ABSTRACT/SUMMARY The Overall Goal of this application is to identify shared, conserved mechanisms that induce precancerous lesions like metaplasia. Our preliminary and published data indicate that pseudopyloric or so- called SPEM-type metaplasia in stomach is the manifestation of a conserved regeneration program induced by large-scale injury. The metaplastic cells themselves are characterized by a “dedifferentiated” phenotype wherein they express embryonic-like or early developmental markers that proliferate to repair the tissue damage. We hypothesize that the metaplastic/regenerative process is fueled by expansion of a population of embryonic-like progenitor cells (EPCs). EPCs arise in large part from mature secretory that become progenitor-like by following a stepwise, conserved cellular program we call “paligenosis”. Here, we will show preliminary data that Hippo signaling via Nf2 (Merlin) and downstream transcription factors YAP1/TAZ may be a critical, conserved modulator of EPC expansion. In Aim 1, we will test necessity/sufficiency of Nf2 and Yap1/Taz in gastric metaplasia in mouse models and in human and mouse organoids. We will perform discovery based RNA-Seq experiments to uncover new Hippo targets modulating EPCs and metaplasia. In Aim 2 we will look at how these Hippo pathway components interact with the stages of paligenosis we have characterized, whether they can overcome molecular checkpoints between Stages 1 and 2 and between 2 and 3. We will also determine how Hippo signaling interacts with the conserved, paligenosis-dedicated gene Ifrd1, which we will show is required to suppress p53 as cells upregulate mTORC1 to reenter the cell cycle in Stage 3 paligenosis. In Aim 3, we will test whether increasing EPC formation and metaplasia via the Hippo pathway increases tumorigenesis by combining Hippo mutants with: 1) the mutagen MNU; or 2) additional gastric-cancer- related mutant alleles p53 and Cdh1; or 3) by increasing chronic inflammation with the human gastric-cancer- predisposing bacterium H pylori. Experiments were designed to be appropriately powered in collaboration with our biostatistician, Dr. Yan Yan. State-of-the-art imaging (eg. AiryScan live-cell, confocal on organoids; FIB-SEM 3-D ultrastructural nanotomography) will be performed with Dr. James Fitzpatrick in our institutional imaging core; organoid support, and gene editing will be in collaboration with Dr. Blair Madison and our shared organoid core; bioinformatic analysis including synergy with data repositories will be via our collaboration with Dr. Bo Zhang, who directs the institutional bioinformatics core for the Center for Regenerative Medicine.