PROJECT SUMMARY / ABSTRACT Paligenosis is a recently described evolutionarily conserved repair mechanism that tissues use for regeneration in response to injury. Paligenosis is characterized by the recruitment of differentiated cells back into the cell cycle through a defined 3-stage process, and is thus an important repair pathway when adult stem cells are insufficient. Aberrant paligenosis underlies chronic metaplasia and tumorigenesis. In prior work, the investigators have used both a high-dose tamoxifen (HDT) acute model and a chronic inflammation model incorporating Helicobacter pylori (HP) to induce zymogenic chief cells to reprogram into spasmolytic-polypeptide expressing metaplasia (SPEM) via the paligenosis pathway. They showed that ATF3 and IFRD1 play crucial roles in paligenosis. Additional preliminary data indicates that the early stages of paligenosis is activated by the unfolded-protein response (UPR) wing of the Integrated Stress Response (ISR), which is governed by the PERK kinase, whereas the dsRNA-responding (ISR) enzyme PKR contributes to the later stages of paligenosis. Preliminary data also indicate that early disassembly of rough endoplasmic reticulum (rER) is an early paligenosis event. This supports a hypothesis that the early events of paligenosis are driven by the PERK wing of the ISR and the dynamic regulation and autophagy of rER. Aim 1 of this project thus seeks to detail activation of PERK over a granular timecourse early in paligenosis in HDT and Helicobacter models, and then test the PERK requirement using PERK and ISR inhibitors, ISR-deficient Eif2aS51A mice, and Perkflox/flox mice crossed to chief cell-specific promoter mice. Sufficiency will be tested by inducing UPR and by drug-induced activation of PERK. Aim 2 will detail paligenotic ER remodeling in HDT and HP models, as well as investigate the impact of blocking autophagolysome activity in paligenotic-lysosome-defective Atf3−/− mice. Using ER-phagy defective mice (Ccpg1flox/flox), the effects of ISR and ER-phagy deficiency on tumorigenesis will also be examined in an established mouse model of paligenosis and tumor formation. Aim 3 will expand on the sample size and timepoints of our pilot phosphoproteome data, and localize protein and phosphoprotein expression in paligenotic cells by corroboration with single-cell RNA-sequencing. This multi-omic approach enables generation of a comprehensive map of the protein and kinase activity that governs the early stages of paligenosis, while also opening a pipeline for future discovery of paligenosis genes that may be novel therapeutic targets for prevention of metaplasia and its associated complications in various tissues.