Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. LS is caused by inherited mutations in the DNA mismatch repair (MMR) genes, defects in which also underlie 15-30% of sporadic colorectal cancers. Loss of MMR function is associated with a 1,000-fold increase in mutation rate likely increasing the risk of mutation to important oncogenes and tumor suppressors. The MMR pathway also activates cell cycle checkpoints and cell death in response to exogenous DNA damage, however, the role of this damage response in preventing tumorigenesis is not known. We hypothesize that colonic stem cells (CSCs) that lose this MMR-dependent damage response will gain a selective advantage over neighboring MMR-proficient cells, particularly in a mutagenic environment such as may be found in the colon. We predict that loss of MMR will enhance survival under conditions of increased DNA damage, favoring these cells in a competition for stem cell niche occupancy. Ultimately, this will lead to the production of more hypermutable intestinal cells, increasing the penetrance of the cancer phenotype. Testing this hypothesis has been problematic previously due to lack of a suitable model system. However, the recent development of human colonic organoid and enteroid models now allow us to study the effects of MMR loss on CSC dynamics via the following aims: 1) Determine whether loss of MMR function in human embryonic stem cells (hESCs) leads to an immediate advantage in the absence or presence of exogenous DNA damaging agents. We will use CRISPR/Cas9-mediated gene editing to knock out the MMR genes in hESCs and examine their growth and damage response as well as determine the mechanism underlying those responses. 2) Determine whether MMR-deficient CSCs have a selective advantage in colonic organoids and colonic enteroids. For this purpose, we will differentiate MMR-proficient and deficient hESCs into colonic organoids. As a complementary model, we will also create MMR knock out enteroids from human adult colon tissue samples. Using both systems, we will compare the response to exogenous DNA damaging agents or oncogenic stress. We will also create mixed organoids containing MMR-proficient and deficient cells and test whether the MMR-deficient cells have a growth or survival advantage over time in the presence or absence of DNA damage. 3) Determine whether MMR loss leads to a selective advantage for CSCs in vivo. We will use an inducible, stem cell specific knockout mouse model of Msh2 to create mosaic intestinal crypts and test whether MMR-deficient CSCs outcompete wild-type CSCs. Together, these aims utilize novel approaches to study the mechanism by which loss of MMR function contributes to tumorigenesis providing information that may help explain disease penetrance while guiding the diagnosis, prevention and treatment of LS-associated cancers.