Grant: NCI R01CA190558-06A1, Verzi, PI Parent project abstract: A lack of knowledge about the initiating moments of tumorigenesis leaves large gaps in our ability to detect cancer early or develop prevention strategies. Our primary objective of this competitive renewal is to define, in vivo, the immediate consequences of oncogenic mutations in BRAF during Serrated colon cancer formation. We will pursue these studies in colon stem cells, the presumed cell-of-origin of colon cancer. Work from the previous grant identified new genetic modifiers of Serrated tumorigenesis. We found that oncogenic BRAFV600E mutations paradoxically cause stem cells to differentiate rather than form tumors. However, when we inactivated tumor suppressor transcription factors CDX2 or SMAD4, Serrated tumorigenesis was markedly restored. Excitingly, we found that CDX2 and SMAD4 target genes can be used to stratify human patients for their susceptibility to Serrated tumors. The logical extension of these studies is to understand the molecular mechanisms that occur in stem cells in response to BRAFV600E mutations. We predict that pERK takes advantage of accessible chromatin upon CDX2 loss. Aim 1 will compare the activity of pERK in wild type stem cells, and BRAFV600E or CDX2-mutant stem cells. State-of-the-art –omics approaches will allow first-of-a-kind measurements of pERK on stem cell chromatin, within hours after the BRAFV600E mutation is expressed from its endogenous locus. We will measure pERK binding activity, nuclear localization, and dynamic interactions with its protein partners. Aim 2 will look at the downstream gene regulatory consequences of the BRAFV600E mutation in stem cells lacking SMAD4. We predict that SMAD4 works with pERK to promote differentiation and suppress stem cell renewal. Epigenomics approaches will map pERK binding to the genome, and ATAC-seq and RNA-seq will profile changes to chromatin and the transcriptome. Finally, Aim 3 will functionally test a model that the injury/repair cycle in the colon can create a susceptible environment for BRAF-pERK to drive tumorigenesis, with the prediction that an altered transcriptional network is permissive to Serrated tumor formation during the injury/repair cycle. These studies would provide important pre-clinical models to help explain and test therapeutic strategies to suppress Serrated tumor initiation in humans. These studies are innovative with cutting edge -omics applications and GEMMs and significant in exploring untested mechanisms of oncogenic pERK in stem cells. Successful completion of these studies will have us poised to test therapeutic strategies for the prevention or early detection of Serrated Colon Cancer.