Colorectal cancer (CRC) is growing global threat, especially among younger adults. Impaired differentiation is a critical mechanism by which CRC cells unlock cell state plasticity; it enables neoplastic cells to persist rather than turnover in the rapidly renewing intestinal epithelium and eventually acquire malignant features. The molecular underpinnings of impaired differentiation are complex and challenging to unravel. Recent studies from our group spotlight the transcription factor (TF) SOX9 as a pivotal functional mediator of impaired differentiation in CRC. Disrupting SOX9 induced differentiation and reduced tumor burden in several CRC models, sparking deeper inquiry. Our preliminary data support a compelling link between aberrant SOX9 activity and reactivation of fetal intestinal genes in mouse and human models of CRC. Based on these findings, our overall hypothesis is that SOX9 impairs differentiation by facilitating developmental reprogramming in CRC. To investigate this hypothesis, we have developed powerful in vitro and in vivo systems to address two Specific Aims. In Aim 1, we will apply unbiased topic modeling analyses to paired singe cell RNA and ATAC sequencing data from genetically engineered mice to nominate regulators of fetal reprogramming in neoplastic cells (1A). By integrating mRNA, open chromatin, and TF binding profiles from our mouse models and patient-derived organoids, we will investigate if SOX9 directly regulates fetal genes via pioneer activity (1B). We will determine if SOX9 is required for fetal gene reactivation in vivo through histopathology and single cell characterization of a genetic mouse model (1C). Finally, we will leverage a newly engineered cellular platform that enables drug- induced rapid degradation of endogenous SOX9 (dTAG system) to study its role in chromatin access, histone mark deposition, and fetal gene expression (1D). We observed new chromatin accessibility at TACSTD2 and aberrant expression of its protein product TROP2, a fetal glycoprotein, in human CRC. Our preliminary data also indicate that SOX9 directly regulates transcription of TACSTD2. In Aim 2, we will test the hypothesis that TROP2 is a functional component of the SOX9-mediated differentiation block in CRC by evaluating its necessity (2A) and sufficiency (2B). Beyond TROP2, we will systematically evaluate short-listed developmental factors using a custom engineered endogenous differentiation reporter system, asking whether each candidate is required for impaired differentiation in a positive selection genetic screen (2C). Through the functional investigation of TROP2, we will strengthen a key link between SOX9 and developmental reprogramming. Overall, armed with robust model systems, rigorous scientific methods, and a hypothesis-driven approach, our proposal will define basic mechanisms by which aberrant SOX9 activity promotes CRC. By offering deeper insights into impaired differentiation and aberrant cell state plasticity, we aim...