The risk of colorectal cancer (CRC) is substantially higher in populations consuming Western diets (WDs) and animal models confirm this association. WD-induced tumor promotion requires epidermal growth factor receptor (EGFR) signals that are driven by increased EGFR ligands. EGFR ligand release is controlled by proteinase ADAM17. Colonocyte deletion or pharmacological blockade of ADAM17 suppressed tumorigenesis. Chemokine receptor CXCR4 is expressed on colonocytes up-regulated with cancer progression and can activate ADAM17. The role of CXCR4 in diet-promoted tumorigenesis has not been elucidated. We found that WD up-regulated both CXCR4 and its ligand Sdf1α (aka CXCL12) in colonic mucosa. Together, these data suggest that CXCR4 is a targetable upstream regulator of WD-promoted tumor development by signaling transactivated colon cancer cell EGFR via an ADAM17-dependent mechanism. While CXCR4 is rarely mutated in cancer, its expression is up-regulated by transcription factors (TFs) and epigenetic changes, further evidence that factors such as diet regulate its activity. In preliminary studies, MSX-122, a CXCR4 inhibitor well tolerated in clinical trials, reduced colon tumor incidence by nearly 70% in azoxymethane (AOM)-treated Apc+/Min mice. Moreover, mice deleted of colonocyte CXCR4 developed fewer tumors than CXCR4+/+ mice on an Apc-deficient background. We hypothesize that CXCR4 is required for WD-promoted tumorigenesis, and that natural product (NP) inhibition of CXCR4 is a promising chemopreventative strategy. Studies that dissect CXCR4 regulation and effector signals and identify novel NP inhibitors could converge to uncover new targets for CRC prevention. To study the role of CXCR4 in tumor promotion by WDs and identify novel inhibitors, we propose three specific aims: Aim 1 To determine if CXCR4 activity is required for WD-promoted tumorigenesis. 1a)To assess the ability of CXCR4 inhibitor MSX-122 to suppress AOM tumorigenesis. We will compare MSX effects in Std diet and WD in premalignant and malignant phases and assess effector signals by RNAseq and EGFR activation. 1b) To genetically assess the role of CXCR4 in CRC, comparing colonocyte null CXCR4Δ/Δ mice to CXCR4+/Δ and CXCR4+/+ mice (all on Apc deficient background) and measure end points described in aim 1a. Aim 2a: To determine effects WD and neoplastic progression on TFs and histone modifications regulating Sdf1α and CXCR4 locally using ChIP assays and assess global effects of diet and tumorigenesis using chromatin ATAC-seq and DNA methylation by nano-5hmC-seal using organoids from conditional Apc-CXCR4 model. 5mC and 5hmC signals discovered globally, for example, regulate local histone modifications. 2b) To assess Sdf1αCXCR4 gene regulation and effector signals in human colonic tumorigenesis using organoids as in 2a. Aim 3a To screen NP libraries for CXCR4 inhibitory activity. CXCR4 reporter assays (Ca2+ transients, migration assays and ligand-binding) will be used to confirm computationally predicted NP hits 3b Promising agents identified in aim3a will be prioritized and tested for chemopreventive efficacy in mouse models.