Project Summary Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. The long-term objective of my research on this award will be to understand the cell-type specificity and molecular mechanisms through which inactivation of the APC tumor suppressor drives the ontogeny of colorectal cancer. APC is mutated in the vast majority of CRC and is broadly considered the initiating event in most of these cancers. Because of this, there has been a large amount of research into the molecular mechanisms through which APC functions. APC is a well-established negative regulator of the canonical Wnt signaling pathway, and consequently a wealth of evidence exists demonstrating that constitutive Wnt pathway activation downstream of APC loss is necessary for initiation and maintenance of CRC. Recent data however suggests that this body of research has largely overlooked several additional important functions of this tumor suppressor, and that such functions will represent valuable points for preventative action and therapeutic intervention in CRC. Specifically, I have uncovered additional oncogenic pathways activated upon APC loss that are also necessary for initiation and maintenance of CRC. In particular, I recently published data demonstrating that the Msi family of RNA binding proteins are activated upon APC loss in a pathway that appears to lie in parallel to and not downstream of -CATENIN. I went on to show that Msi activity is necessary for CRC initiation and maintenance, and that Msi gain of function alone is sufficient to transform the intestinal epithelium in a Wnt-independent manner. At the cellular level, my colleagues and I found that Msi activity acts specifically to drive metabolic activation of quiescent (in G0) intestinal stem cells (ISCs) resulting in cell cycle entry, proliferation, and a block in their differentiation. In contrast, Msi activity has no discernable molecular effect on the active ISC population driven by Wnt pathway activity and previously posited to be the cell-of-origin in CRC. Previous studies have found that these quiescent ISCs are refractory to canonical Wnt pathway stimulation in their dormant state. Therefore, the central hypothesis of my work in the Lengner lab moving forward is that loss of APC can initiate tumorigenesis through promiscuous activation of quiescent intestinal stem cells via Wnt-independent mechanisms. I propose that APC loss initiates a number of oncogenic pathways independent of the canonical Wnt pathway activation (including Msi induction). I will test whether APC loss can initiate CRC by driving quiescent ISCs out of G0 and into the cell cycle, thus establishing quiescent ISCs as a cell-or-origin in colorectal cancer. I will address this hypothesis using a combination of genomic and genetic techniques with the ultimate goal of testing whether Wnt- independent pathways activated downstream of APC loss are viable points for therapeutic intervention in CRC.