Project Summary The incidence of esophageal adenocarcinoma and its premalignant metaplastic precursor Barrett's esophagus (BE) have both displayed marked increases in incidence in the US in recent decades. The progression to esophageal adenocarcinoma occurs through a transformation in which non-dysplastic BE becomes dysplastic BE and acquires the earliest histological features of cancer. Currently, we do not know the molecular drivers of this transition and how it occurs in human patients. The primary objective of this proposal is to identify the earliest events underlying the transition to dysplasia as these will yield new therapeutic and diagnostic targets. Our proposal leverages technological advances in single-cell biology using mitochondrial mutation lineage tracing combined with RNA-sequencing to track clonality and RNA profiles in single cells. Our preliminary data demonstrates the technological approach and reveals that non-dysplastic BE emerges through multiple clones that can generate all BE cell types. However, our preliminary data from dysplastic BE revealed the opposite result. Dysplasia emerged from non-dysplasia BE through a single clone with a distinguishing transcriptional profile. Using our single-cell and clonal tracking data, we identified the Wnt pathway, including Wnt genes LGR5, NOTUM, and DKK1, as differentially expressed in dysplasia. Further supporting the Wnt pathway, we also uncovered an APC mutation occurring in the same sample. Thus, our central hypothesis is that the transformation from non-dysplastic to dysplastic Barrett's esophagus emerges from a single clone with genetic and transcriptional changes that can be tracked to identify new targets for dysplasia. To test this hypothesis, we will implement a comprehensive and rigorous experimental approach that couples single-cell RNA-Seq, mitochondrial mutation-based lineage tracing, and 3D organoids for functional investigation into the mechanisms of dysplasia. The proposed studies will leverage these techniques to track the clonal evolution and RNA profiles of single cells in non-dysplasia and dysplastic BE (Aim 1) and to provide mechanistic exploration into the role of the Wnt pathway in the emergence of dysplasia (Aim 2). This study leverages the research team's diverse and complementary expertise in cancer single cell genomics and bioinformatics, clinical and basic approaches to BE, and in vitro epithelial model systems to develop a robust platform for tracking cell fate in human tissues that will provide unprecedented molecular insight into the development of dysplasia in BE. Our expected outcomes are a molecular characterization of the earliest changes underlying the transformation to dysplasia in BE. Our work will have broad positive impact as it will reveal both therapeutic targets as well as diagnostic markers for patients with BE who will progress to esophageal adenocarcinoma. This work ultimately aims to dramatically decrease the number of patients who are ...