Abstract B11: Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila

Colorectal cancer (CRC) is an extremely complex and genetically diverse disease with few available models that recapitulate the key features of heterogeneity. The Cancer Genome Atlas has identified 30 recurrent mutated genes as drivers of CRC. Individual tumors can carry any number of combinations of these mutations. A critical challenge in research has been the ability to model these genetically heterogenous tumors in animal systems. Highly sophisticated genetic tools in Drosophila make it an ideal model system to create and study different genome-based CRC tumors that well complement other animal models. Highly conserved signaling pathways and high similarity in gut homeostasis also make it a powerful and representative system to better understand CRC tumors and their progression. We have previously generated a panel of genetically complex Drosophila models that reflect the genomic landscape of sequenced colon tumors. The most complex model in this panel carried transgenic constructs to knock down Drosophila orthologs of tumor suppressors TP53, APC, SMAD4, and PTEN as well as to express oncogenic KRASG12V. However, creating more complex models using standard Drosophila crosses has been challenging. To address this, we have generated a multigenic vector that carries three different multiple cloning sites (MCSs) designed for cloning three separate transgenes. Two are designed for inducible protein expression to model oncogenic activation while the third is for a micro-RNA inspired synthetic multi-hairpin cluster to knock down multiple tumor suppressors. Here, we will report our efforts to further validate and expand this technology to build more complex cancer models. Using a series of test constructs, we show that transgenic flies carrying synthetic clusters of as many as 16 hairpins can be generated. Hairpins expressed in the context of synthetic clusters are as effective as single hairpins with no detectable positional effects. Finally, by introducing additional design elements such as intron-mediated hairpin cluster expression and bicistronic protein expression using a T2A self-cleaving peptide design, we have generated transgenes that express two proteins and up to 16 hairpins from a single transcript. We then used this updated platform to build a proof-of-concept colorectal cancer model that reflects IRS2 amplification, oncogenic KRASG12V, and loss of tumor suppressors TP53, APC, SMAD4, and SMAD2 from a single transcript designed for inducible expression. This new design frees up the remaining two cloning sites in our multigenic vector for additional genetic manipulations and pushes the limits of our technology to build a new generation of colorectal cancer models to study features of human tumors that have been traditionally challenging to capture in experimental models. Using this technology, we hope to better understand tumor development and find potential therapies that can specifically target these unique, personalized CRC genome-based models. Citation Format: Maria Quintero, Alexander Teague, Ross Cagan, Erdem Bangi. Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B11.