in tumors compared to organoids before transplantations are likely to be a novel tumor suppressor gene. So far we validated 30 candidate tumor suppressor genes by this approach and pick up Arid2, Acvr2a and Acvr1b. These 3 genes were mutated in ~5% of human CRC. Since single gene knockout for each of 3 genes in AK organoids also caused tumor development, we were able to confirm that these 3 genes were truly tumor suppressor genes in CRC, We are now validating using human CRC organoid. In summary, we have established the experimental system to validate candidate tumor suppressor genes in CRC using the CRISPR-Cas9 system and the organoid culture system. So far we have succeeded in identifying novel tumor suppresser genes such as Arid2, Acvr2a and Acvr1b. We will validate all candidate tumor suppressor genes and reveal the whole driver genes involved in CRC. Our results will give an important insight in finding new drug targets to cure cancers. Citation Format: Haruna Takeda. Sleeping Beauty transposon mutagenesis identifies genes involved in colorectal cancer progression [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 4078.