PROJECT SUMMARY The ability to perform sophisticated genetic manipulations and large-scale exploratory studies is a key strength of Drosophila as a model system. This proposal leverages this strength and pushes the limits of Drosophila genetics to establish a novel, innovative and ambitious platform that allows sequential introduction of genetic manipulations —each coupled with a different fluorescent protein— into individual cells. My laboratory is interested in using this platform to build and study tumors in the adult Drosophila intestine composed of genetically heterogeneous cell populations with distinct mutation profiles (i.e. subclones). We have previously used tumor sequence data as blueprints to build several colorectal cancer models by genetically manipulating Drosophila orthologs of human cancer driver genes in the adult fly intestine. We now seek to expand these efforts to building new cancer models that reflect tumor subclonal architectures identified by sequencing and computational studies. Tumors arise by sequential accumulation of genomic alterations in cancer driver genes. Consequently, most solid tumors are composed of subclones with distinct mutation profiles in constant competition for limited space and resources. Therapy often alters subclonal dynamics by introducing additional selective pressures, eventually leading to the expansion of resistant subclones and therapy failure. Building subclonal tumors is a technically challenging problem that requires sophisticated genetic manipulations and represents an important area of unmet medical need. Drosophila is a useful cancer model that captures several key hallmarks of human tumors, including colorectal cancer. The adult Drosophila intestine is a well characterized tissue with multipotent intestinal stem cells where signaling pathways altered in colon tumors are remarkably conserved and well characterized. The platform proposed here, combined with practical advantages of Drosophila, provides a unique opportunity to generate tumors with diverse subclonal architectures observed in human tumors. If successful, this technology will complement mammalian cancer models by providing an experimental platform to functionally explore yet another layer of complexity of human tumor genome landscapes revealed by big cancer data. We also hope to use this platform to explore the impact of altering the order of cancer driving genomic alterations on tumorigenesis and the potential of different cell types of the intestine as tumor cells of origin. The platform proposed here is a flexible technology that can easily be combined with other genetic tools to generate populations of cells carrying different genetic manipulations barcoded with unique fluorescent protein combinations in any tissue of interest. If successful, it could open up promising opportunities in other research areas including developmental biology, stem cell biology and neuroscience by providing a platform to explore mechanisms o...