Genetic technology development for elucidating lineage plasticity mechanisms in cancer - PROJECT SUMMARY Lineage identity is an important attribute of human cancer cells, which can influence many aspects of tumor biology, including metastasis and the response to targeted therapies. In some cancers, such as leukemia and sarcoma, there exists a powerful block in cell differentiation which can be overcome through pharmacological modulation of oncoproteins and their epigenetic cofactors. In human carcinomas (e.g. of the pancreas and lung), tumor cells have a malleable identity and can adopt different cell fates to enhance their fitness in vivo and in response to therapy. As the Senior Research Investigator of the Vakoc laboratory, I have been responsible for developing technology for probing lineage identity in cancer using high-throughput genetic screening. This important research builds upon my deep expertise in cell biology and genetics obtained during my doctoral and post-doctoral training, with my skillset continuing to expand as I contribute to several ongoing and completed projects in the Vakoc lab. My technological accomplishments to date include: 1) developing a paralog co- targeting CRISPR screening methodology, which allows for high-throughput `double knockout' screens in search of redundant epigenetic and signaling regulators in cancer 2) developing several different marker-based genetic screening platforms, which we have applied broadly in human cancer models to reveal novel epigenetic regulators of cell fate and 3) Deep-mutational scanning technology, which allows for saturation mutagenesis of transcriptional regulators to map all of functional and non-functional mis-sense mutations. As evidenced by our recent publications, my exploratory screening efforts have yielded compelling and novel modes of epigenetic and signaling regulation, which I have characterized biochemically and using epigenomics in collaboration with the students and postdocs in our group. Outlined in this proposal is my plan for the next 5 years that describes how I will continue to apply advanced genetic screening techniques to address fundamental epigenetic mechanisms related to our NCI-funded projects focused on pancreatic ductal adenocarcinoma, small cell lung cancer, and rhabdomyosarcoma. First, I will develop genetic screens for revealing upstream activators of the MARK kinases, which I recently identified as co-dependencies of YAP/TAZ using paralog co-targeting CRISPR screens. Second, I will also make effective use of a newly developed deep mutational scanning technique, which I will apply to two high-value oncology targets KLF5 and POU2F3 in search of functional surfaces of these transcription factors that engage with their coactivators. Third, I will perform a base editing scan of MED12, in an effort to pinpoint how this protein supports the function of ∆Np63 in specifying basal identity in pancreatic cancer. Finally, I will expand our genetic screens in rhabdomyosarcoma to find regulators that sustain the block in myo-differentiation present in this disease. My technology-driven pursuits are vital to much of the research in our lab, as it seeks to identify targets for epigenetic therapy in human cancer.
