PROJECT SUMMARY Stem and progenitor cells at the gastroesophageal junction (GEJ) have been identified as crucial to the development of adenocarcinoma of the distal esophagus, gastroesophageal junction, and proximal stomach. Combined, these cancers have over 20,000 new cases per year in the U.S., are associated high mortality, and represent a major public health burden. Our group has identified both gastric cardia as well as GEJ transitional basal stem cells as likely cells of origin for precancerous states in this region. However, defining the mechanisms and effectors that drive GE junction stem cell fate and promote cancer development remains a critical gap in knowledge. Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) represent the prototype for neoplasia arising from GE junction stem cells. We have extensive preliminary data demonstrating that circulating secondary bile acids derived from gut bacteria directly promote the development of BE and EAC, treating our L2-IL1B mouse model of BE/EAC with deoxycholic acid (DCA) accelerates neoplasia, and treatment with obeticholic acid, an agonist of nuclear bile acid receptor FXR (farnesoid X receptor), decreases proliferation, GEJ stem cell numbers, and dysplasia. However, the exact mechanisms by which secondary bile acids impact GEJ stem cells and the associated microenvironment have not been elucidated. We hypothesize that circulating secondary bile acids produced by gut bacteria promote early cancer development via direct effects on GE junction stem cells through FXR antagonism and by inducing pro-inflammatory microenvironment alterations. Using highly novel techniques and approaches (including scRNA-Seq and CyTOF), we will perform a series of experiments using mouse models, mouse and human organoids, and with validation of findings in a prospective study of patients, to address the following specific aims: Aim 1. To determine the role of circulating secondary bile acids in GEJ epithelial stem cell fate and early cancer promotion; Aim 2. To assess the effects of circulating secondary bile acids on the GEJ epithelial stem cell microenvironment; Aim 3. To determine whether targeted microbiome modification that regulates the circulating bile acid pool modifies GEJ cancer development. To achieve these aims we will use our unique L2-IL1B mouse model with FXR knockout in stem cells (L2- IL1B/Fxrfl/fl), allowing us to assess the effects of secondary bile acids on GEJ stem cells as well the L2-IL1B/Nlrpfl/fl model to explore inflammasome activation in stem cells and assess for cross-talk with the microenvironment. Ultimately, we will perform experiments treating with distinct consortia of highly characterized bacterial strains to modulate the secondary bile acid producing capacity of the gut microbiome and determine the effects on cancers arising from GE junction stem cells. Elucidation of the specific mechanisms by which secondary bile acids interact with GEJ stem cells and modify the microenvironment to promote cancer development may lead to the identification of novel therapeutic targets, including the potential for rationally designed probiotic therapy, which would have a major public health impact.

PROJECT NARRATIVE Deaths from cancers arising from the gastro-esophageal junction represent a major public health burden, yet the factors that drive early changes in this region remain poorly understood. We aim to gain a clearer understanding of the effects of secondary bile acids produced by bacteria in the gut on stem cells in gastro-esophageal region. This knowledge can in turn be translated to future efforts to target the gut microbiome to prevent gastro- esophageal cancer.